title: Adipocyte lipin 1 is positively associated with human metabolic health and regulates mouse systemic metabolism

status: Public on Jul 15 2024

city: Kansas City

first: Andrew

last: Lutkewitte

state: Kansas

address: 3901 Rainbow Blvd

institute: The University of Kansas Medical Center

summary: Dysfunctional adipose tissue is believed to promote the development of hepatic steatosis and systemic insulin resistance, but many of the mechanisms involved are still unclear. Lipin 1 catalyzes the conversion of phosphatidic acid to diacylglycerol (DAG), the penultimate step of triglyceride synthesis, which is essential for lipid storage. Herein we found that adipose tissue LPIN1 expression is decreased in people with obesity compared to lean subjects and low LPIN1 expression correlated with multi-tissue insulin resistance and increased rates of hepatic de novo lipogenesis. Comprehensive metabolic and multi-omic phenotyping demonstrated that adipocyte-specific Lpin1-/- mice had a metabolically-unhealthy phenotype, including liver and skeletal muscle insulin resistance, hepatic steatosis, increased hepatic de novo lipogenesis, and transcriptomic signatures of nonalcoholic steatohepatitis that was exacerbated by high-fat diets. We conclude that adipocyte lipin 1-mediated lipid storage is vital for preserving adipose tissue and systemic metabolic health and its loss predisposes mice to nonalcoholic steatohepatitis.

overall_design: 2 x 2 design. Littermate wildtype mice (Controls) and adipocyte-specific Lpin1 knock out mice were fed a low fat diet or a high fat diet for 16 weeks. Mice were fasted for four hours proir to euthanasia where liver tissue was excised and flash frozen. RNA was isolated from livers and submitted for sequencing.

first: Brian

last: Finck

first: Trevor

last: Shew

first: Jason

last: Singer

first: Daniel

last: Ferguson

last: LaPoint


title: Epigenetic regulation of p63 blocks squamous-to-neuroendocrine transdifferentiation in esophageal development and malignancy [ChIP-seq]

status: Public on Jul 26 2024

city: New York

first: Dimitris

last: Karagiannis

state: NY

country: USA

institute: Columbia University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: While cell fate determination and maintenance are important in establishing and preserving tissue identity and function during development, aberrant cell fate transition leads to cancer cell heterogeneity and resistance to treatment. Here, we report an unexpected role for the transcription factor p63 (Trp63/TP63) in the fate choice of squamous versus neuroendocrine lineage in esophageal development and malignancy. Deletion of p63 results in extensive neuroendocrine differentiation in the developing mouse esophagus and esophageal progenitors derived from human embryonic stem cells. In human esophageal neuroendocrine carcinoma (eNEC) cells, p63 is transcriptionally silenced by EZH2-mediated H3K27 trimethylation (H3K27me3). Upregulation of the major p63 isoform ΔNp63α, through either ectopic expression or EZH2 inhibition, promotes squamous transdifferentiation of eNEC cells. Together these findings uncover p63 as a rheostat in coordinating the transition between squamous and neuroendocrine cell fates during esophageal development and tumor progression.

overall_design: ChIP-seq of H3K27me3 in DMSO-treated TYUC-1 cells for 6 days.

first: Yongchun

last: Zhang

first: Xiao

last: Chen

first: Jianwen

last: Que

first: Chao

last: Lu


title: Single-cell atlas of dura cells from non-tumor or leptomeningeal metastasis mice

status: Public on Jul 24 2024

city: Shanghai

first: Yudan

last: Chi

address: 131 Dongan Road

country: China

institute: Fudan University

type: Expression profiling by high throughput sequencing

summary: We performed scRNA-seq on the cells isolated from the dura mater in non-tumor control mice and leptomeningeal metastasis (LM) mice with LLC-LeptoM cell inoculation for 7 or 12 days.

overall_design: Dura cells were isolated from both control mice and LM mice at stage day 7 and day 12. Total  10 samples were analyzed using scRNAseq, including three samples from control mice, four samples from LM mice at day 7, and three samples from LM mice at day 12.

first: Jiaxu

last: Zhao

first: Rui

last: Zeng


title: Epigenetic regulation of p63 blocks squamous-to-neuroendocrine transdifferentiation in esophageal development and malignancy [RNAseq_ip63]

overall_design: We performed RNA-sequencing of dmso- or doxycycline-treated TYUC-1 cells for induction of Tap63 and ΔΝp63 expression. We assessed 3 biological replicates.


title: High-Throughput Screen Identifies Non-Inflammatory Small Molecule Inducers of Trained Immunity

status: Public on Jul 08 2024

city: Chicago

first: Luis

middle: Bruno

last: Barreiro

state: IL

address: 900 E 57th St

department: Genetic Medicine

institute: University of Chicago

summary: Trained immunity is a form of innate immune memory characterized by epigenetic and metabolic reprogramming in response to specific stimuli. This rewiring can result in increased cytokine and effector responses to pathogenic challenge, providing non-specific protection against disease. It may also improve immune responses to established immunotherapeutics and vaccines. Despite the promise of training for next-generation therapeutic design, most current understanding and experimentation is conducted with complex and heterogeneous biologically derived molecules, such as β-glucan or the BCG vaccine. This limited collection of training compounds also limits study of the genes most involved in training responses as each molecule has both training and non-training effects. Small molecules with tunable pharmacokinetics and delivery modalities would both assist in the study of trained immunity and its future application for therapeutics. To identify novel small molecule inducers of trained immunity, we screened a library of 2000 drugs and drug-like compounds. Identification of well-defined compounds can improve our understanding of innate immune memory and broaden the scope of its clinical applications. We identified over 2 dozen small molecules in several chemical classes, including the traditionally immunosuppressive glucocorticoids, that induce a training phenotype in the absence of initial immune activation – a current limitation of reported inducers of training. We chose 7 of these top candidates to characterize and establish training activity in vivo. In this work, we expand the number of compounds known to induce trained immunity, creating new avenues for the study and application of innate immune training.

overall_design: In order to discover epigentic changes initiated by these novel small molecules, we performed assay for transposase accessible chromatin sequencing (ATAC-seq) on bone marrow derived macrophages (BMDMs) from a single female mouse. In a 96 well plate, we treated 100,000 BMDMs with 10 μM each of seven small molecules found to induce innate immune training in a high throughput screen. As a positive control for training, we also treated BMDMs with 10 μM of Beta Glucan. As negative control for training, we treated BMDMs with phosphate buffered saline (PBS). Three biological replicates each were sequenced for the beta glucan treated BMDMs and for the seven experimental small molecules. We sequenced six biological replicates of the PBS treated BMDMs to increase statistical power. Two sequencing runs were performed on the same samples to increase sequencing depth (see raw files from sequencing run #1 labeled "AEK-RK..." vs from sequencing run #2 "AEK-HRK...").

first: Hannah

middle: R

last: Knight

first: Ellen

last: Ketter

last: Ung

first: Adam

last: Weiss

first: Jainu

last: Ajit

first: Qing

first: Jingjing

last: Shen

first: Ka

middle: M

last: Ip

first: Chun-yi

last: Chiang

first: Aaron

last: Esser-Kahn


title: A Metabolic Switch Controlling Liver Cancer Evolution from Senescent NASH Hepatocytes

city: Chongqing

first: Yahui

last: Zhu

address: ACampus, Chongqing University,Shapingba District, Chongqing, China

institute: Chongqing University

summary: Hepatocellular carcinoma (HCC), the third most common cancer, originates from differentiated hepatocytes undergoing compensatory proliferation in livers damaged primarily by viruses or nonalcoholic steatohepatitis (NASH)1,2. While increasing HCC risk3, NASH also induces TP53-dependent hepatocyte senescence4. How this tumor-suppressive response is activated but eventually bypassed to license HCC progression is unknown. We identified the gluconeogenic enzyme fructose-1,6-bisphosphatase 1 (FBP1) as a TP53 target, induced in senescent NASH hepatocytes but downregulated in most human HCCs. Initial FBP1 downregulation in disease-associated hepatocytes, whose accumulation precedes HCC development5, activates AKT to inhibits GSK3 substrate binding, thereby augmenting activation of NRF2 which accelerates FBP1 and TP53 degradation. Intrinsic NRF2 activation and FBP1 loss trigger overlapping transcriptomic responses that suppress senescence, enhance hepatocyte proliferation and metabolism, and enable NRAS- or NASH-induced hepatocarcinogenesis. This AKT-dependent metabolic switch, operative in mice and humans, controls NASH to HCC progression. Our results further suggest that NASH-related hepatocyte senescence is triggered by hypernutrition-induced single strand DNA breaks. Senescence reversal enables HCC progenitor expansion and somatic mutagenesis.

overall_design: For HCC induction by hydrodynamic injection, Fbp1ΔHepor Nfe2l2Act-Hep mice were intravenously(i.v.) injected with NRASG12V (12.5 μg/ml) and Sleeping Beauty transposase (SB13) (2.5 μg/ml) plasmids diluted in Ringer’s solution at 0.1 ml/g body weight in 5-7 seconds.

first: Li

last: Gu

first: Shuvro

middle: P

last: Nandi

first: Michael

last: Karin


title: Personalized dendritic cell vaccine facilitates the preparation of neoantigen-reactive T cells for adoptive transfer (scTCR-Seq)

status: Public on Jul 05 2024

city: chengdu

first: Hao

state: sichuan

address: 37 Guoxue Alley

institute: West China Hospital

summary: Adoptive cell transfer (ACT) using neoantigen-specific T cells is an effective immunotherapeutic strategy. However, the difficulty in identifying and screening neoantigen-specific T cells limits its widespread application. Here, we prepared neoantigen-reactive T cells (NRTs) after immunization with a tumor lysate-loaded dendritic cell (DC) vaccine (OCDC) for ACT. Our results demonstrated that the OCDC vaccine could induce a neoantigen-specific immune response, and it was feasible to prepare NRTs by loading immunogenic neoantigens onto DCs and coculturing them with spleen lymphocytes from mice immunized with the OCDC vaccine. We then transferred these NRTs back to the LL/2 tumor-bearing mice after OCDC vaccine immunization and found that OCDC vaccine and NRTs adoptive transfer combination treatment could induce a stronger antitumor response. Furthermore, we found that infused NRTs could migrate into the tumor microenvironment to exert antitumor effects. Our research provides a new and convenient method of preparing NRTs for ACT. The clinical translation of this approach has the potential to increase ACT efficacy.

overall_design: We sequenced five groups of mice treated differently through scRNA-seq and scTCR-seq.The sequencing was performed by the 10x Genomics single-cell 5′ V(D)J library platform. The cell suspension was loaded into Chromium microfluidic chips with V3 chemistry and barcoded with a 10× Chromium Controller (10X Genomics). Single-cell gene expression and TCR libraries were generated according to the manufacturer’s instructions. RNA from the barcoded cells was subsequently reverse-transcribed, and sequencing libraries were constructed with reagents from a Chromium Single Cell 30 v3 reagent kit (10X Genomics) according to the manufacturer’s instructions. Sequencing was performed with Illumina according to the manufacturer’s instructions (Illumina).

last: Yang


title: Epigenetic regulation of p63 blocks squamous-to-neuroendocrine transdifferentiation in esophageal development and malignancy [CUTnTag_ip63]

first: Dimitris

last: Karagiannis

overall_design: CUT&Tag of p63α in doxycycline-treated TYUC-1 cells for induction of ΔΝp63 expression. Included are 1 IgG control sample and 2 p63α technical replicates.

first: Chao

last: Lu


title: Personalized dendritic cell vaccine facilitates the preparation of neoantigen-reactive T cells for adoptive transfer (scRNA-Seq)

last: Zeng


title: The specificity of hpCasMINI for gene editing.[Tag-seq]

status: Public on Jun 29 2024

city: Guangzhou

first: Kechen

last: Chen

state: Guangdong

address: Shatai South Road

institute: Southern Medical University

summary: The hpCasMINI exhibits high specificity for gene editing. Compared to SpCas9 and LbCas12a, hpCasMINI also exhibited higher specificity for genomic DNA cleavage at the tested sites.

overall_design: We evaluated the specificity of hpCasMINI-mediated gene editing in human cells using Tag-seq. Within the 10 tested sites, hpCasMINI exhibited about 2.5-fold increased activity and similar specificity compared to CasMINI. we also compared the DNA cleavage activity of SpCas9, LbCas12a, CasMINI, and hpCasMINI across four genomic sites at the same location and five sites at adjacent locations, hpCasMINI also exhibited higher specificity for genomic DNA cleavage at the tested sites.

first: Shufeng

last: Ma

first: Ying

last: Lin

first: Zhili

last: Rong


title: Estrogen-triggered Monkey Multi-Organ Cell Atlas [scATAC-seq]

status: Public on Jul 25 2024

city: Nan Jing

first: Fang

last: Wen

address: Xianlin street

institute: Nanjing University

summary: Awareness of estrogen's effects on health is broadening rapidly. The effects of long-term high levels of estrogen on the body involve multiple organs. Here we use both single cell chromatin accessibility and RNA sequencing data to analyze the potential effect of estrogen on major organ at single cell levels.

overall_design: A total of 10 scRNA-seq data and 10 paired scATAC-seq data for different organs and groups were available

first: Yang

last: Sun

first: Jiao

last: Qu

first: Wen

last: Fang

first: Wanjun

last: Zhao

first: Xinran

last: Cao


title: CRISPRi-based screens in iAssembloids to elucidate neuron-glia interactions

status: Public on Jul 12 2024

city: San Francisco

first: Emmy

last: Li

email: emmyli0306@gmail.com, emmy.li@ucsf.edu

state: CA

address: 675 Nelson Rising Lane

institute: UCSF

summary: The sheer complexity of the brain has complicated our ability to understand its cellular mechanisms in health and disease. Genome-wide association studies have uncovered genetic variants associated with specific neurological phenotypes and diseases. In addition, single-cell transcriptomics have provided molecular descriptions of specific brain cell types and the changes they undergo during disease. Although these approaches provide a giant leap forward towards understanding how genetic variation can lead to functional changes in the brain, they do not establish molecular mechanisms. To address this need, we developed a 3D co-culture system termed iAssembloids (induced multi-lineage assembloids) that enables the rapid generation of homogenous neuron-glia spheroids. We characterize these iAssembloids with immunohistochemistry and single-cell transcriptomics and combine them with large-scale CRISPRi-based screens. In our first application, we ask how glial and neuronal cells interact to control neuronal death and survival. Our CRISPRi-based screens identified that GSK3β inhibits the protective NRF2-mediated oxidative stress response in the presence of reactive oxygen species elicited by high neuronal activity, which was not previously found in 2D monoculture neuron screens. We also apply the platform to investigate the role of APOE-𝜀4, a risk variant for Alzheimer’s Disease, in its effect on neuronal survival. We find that APOE-𝜀4 expressing astrocytes may promote more neuronal activity as compared to APOE-𝜀3 expressing astrocytes.  This platform expands the toolbox for the unbiased identification of mechanisms of cell-cell interactions in brain health and disease.

overall_design: Examination of cellular differention and composition in 3D neuron-astrocyte-microglia co-culture models (iAssembloids)

last: Emmy


title: scRNA-seq profiling of lymph node activation in non-human primate Rhesus macaques by aluminum hydroxide and saponin adjuvant SMNP

status: Public on Jul 01 2024

city: Cambridge

first: Darrell

middle: J.

last: Irvine

state: MA

address: 77 Massachusetts Ave

institute: Massachusetts Institute of Technology

summary: Saponin-based vaccine adjuvants are potent in preclinical animal models and humans, but their mechanisms of action remain poorly understood. Using a stabilized HIV envelope trimer as a model immunogen, we carried out studies in non-human primates (NHPs), comparing the most common clinical adjuvant alum and Saponin/MPLA Nanoparticles (SMNP), a novel ISCOMs-like adjuvant. SMNP elicited substantially stronger humoral immune responses than alum and induced strong type I interferon transcriptional signatures, expansion of innate immune cells, and increased antigen presenting cell activation in LNs. These findings indicate that SMNP promotes multiple facets of the early immune response relevant for enhanced immunity to vaccination.

overall_design: Lymph nodes from three NHPs per group were collected at 48 hours following immunization and were processed for scRNA-seq analysis. A contralateral inguinal lymph node without antigen signal from an NHP immunized with alum was collected as the control. CD3lowCD8lowCD20low single cells were flow-sorted to moderately enrich for cells of myeloid lineage using flow cytometry.

first: Yiming

middle: J

last: Zhang

first: Laura

last: Maiorino

first: Parisa

last: Yousefpour

first: J

middle: C

last: Love


title: Effects of ZAKalpha on UVB-dependent trancriptional changes in N/TERT-1 cells

status: Public on Jul 03 2024

city: Singapore

first: Franklin

last: ZHONG

state: Singapore

address: 11 Mandalay Road

country: Singapore

institute: NTU LKC School of Medicine

summary: This study investigates the effect of ZAKalpha kinase in UVB-triggered signaling in an immortalized keratinocyte cell line N/TERT-1. The experiments compared sham-irradiated and UVB-irradiated isogenic Cas9 control and ZAK KO cells in order to define which UVB responsive transcripts are controlled by ZAKalpha.

overall_design: Isogenic Cas9 control and ZAK KO N/TERT-1 cells are either sham irradiated or UVB-irradiated (100mJ2/cm2). All cells were harvested 4 hours after irradiation.

last: Zhong


title: SLAM/SAP signaling regulates discrete γδ T cell developmental checkpoints and shapes the innate-like γδ TCR repertoire

city: Burlington

first: Jonathan

middle: E

last: Boyson

state: Vermont

address: 89 Beaumont Ave.

department: Surgery

institute: University of Vermont

summary: During thymic development, most γδ T cells acquire innate-like characteristics that are critical for their function in tumor surveillance, infectious disease, and tissue repair. The mechanisms, however, that regulate γδ T cell developmental programming remain unclear. Recently, we demonstrated that the SLAM-SAP signaling pathway regulates the development and function of multiple innate-like γδ T cell subsets. Here, we used a single-cell proteogenomics approach to identify SAP-dependent developmental checkpoints and to define the SAP-dependent γδ TCR repertoire. SAP deficiency resulted in both a significant loss of an immatureGzma+Blk+Etv5+Tox2+γδT17 precursor population, and a significant increase inCd4+Cd8+Rorc+Ptcra+Rag1+thymic γδ T cells. SAP-dependent diversion of embryonic day 17 thymic γδ T cell clonotypes into the αβ T cell developmental pathway was associated with a decreased frequency of mature clonotypes in neonatal thymus, and an altered γδ TCR repertoire in the periphery. Finally, we identify TRGV4/TRAV13-4(DV7)-expressing T cells as a novel, SAP-dependent Vγ4 γδT1 subset. Together, the data suggest that SAP-dependent γδ/αβ T cell lineage commitment regulates γδ T cell developmental programming and shapes the γδ TCR repertoire.

overall_design:
Single-cell CITE-Seq and TCR repertoire profiling of γδ T cells across various developmental stages and mouse models shows the influence of SLAM/SAP signaling on discrete γδ T cell developmental checkpoints and the shaping of the innate-like γδ TCR repertoire. Thymus single-cell CITE-seq with TCR repertoire analysis includes embryonic day 17 (E17) B6 and B6.Sh2d1a-/- (SAP-/-) mice with four biological replicates each, along with nine-day-old (D9) neonatal B6 and B6.SAP-/- mice with three biological replicates each. Additionally, the dataset encompasses thymus samples from six-week-old B6 and B6.SAP-/-mice with three replicates per group. Following thymic analysis, single-cell RNAseq with TCR repertoire analysis of adult lung γδ T cellsinvolved pooled cells from three 13-week-old B6 and B6.SAP-/- littermates per group. These experiments generated three libraries per experiments: RNA, VDJ, and cell surface protein (CSP). The cell surface protein (CSP) libraries encompassed both Antibody-derived tags (ADTs)and hashtag oligonucleotides (HTOs). This comprehensive approach sheds light on the molecular mechanisms underlying γδ T cell development and the impact of SLAM/SAP signaling across different developmental stages and mouse models.
---------------------------------------
ADT/HTO barcode sequence hashtagged sample ADT sample
TotalSeq-C0212 anti-mouse CD24 TATATCTTTGCCGCA  E17thy, D9thy, W6thy
TotalSeq-C0073 anti-mouse CD44 TGGCTTCAGGTCCTA  E17thy
TotalSeq-C1063 anti-mouse CD45RB TTGTATCTCCCTTGG  E17thy
TotalSeq-C0209 anti-mouse Vg1 TCGTTTAACCAGCCT  E17thy
TotalSeq-C0211 anti-mouse Vg4 AAGCTGCACCGTAAT  E17thy, D9thy, W6thy, Lung
TotalSeq-C0930 anti-mouse SLAMF6 CGATTCTTTGCGAGT  E17thy, D9thy, W6thy
TotalSeq-C0203 anti-mouse SLAMF1 CAACGCCTAGAAACC  E17thy, D9thy, W6thy
TotalSeq-C0077 anti-mouse CD73 ACACTTAACGTCTGG  E17thy, D9thy, W6thy
TotalSeq-C0301 anti-mouse hashtag 1 ACCCACCAGTAAGAC E17thy_B6-1, D9thy_B6-1, W6thy_B6-1
TotalSeq-C0302 anti-mouse hashtag 2 GGTCGAGAGCATTCA E17thy_B6-2, D9thy_B6-2, W6thy_B6-2
TotalSeq-C0303 anti-mouse hashtag 3 CTTGCCGCATGTCAT E17thy_B6-3, D9thy_B6-3, W6thy_B6-3, Lung_B6
TotalSeq-C0304 anti-mouse hashtag 4 AAAGCATTCTTCACG E17thy_B6-4, D9thy_SAPKO-1, W6thy_SAPKO-1, Lung_SAPKO
TotalSeq-C0305 anti-mouse hashtag 5 CTTTGTCTTTGTGAG E17thy_SAPKO-1, D9thy_SAPKO-2, W6thy_SAPKO-2
TotalSeq-C0306 anti-mouse hashtag 6 TATGCTGCCACGGTA E17thy_SAPKO-2, D9thy_SAPKO-3, W6thy_SAPKO-3
TotalSeq-C0307 anti-mouse hashtag 7 GAGTCTGCCAGTATC E17thy_SAPKO-3
TotalSeq-C0308 anti-mouse hashtag 8 TATAGAACGCCAGGC E17thy_SAPKO-4, D9thy_ADTcontrol, W6thy_ADTcontrol

first: Somen

middle: K

last: Mistri

first: Brianna

last: Hilton

first: Katherine

last: Horrigan

first: Emma

middle: S

last: Andretta

first: Remi

last: Savard

first: Oliver

last: Dienz

first: Kenneth

last: Hampel

first: Diana

middle: L

last: Gerrard

first: Joshua

middle: T

last: Rose

first: Nikoletta

last: Sidiropoulos

first: Devdoot

last: Majumdar


title: Intrinsic Gata4 expression sensitizes the aortic root to dilation in a Loeys-Dietz syndrome mouse model

status: Public on Jun 28 2024

city: Baltimore

first: Tyler

last: Creamer

state: Maryland

address: 733 N Broadway

institute: Johns Hopkins University

summary: Loeys-Dietz syndrome (LDS) is a hereditary aneurysm disorder caused by mutations that impair transforming growth factor-β (TGF-β) signaling. Although LDS patients develop aneurysms throughout the arterial tree, the aortic root is a site of increased risk. In order to identify molecular determinants of this regional vulnerability, we investigated the transcriptional heterogeneity of vascular smooth muscle cells (VSMCs) in the aorta of Tgfbr1M318R/+ LDS mouse models by single cell RNA sequencing (scRNAseq) and spatial transcriptomics. Downregulation of transcripts coding for components of the extracellular matrix-receptor mechanosensing apparatus and upregulation of transcripts related to stress and inflammation were observed in all Tgfbr1M318R/+ VSMCs. However, regardless of genotype, a subset of Gata4-expressing VSMCs predominantly located in the aortic root intrinsically displayed a less differentiated, pro-inflammatory transcriptional profile. A similar population was also identified in a published scRNAseq dataset of the aorta of LDS patients via the Coordinated Gene Activity in Pattern Sets (CoGAPS)/ProjectR pipeline. Postnatal VSMC-specific Gata4 deletion resulted in reduced aortic root dilation in LDS mice, in association with decreased levels of Agtr1a and other pro-inflammatory regulators. We propose that widespread dysregulation of mechanosensitive pathways may act on regionally restricted factors that “prime” specific aortic locations to increased risk of aneurysm.

overall_design: Tgfbr1+/+ and Tgfbr1M318R/+ (The Jackson Laboratory, strain #036511) mice, some bearing the EGFP-L10a (The Jackson Laboratory, strain #024750) conditional tracer allele and a CNC-specific CRE recombinase expressed under the control of Wnt2 promoter (The Jackson Laboratory, strain #003829) were used for scRNAseq at 16 weeks of age. All mice were maintained on a 129-background strain (Taconic, 129SVE).

first: Emily

last: Bramel

first: Wendy

middle: A

last: Espinoza Camejo

first: Leda

last: Restrepo

first: Muzna

last: Saqib

first: Rustam

last: Bagirzadeh

first: Anthony

last: Zeng

first: Jacob

last: Mitchell

first: Genevienve

last: Stein-O'Brien

first: Harry

last: Dietz

first: Albert

last: Pedroza

first: Michael

last: Fischbein

first: Elena

last: Gallo MacFarlane


title: Efficient small DNA fragment sequencing and miRNA, small RNA or csRNA-seq libraries using AVITI

status: Public on Jun 26 2024

city: Pullman

first: Sascha

last: Duttke

state: Washington

address: BLS 147, 100 Dairy Road

department: School of Molecular Biosciences

institute: Washington State University

type: Non-coding RNA profiling by high throughput sequencing

summary: Next-Generation Sequencing (NGS) catalyzed breakthroughs across various scientific domains. Illumina's sequencing by synthesis method has long been essential for NGS but emerging technologies like Element Biosciences' sequencing by avidity (AVITI) represent a novel approach. It has been reported that AVITI offers improved signal-to-noise ratios and cost reductions. However, the method relies on rolling circle amplification which can be impacted by polymer size, raising questions about its efficacy sequencing small RNAs (sRNAs) libraries including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and others that are crucial regulators of gene expression and involved in various biological processes. In addition, capturing capped small RNAs (csRNA-seq) has emerged as a powerful method to map active or “nascent” RNA polymerase II transcription initiation in tissues and clinical samples. Finally, we present an accelerated and optimized protocol for small fragment sequencing with AVITI that aligns seamlessly with available sRNA library kits to meet this need.

sample_organism:
Homo sapiens
Bison bison
Bos taurus

platform_organism:
Homo sapiens
Bison bison
Bos taurus

overall_design: Capped small (cs)RNA-sequencing for human A375 and BT474 cells, as well as blood samples from cattle and bison using Illumina and AVITI sequencing.

first: Anna

last: McDonald

first: Andrew

last: Boddicker

middle: H


title: An integrated clinical morphology and functional genomics approach reveals upregulation of PLEKHA7 and C10orf53 in iris contributes to primary angle closure glaucoma [RNA-seq]

status: Public on Jul 22 2024

city: 津南区

first: An

last: Jiang

state: 天津市

address: 南开大学津南校区综合实验楼D413

institute: 南开大学

summary: Previous Genome-wide association studies (GWASs) have identified susceptibility loci of primary angle closure glaucoma (PACG), but applicating these findings is difficult. Although shallow anterior chamber depth (ACD) and short axial length (AL) are characteristic phenotypes of PACG, current GWAS variants do not show any correlation with these features, calling for the identification of more risk factors . Here, thicker and enlarged iris was identified as a significant independent risk factors. By employing allelic-specific STARR-seq and assay for transposase-accessible chromatin using sequencing (ATAC-seq), we screened disease-related variants in linkage disequilibrium and identified 2 causative variants in iris. Alterations in these variants may contribute to the pathogenic iris phenotype by upregulating the expression of PLEKHA7 and C10orf53, potentially regulating PACG development and progression. Our efforts nominate the important role of iris and identify pathogenic SNP-target gene interactions for PACG, providing a potentially powerful approach for interpreting noncoding variation of diseases.

overall_design: We conducted allelic-specific STARR-seq for PACG candidate variants and ATAC-seq for ARPE-19 cell lines and human iris.

first: Jiaying

last: Li

first: Yun

last: Chen

first: Wenbin

last: Wang

first: Guangsong

last: Su

first: Yuanyuan

last: Zhang

first: Yilong

last: Yao

first: Wange

last: Lu

first: Chunyan

last: Qiao

first: Kunlin

first: Ye

first: Yuwen

last: Liu

first: Ningli


title: Cis-regulatory chromatin loops analysis identifies GRHL3 as a master regulator of surface epithelium commitment

status: Public on Jun 25 2024

first: Huaxing

last: Huang

address: 7 Jinsui Road

department: Zhongshan Ophthalmic Center

institute: Sun Yat-sen University

summary: Understanding the regulatory network of cell fate acquisition remains a major challenge. Using the induction of surface epithelium (SE) from human embryonic stem cells as a paradigm, we show that the dynamic changes in morphology-related genes (MRGs) closely correspond to SE fate transitions. The marked remodeling of cytoskeleton indicates the initiation of SE differentiation. By integrating promoter interactions, epigenomic features, and transcriptome, we delineate an SE-specific cis-regulatory network and identify grainyhead-like 3 (GRHL3) as an initiation factor sufficient to drive SE commitment. Mechanically, GRHL3 primes the SE chromatin accessibility landscape and activates SE-initiating gene expression. In addition, the evaluation of GRHL3-mediated promoter interactions unveils a positive feedback loop of GRHL3 and bone morphogenetic protein 4 on SE fate decisions. Our work proposes a concept that MRGs could be used to identify cell fate transitions and provides insights into regulatory principles of SE lineage development and stem cell?based regenerative medicine.

overall_design: ChIP-seq for transcription factors (GRHL3, GRHL2, TFAP2C and GATA3) and histone modifications (H3K27ac, H3K4me1, H3K4me3 and H3K27me3)

first: Jiafeng


title: Human birth tissue products as a regenerative medicine for post-surgical pain through multi-modal action

first: Chi

state: MD

address: 720 Rutland Avenue

summary: Post-surgical pain causes significant suffering. Extracts of the human amniotic membrane (AM) may be novel regenerative matrices, but little is known about their use in pain treatment. Locally applying FLO (particulates of AM) in mice acutely attenuated post-surgical pain hypersensitivity and inhibited its transition to a prolonged state after plantar-incision. Mechanistically, this was achieved through direct nociceptive neuronal inhibition via CD44-dependent mechanisms and indirect anti-pain effect by attenuating immune cell recruitment and promoting wound healing. We purified heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) from human AM as a major bioactive matrix component for pain relief, as it mirrored FLO-induced analgesia and nociceptive neuronal inhibition. Strikingly, HC-HA/PTX3 triggered acute cytoskeleton rearrangement to inhibit ion channels on nociceptive neurons, presenting a novel cellular mechanism for pain control. Moreover, long-term drug treatment broadly changed neuronal gene expression. Our study demonstrated the potential and mechanisms of a naturally occurring biologic from human birth tissues as non-opioid treatments for post-surgical pain.

overall_design: The injured nerves were exposed to FLO for hours with the previously described intra-operative treatment, prolonging attenuation of post-operative pain. Therefore, we investigated whether long-term HC-HA/PTX3 treatment altered gene expression in DRG neurons, possibly rendering prolonged changes in neuronal function. Changes in distinct gene networks in DRG neurons after 24 hours treatment with HC-HA/PTX3 (15 µg/mL) were examined by using unbiased bulk RNA-sequencing

first: Shao-Qiu

last: He


title: The DNA cleavage efficiency of the hpCasMINI system [Deep-seq]

first: Kechen

summary: The fusion of an α-helix to the N-terminus of CasMINI (hpCasMINI) could indeed enhance the DNA cleavage efficiency in mammalian cells.

overall_design: To investigate the potential of hpCasMINI in genomic DNA cleavage, we transfected CasMINI or hpCasMINI or SpCas9 or LbCas12a into MCF7 cells with gRNA targeting different genomic sites. The results indicated that hpCasMINI significantly improved editing efficiency at all the tested sites compared to CasMINI.When compared to SpCas9 and LbCas12a, hpCasMINI displayed higher DNA cleavage specificity although with lower activity at the tested sites.

first: Shufeng

last: Ma

first: Ying

last: Lin

first: Zhili

last: Rong


title: The specificity of gene activation was revealed by RNA-seq in the HEK293T cells transfected with the dCasMINI-VPR or dhpCasMINI-VPR system targeting IL1RN [RNA-seq]

summary: The specificity of gene activation, both the dCasMINI-VPR and dhpCasMINI-VPR significantly and obviously activated IL1RN and only a few genes showed expression change, indicating high genome-wide targeted transcriptional specificity.

overall_design: We performed transcriptome profiling by RNA-seq in HEK293T cells co-transfected with the dCasMINI-VPR or dhpCasMINI-VPR system targeting IL1RN for assessing the gene activation specificity. The total amount of DNA was 500ng per well for a 24-well plate, and plasmids encoding the dCasMINI-VPR or dhpCasMINI-VPR and sgRNA were transfected at a 1:49 ratio. Approximately 1 × 105 cells were plated in 24-well plates 1 day before transfection, and analyzed 2 days post-transfection for transcriptome profiling by RNA-seq.


title: Transcriptome analysis of embryos of STAT3-K177R/K180R mutant mice

status: Public on Jul 20 2024

city: HangZhou

first: LI

last: XU

state: Zhejiang Province

address: Binwen 7-3-1101,Binwen Road

department: School of Basic Medical Science

institute: Zhejiang Chinese Medical University

summary: Total RNA from embryos (12 days) of STAT3 homozygous and  heterozygote mutant mice (K177/180R) and STAT3 wild type mice which come from the same womb were extracted using TRIzol reagent (Invitrogen) following the manufacturer’s protocols.  RNA quantity, quality and integrity were analyzed with NanoDrop ND-1000 and standard denaturing agarose gel electrophoresis.  Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol of Agilent Technology. The Whole Mouse Genome Oligo Microarray (Agilent 4*44k) represents all known genes and transcripts in the mouse genome (Kyoto Encyclopedia of Genes and Genomes, KEGG).  All the microarray experiments were performed by Kangchen Biotechnology Limited (Shanghai, China).

overall_design: Gene expression in embryos (12 days) of STAT3 homozygous and  heterozygote mutant mice (K177/180R) and STAT3 wild type mice  were detected by the Whole Mouse Genome Oligo Microarray (Agilent 4*44k) .

first: Li

last: Xu


title: Acod1-mediated effect on osteoclast differentiation

city: Erlangen

first: Katerina

last: Kachler

address: Glueckstraße 6

department: Medicine 3 - Rheumatology and Immunology

country: Germany

institute: University Clinic Erlangen

summary: Enhanced osteoclast-mediated bone erosion is a prominent hallmark of rheumatoid arthritis. Emerging evidence suggests that this process is facilitated by metabolic dysregulation of osteoclasts. The mitochondrial enzyme aconitate decarboxylase 1 (Acod1, also known as immune responsive gene 1 [Irg1]) serves as a mediator between the metabolic condition and the functional state of different types of cells. Acod1-deficient mice are characterized by enhanced osteoclast differentiation and bone erosion in an inflammatory arthritis model, while therapeutic treatment with the itaconate-derivative 4-octyl-itaconate (4-OI) alleviates the disease phenotype in experimental arthritis. To ascertain the influence of the Acod1-itaconate axis on the genomic transcriptional network of osteoclasts, we performed a whole transcriptome RNA sequencing analysis with fully differentiated osteoclasts from WT and Acod1-deficient mice that were cultured in the presence or absence of 4-OI.

overall_design: Comparative gene expression analysis of RNAseq data was performed for the following 3 groups: unstimulated WT osteoclasts (WT CTR), unstimulated Acod1-deficient osteoclasts (KO CTR) and 4-OI stimulated Acod1-deficient osteoclasts (KO 4-OI). Each group contained 3 replicates.

first: Aline

last: Bozec


title: Transcriptome-wide mapping of internal mRNA N7-methylguanosine in sporulated and unsporulated oocysts of Eimeria tenella reveals stage-specific signatures

status: Public on Jun 23 2024

city: Jinzhong

first: Qing

address: Taigu, Shanxi Province, PR China

institute: Shanxi Agricultural university

summary: Growing evidence indicates that 7-methylguanosine (m7G) modification plays critical roles in epigenetic regulation. However, no data regarding m7G modification are currently available in Eimeria tenella, a highly virulent species causing coccidiosis in chickens. Thus, in the present study, we explored the distribution of internal mRNA m7G modification in sporulated and unsporulated oocysts of E. tenella as well as its biological functions during oocyst development. Our data showed that m7G peaks were detected throughout the whole mRNA body, and the coding DNA sequence (CDS) region displayed the most methylation modification. Compared with unsporulated oocysts, 7799 hypermethylated peaks and 1945 hypomethylated peaks were identified in sporulated oocysts. Further combined analysis of differentially methylated genes (DMGs) and differentially expressed genes (DEGs) showed that there was a positive correlation between m7G modification levels and gene transcript abundance. Unsurprisingly, the mRNA sequencing (RNA-seq) and methylated RNA immunoprecipitation sequencing (MeRIP-seq) data showed a good consistence with the results of the quantitative reverse transcription PCR (RT-qPCR) and MeRIP-qPCR, respectively. Gene Ontology (GO) and pathway enrichment analysis of DEGs with altered m7G-methylated peaks were involved in diverse biological functions and pathways, including DNA replication, RNA transport, spliceosome, autophagy-yeast, and cAMP signaling pathway. Altogether, our findings revealed the potential significance of internal m7G modification in E. tenella oocysts, providing some directions and clues for later in-depth research.

overall_design: MeRIP-seq and RNA-seq of sporulated and unsporulated oocysts of E. tenella.

first: Qing-Xin

last: Fan

first: Zi-Rui

first: Jin-Long

first: Yu-Xuan

first: Ze-Dong

first: Lin-Mei

last: Yu

first: Tao

last: Jia

first: Xing-Quan

last: Zhu


title: Samd7 preserves cell identity and enforces the ‘one neuron-one receptor’ rule in vertebrate photoreceptors

status: Public on Jun 21 2024

city: St. Louis

first: Ramiz

last: Somjee

state: Missouri

address: 660 S Euclid ave

institute: Washington University

summary: The exclusive expression of single sensory receptors in individual neurons (the ‘one-neuron-one receptor’ rule) is essential for vision and other sensory systems. Here, we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7-/- retina, red cones are transformed to hybrid red/UV-sensitive cones, green cones are transfated to blue cones, and the number of rods is greatly reduced. In the mouse Samd7-/- retina, dorsal M-cones are transformed to hybrid M/S-cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S-opsin. Altogether, Samd7 acts to repress short-wavelength cone gene expression in long-wavelength-sensitive cones, thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision.

overall_design: Compare the transcriptomes of wild-type and Samd7 KO mouse retina using RNA-sequencing, with three replicates per condition.


title: Systematic surveillance of SARS-CoV-2 reveals dynamics of variant mutagenesis and transmission in a large urban population

status: Public on Jul 30 2024

city: Toronto

first: Jeff

last: Wrana

state: Ontario

address: 600 University Avenue

country: Canada

institute: Lunenfeld-Tanenbaum Research Institute

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

sample_organism: Severe acute respiratory syndrome coronavirus 2

overall_design: Refer to individual Series


title: Samd7 preserves cell identity and enforces the ‘one neuron-one receptor’ rule in vertebrate photoreceptors [5dpf_td]

city: Saint Louis

first: Joseph

last: Corbo

state: MO

address: 660 S. Euclid

department: Pathology and Immunology

institute: Washington University School of Medicine

overall_design: To perform RNA-seq on 5-dpf thrb:tdTomato+ cells, the following crosses were performed: WT × WT;thrb:tdTomato+/- or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/-. A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40-50 eyes from 20-25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS as described in another section. 10,000-25,000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit (Qiagen); RNA was then extracted using the Rneasy Micro Kit (Qiagen), and on-column DNAse treatment performed using the Rnase-Free Dnase Set (Qiagen). RNA concentrations ranged from 40-190 pg/µl (0.3-1.8 ng total), with RNA integrity (RIN) scores ranging from 7.4-8.4.


title: Samd7 preserves cell identity and enforces the ‘one neuron-one receptor’ rule in vertebrate photoreceptors [adult]

overall_design: To perform RNA-seq on 3 month-old adult retinas, samd7stl888/+ heterozygotes were intercrossed to produce mixed genotype offspring and grown to adulthood. After adults were genotyped, pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate, and RNA was extracted using the RNEasy Mini Kit (Qiagen) with on-column DNAse treatment using the Rnase-Free Dnase Set (Qiagen). RNA concentrations ranged from 20-37 ng/µl (600-1100 ng total), with RIN scores ranging from 9-9.5; there was minimal RNA degradation as shown by Bioanalyzer traces.


title: Tissue-specific transcriptomic profiling of ‘Candidatus Liberibacter asiaticus’ in citrus reveals novel insights into Huanglongbing pathogenesis

status: Public on Jun 30 2024

city: Norwich

first: Wenbo

address: Norwich Research Park

country: United Kingdom

institute: The Sainsbury Laboratory

summary: ‘Candidatus Liberibacter asiaticus’ (Las) is a gram-negative bacterial pathogen associated with citrus huanglongbing (HLB) or greening disease. Las is transmitted by the Asian citrus psyllid (ACP) where it colonizes the phloem tissue, resulting in substantial economic losses to citrus industry worldwide. Despite extensive efforts, effective management strategies against HLB remain elusive, necessitating a deeper understanding of the pathogen’s biology. Las undergoes cell-to-cell movement through phloem flow from source to sink tissues and these tissues have varying responses to Las infection. Here, we investigate the transcriptomic landscape of Las in citrus sink tissues, particularly seed coat vasculatures, revealing more complete gene expression profiling of Las and unique transcriptomic pattern compared to previous studies using midrib tissues. Comparative transcriptomics between citrus seed coat vasculature and midrib tissues and ACP allowed the identification of tissue-specific responses and metabolic states of Las in planta. Furthermore, analysis of putative regulatory elements uncovers the potential role of LuxR-type transcription factors in regulating the expression of effector genes in citrus. We characterized two Las virulence factors that exhibit increased expression in seed coat vasculature tissue. Their ability to suppress different immune outputs was accessed and we demonstrate that they may contribute to the suppression of callose deposition during Las colonization. Moreover, this work provides novel insights into the pathogenesis of the devastating citrus HLB.

overall_design: Samples collected from seed coat vasculature tissues of HLB-infected citrus (grapefruit). Some RNA samples underwent a pre-enrichment bacterial isolation method for CLas transcripts

first: Amelia

last: Lovelace


title: Effect of influenza A virus infection during pregnancy on the developing fetal brain

status: Public on Jun 20 2024

city: Urbana

first: Adrienne

last: Antonson

address: 1201 W Gregory Dr

institute: University of Illinois Urbana-Champaign

summary: Epidemiological studies link NDDs with exposure to maternal viral infection in utero. As the immature brain is vulnerable to insult, it is thought that prenatal inflammation alters neurodevelopmental trajectories, leading to NDD pathologies. Using a biologically relevant mouse model of live influenza (flu) infection during pregnancy, we aim to determine if gestational flu infection triggers detrimental maternal cell signaling that leads to NDD-relevant pathologies in offspring. Overall, this work will improve translation to the clinic and will build a foundation for developing targeted therapeutics for NDDs that can be delivered during gestation.

overall_design: On GD9.5, pregnant dams were randomly assigned to infected or control groups. They were then anesthetized by inhalation of isoflurane before intranasal inoculation with mouse-adapted IAV (subtype H3N2, strain X31) 104 tissue culture infectious dose (TCID50) in sterile saline. Control animals were inoculated with sterile saline.

first: Ashley

last: Otero

first: Meghan

last: Connolly

first: Jenny

last: Drnevich

first: Chris

last: Wright

first: Alvaro

last: Hernandez


title: The Greatwall-Endosulfine-PP2A/B55 pathway controls entry into quiescence by promoting translation of Elongator-tuneable transcripts

status: Public on Jun 17 2024

city: Salamanca

first: Sergio

last: Moreno

email: smo@usal.es, ibfg_smp@usal.es

state: Salamanca

address: Zacarias Gonzalez 2

department: IBFG

country: Spain

institute: Institute of Functional Biology and Genomics (IBFG) (CSIC)

summary: Here, we report that the Greatwall-Endosulfine-PPA/B55 pathway connects the downregulation of TORC1 with the upregulation of TORC2, resulting in the activation of Elongator-dependent tRNA modifications essential for sustaining the translation programme during entry into quiescence. This process promotesU34 and A37 tRNA modifications at the anticodon stem loop, enhancing translation efficiency and fidelity of mRNAs enriched for AAA versus AAG lysine codons. Notably, some of these mRNAs encode inhibitors of TORC1, activators of TORC2, tRNA modifiers, and proteins necessary for telomeric and subtelomeric functions. Therefore, we propose a novel mechanism by which cells respond to nitrogen stress at the level of translation, involving a coordinated interplay between the tRNA epitranscriptome and biased codon usage.

overall_design: We performed a comparative gene expression profiling analysis of RNA-seq data between wild type cells and the mutants igo1∆ and nmt41:GST:pab1+ igo1∆ growing in minimal medium lacking nitrogen (MM-N) after 4 hours .

first: Javier

last: Encinar del Dedo

first: Rafael

last: López-San Segundo

first: Alicia

last: Vázquez-Bolado

first: Belén

last: Suárez


title: The transcriptional response to low temperature is weakly conserved across Enterobacteriales

status: Public on Jun 11 2024

city: Claremont

first: Daniel

last: Stoebel

address: 301 Platt Blvd

department: Biology

institute: Harvey Mudd College

summary: Bacteria respond to changes in their external environment like temperature by changing the transcription of their genes, but we know little about how these regulatory patterns evolve. We used RNA-seq to study the transcriptional response of a shift from 37°C to 15°C in wild-type Escherichia coli, Salmonella enterica, Citrobacter rodentium, Enterobacter cloacae, Klebsiella pneumoniae, and Serratia marcescens, as well as ∆rpoS strains of E. coli and S. enterica. We found that these species change the transcription of between 626 and 1057 genes in response to the temperature shift, but there are only 16 genes differentially expressed in wild-type strains of all six species. GO enrichment of regulated genes suggests many species-specific phenotypic responses to temperature changes, but terms involved in iron metabolism, central metabolism, and response to osmotic stress are implicated in at least half of the species. The alternative sigma factor RpoS regulates about 200 genes at 15°C in E. coli and S. enterica, with only 83 genes in common between the two species. Divergence in the RpoS-regulon between the two species is due to both species-specific genes in each genome as well as differences in regulation of shared genes. Overall, there is limited conservation of the response to low temperature generally, or the RpoS-regulated part of the response specifically, due both to some genes being species-specific, as well as the species-specific regulation of shared genes. Regulatory responses to a common stress evolve rapidly between closely related species.

sample_organism:
Enterobacter cloacae
Escherichia coli
Klebsiella pneumoniae
Serratia marcescens
Salmonella enterica
Citrobacter rodentium

overall_design: This experiment measured the how six species of bacteria responded to a shift in tempterature from 37°C to 15°C. Bacteria were growing exponentially at 37°C in LB media, then shifted to 15°C for 3 hours. 4 replicates are present for each strain for each of the two temperatures.

first: Johnson

last: Hoang


title: The results of RNA-seq for EhracM gene silencing strain and EhracJ gene silencing strain

city: Bunkyo-ku

first: Misato

last: Shimoyama

state: Tokyo

address: Hongo 7-3-1

department: School of Molecular and Cellular Biology, Graduate School of Medicine

country: Japan

institute: The University of Tokyo

summary: To know the role of Rho small GTPases of enteric protozoan parasite Entamoeba histolytica, ten Rho small GTPases were specifically silenced and examined the effect on virulence-related phenotypes. Among them, EhRacM gene silence caused an enhancement in macropinocytosis. To clarify transcriptomic difference in EhRacM gene silenceing strain, RNA-seq analysis was conducted.

overall_design: To know the role of Rho small GTPases of enteric protozoan parasite Entamoeba histolytica, ten Rho small GTPases were specifically silenced and examined the effect on virulence-related phenotypes. Among them, EhracM gene silencing caused an enhancement in macropinocytosis. Also, EhracJ gene silencing caused a slight decrease in trogocytosis. To clarify the transcriptomic differences in EhracM gene silenced strain and EhracJ gene silenced strain, RNA-seq analysis was conducted.

first: Kumiko

last: Nakada-Tsukui

first: Tomoyoshi

last: Nozaki


title: ChEC-seq2 of RNA Polymerase II and Preinitation Complex in S. cerevisiae

status: Public on Jun 18 2024

city: Evanston

first: Jason

last: Brickner

state: IL

address: Hogan 1-500, 2205 Tech Drive

institute: Northwestern University

summary: Genome occupancy of RNA Polymerase II and its phosphorylated forms were determined by ChEC-seq2 in S. cerevisiae. We test the dependency of RNAPII interactions on TFIIB, TFIIH Kinase, and the transcription factor GCN4.

overall_design: ChEC-seq2 with Rpb1-MNase and Rpb3-MNase (RNA Polymerase II).  In addition, ChEC-seq2 was performed on components on the preinitiation complex, transcription activators, and coactivators.  Changes in Rpb1-MN occupancy in response to TFIIB degradation, Kin28 inhibition (kin28is), and gcn4 deletion or mutation (gcn4-pd) were measured.  In addition, SLAM-seq (RNA)was was performed under a subset of conditions

first: D

last: VanBelzen


title: A Constitutive Heterochromatic Region Shapes Genome Organization and Impacts Gene Expression in Neurospora crassa [ChIP-Seq]

status: Public on Jun 10 2024

city: Colorado Springs

first: Andrew

middle: David

last: Klocko

state: Colorado

address: 278 Centennial Hall, 1420 Austin Bluffs Pkwy

department: Chemistry and Biochemistry

institute: University of Colorado Colorado Springs

summary: Genome organization is essential for proper function, including gene expression. In metazoan genome organization, chromatin loops and Topologically Associated Domains (TADs) facilitate local gene clustering, while chromosomes form distinct nuclear territories characterized by compartmentalization of silent heterochromatin at the nuclear periphery and active euchromatin in the nucleus center. A similar hierarchical organization occurs in the fungus Neurospora crassa where its seven chromosomes form a Rabl conformation, where heterochromatic centromeres and telomeres independently cluster at the nuclear membrane, while interspersed heterochromatic loci in Neurospora aggregate across megabases of linear genomic distance for forming TAD-like structures. However, the role of individual heterochromatic loci in normal genome organization and function is unknown. Here, we examined the genome organization of a Neurospora strain harboring a ~41 kilobase facultative (temporarily silent) heterochromatic region deletion, as well as the genome organization of a strain deleted of a ~110 kilobase permanently silent constitutive heterochromatic region. While the facultative heterochromatin deletion had little effect on local chromatin structure, the constitutive heterochromatin deletion altered local TAD-like structures, gene expression, and the predicted 3D genome structure by qualitatively repositioning genes into the nucleus center. Our work elucidates the role of individual heterochromatic regions for genome organization and function.

overall_design: Chromatin Immunoprecipitation-sequencing (ChIP-seq) of the trimethylation of lysine 9 on histone H3 (H3K9me3) and di- and trimethylation of lysine 27 on histone H3 (H3K27me2/3) from the filamentous fungus Neurospora crassa

last: Reckard

first: Abhishek

last: Pandeya

first: Jacob

last: Voris

first: Carlos

middle: G

last: Gonzalez Cruz

first: Oluwatosin

last: Oluwadare

middle: D


title: A Constitutive Heterochromatic Region Shapes Genome Organization and Impacts Gene Expression in Neurospora crassa [Hi-C]

overall_design: Chromosome conformation capture coupled with high-throughput sequencing of strains of the filamentous fungus Neurospora crassa deleted of heterochromatic regions, including the N4933 strain deleted of a 47.4 kb facultative heterochromatic region enrichched wiith H3K27me2/3 and the NKL2 (delta LGIK9het25) strain deleted of a 110 kb constitutiive heterochromatic region enriched with H3K9me3.


title: A Constitutive Heterochromatic Region Shapes Genome Organization and Impacts Gene Expression in Neurospora crassa [RNA-Seq]

overall_design: Polyadenylated messenger RNA-sequencing (polyA mRNA-seq) from strains of the filamentous fungus Neurospora crassa, including a wild type strain, a deletion of a large constitutiive heterochromatic region (delta LGIIK9het25) strain, and a delta dim-5 (complete loss of H3K9me3) strain.


title: A Constitutive Heterochromatic Region Shapes Genome Organization and Impacts Gene Expression in Neurospora crassa


title: Effect of insulin on West Nile virus (WNV) infection in Culex quinquefasciatus

status: Public on Jun 06 2024

first: Alan

middle: G.

last: Goodman

state: WASHINGTON

address: 100 Dairy Rd., BLS 202

summary: Adult mosquitoes were fed blood containing 1e7 PFU WNV (kunjin strain) containing +/- 170 pM bovine insulin for 24 h. We then performed gene expression profiling analysis using data obtained from RNA-seq.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for WNV-infected adult mosquitoes +/- insulin. Raw gene expression data was used to determine the effect of insulin on Culex responses during WNV infection.


title: CXXC5 stabilizes DNA methylation patterns in mouse embryonic stem cells [RNA-seq]

status: Public on Jun 04 2024

city: Grand Rapids

first: Gerd

middle: P.

last: Pfeifer

state: Michigan

address: 333 Bostwick Ave. NE.

department: Center for Epigenetics

institute: Van Andel Research Institute

summary: Mammalian genomes encode a family of 12 proteins that contain a CXXC zinc finger domain. These domains are known to bind to unmethylated DNA sequences rich in CpG dinucleotides. Most members of this family are large multi-domain proteins that function in the control of DNA methylation and histone methylation patterns. CXXC5 is a smaller member of the family, along with its closest homologue CXXC4. These two proteins lack known catalytic domains. Here we have characterized CXXC5 in mouse embryonic stem (ES) cells. We show that CXXC5 is a nuclear protein that interacts with 5-methylcytosine oxidases (TET proteins). Removal of CXXC5 from ES cells leads to very few changes in gene expression. CXXC5 extensively colocalizes with TET1 and TET2 at CpG islands. CXXC5 inactivation leads to a substantial reduction of DNA methylation levels that affects all genomic compartments including genic and intergenic regions and CpG island shores. No hypermethylation of CpG islands was observed. We propose a model in which CXXC5 serves as an anchor for TET proteins at CpG islands. In the absence of CXXC5, the 5-methylcytosine oxidases become dislodged from CpG islands and are enabled to induce genome-scale DNA demethylation. Thus, CXXC5 serves as a stabilizer of DNA methylation patterns.

overall_design: To gain insight into a role of CXXC5 in mouse embryonic stem cells, we established CXXC5 single or CXXC4 and CXXC5 double knockout mES clones by CRISPR/Cas9 system and observed gene expression profile through RNA-seq.

first: Seung-Gi

last: Jin


title: CXXC5 stabilizes DNA methylation patterns in mouse embryonic stem cells [ChIP-Seq]

overall_design: To gain insight into the genomic targets of CXXC5 in mouse embryonic stem cells, we performed chromatin immunoprecipitation sequencing (ChIP-seq).


title: DNA methylation profile in matched oral cavity squamous cell carcinoma and normal adjacent tissue samples

status: Public on Jun 07 2024

city: Sao Paulo

first: Fabio

middle: Albuquerque

last: Marchi

address: Av Dr Arnaldo

department: CTO

country: Brazil

institute: ICESP

summary: Oral cavity squamous cell carcinoma (OSCC) is a complex and dynamic disease characterized by clinicopathological and molecular heterogeneity. Spatial and temporal heterogeneity of cell subpopulations has been associated with cancer progression and implicated in the prognosis and therapy response. Emerging evidence indicates that aberrant epigenetic profiles in OSCC may foster an immunosuppressive tumor microenvironment by modulating the expression of immune-related long non-coding RNAs (lncRNAs). DNA methylation analysis was performed in 46 matched OSCC and normal adjacent tissue samples using a genome-wide platform (Infinium HumanMethylation450 BeadChip). Reference-based computational deconvolution (MethylCIBERSORT) was applied to infer the immune cell composition of the bulk samples. The expression levels of genes encoding immune markers and differentially methylated lncRNAs were investigated using The Cancer Genome Atlas dataset. OSCC specimens presented distinct immune cell composition, including the enrichment of monocyte lineage cells, natural killer cells, cytotoxic T-lymphocytes, regulatory T-lymphocytes, and neutrophils. In contrast, B-lymphocytes, effector T-lymphocytes, and fibroblasts were diminished in tumor samples. The hypomethylation of three immune-associated lncRNAs (MEG3, MIR155HG, and WFDC21P) at individual CpG sites was confirmed by bisulfite-pyrosequencing. Also, the upregulation of a set of immune markers (FOXP3, GZMB, IL10, IL2RA, TGFB, IFNG, TDO2, IDO1, and HIF1A) was detected. The immune cell composition, immune markers alteration, and dysregulation of immune-associated lncRNAs reinforce the impact of the immune microenvironment in OSCC. These concurrent factors contribute to tumor heterogeneity, suggesting that epi-immunotherapy could be an efficient alternative to treat OSCC.

overall_design: Genome-wide DNA methylation analysis was performed in 46 matched oral cavity squamous cell carcinoma (OSCC) and normal adjacent tissue samples. Differentially methylated probes were determined by comparing OSCC with normal samples.

first: Naiade

last: Calanca

first: Ana

last: Francisco

first: Luiz

last: Kowalski

first: Silvia

middle: R

last: Rogatto


title: Sex-dependent APOE4 Neutrophil-microglia interactions drive cognitive impairment in Alzheimer's Disease

city: Boston

first: Neta

last: Rosenzweig

state: Massachusetts

address: 60 Fenwood Road, 10002K, Brigham and Women’s Hospital

institute: Brigham and Women's Hospital, Harvard Medical School

summary: APOE4 is the strongest genetic risk factor for Alzheimer's disease (AD), with increased odds ratios in females. Targeting amyloid plaques shows modest improvement in male non-APOE4 carriers. Leveraging single-cell transcriptomics across APOE variants in both sexes, multiplex flow cytometry, and validation in two independent cohorts of APOE4 female AD patients, we identify a new subset of neutrophils interacting with microglia associated with cognitive impairment. This phenotype is defined by increased IL-17 and IL-1 co-expressed gene modules in blood neutrophils and in microglia of cognitively impaired female APOE e4 carriers, showing increased infiltration to the AD brain. APOE4 female IL-17+ neutrophils upregulated the immunosuppressive cytokines IL-10 and TGFB and immune checkpoints, including LAG-3 and PD-1, associated with accelerated immune aging. Deletion of APOE4 in neutrophils reduced this immunosuppressive phenotype and restored the microglial response to neurodegeneration (MGnD), limiting plaque pathology in AD mice. Mechanistically, IL-17F upregulated in APOE4 neutrophils interacts with microglial IL-17RA to suppress the induction of MGnD phenotype, and blocking this axis supported cognitive improvement in AD mice. These findings provide a translational basis to target IL-17F in APOE e4 female carriers with cognitive impairment.

overall_design: Nuclei were isolated from frozen human brain tissue and enriched for microglia and astrocytes using Fluorescence-activated nuclei sorting (FANS) and analysed using snRNAseq. Donors included both males and females from an Alzheimer's diseased (AD) cohort and non-demented control (CTRL) cohort that have the APOE 3/3 or 3/4 genotype

first: Kilian

last: Kleemann

first: Thomas

last: Rust

first: Madeline

last: Grucci

first: Michael

last: Aronchik

first: Nieske

last: Brouwer

first: Joya

last: Cooper-Hohn

first: Malvika

last: Iyer

first: Rajesh

last: Krishnan

first: Isabel

last: Valenbreder

first: Madison

last: Carpenter

first: Sivanathan

last: Kisha

first: Jean

middle: P

last: Chadarevian

first: Wesley

last: Brandão

first: Taha

last: Yahya

last: Durao

first: Zhuoran

last: Yin

last: Properzi

first: Hyun-Sik

last: Yang

first: Roni

last: Norwaski

first: Hayk

last: Davtyan

first: Mathew

middle: M

last: Blurton-Jones

first: Reisa

last: Sperling

first: Bart

last: Eggen

first: Oleg

last: Butovsky


title: Ex Vivo Expansion and Hydrogel-Mediated In Vivo Delivery of Tissue Resident Memory T Cells for Immunotherapy

city: Philadelphia

first: Hanzhi

last: Wang

address: 422 Curie Blvd

institute: University of Pennsylvania

summary: Tissue-resident memory (TRM) T cells preferentially reside in peripheral tissues, serving as key players in tumor immunity and immunotherapy. Currently, the lack of effective approaches for expanding TRM cells and delivering these cells in vivo hinder the exploration of TRM cell-mediated cancer immunotherapy. Here, we report a nanoparticle artificial antigen-presenting cell (nano-aAPC) ex vivo expansion approach and an in vivo delivery system for TRM cells. Using the nano-aAPC platform, we expanded functional antigen-specific murine and human TRM-like CD8+ T cells ex vivo. We also developed an injectable macroporous hyaluronic acid (HA) hydrogel for delivery of TRM-like cells. TRM-like cells delivered in the optimized HA hydrogel trigger robust local and systemic antitumor immunity and show synergistic effect with anti-PD1 treatment. Our findings suggest that nano-aAPC-induced TRM-like cells, coupled with a hydrogel delivery system, offer an efficient way to advance the understanding of TRM cells-mediated cancer therapy.

overall_design: CD8+ T cells were isolated from OT-I  transgenic mouse lymph nodes and spleens using CD8+ T cells isolation kits and magnetic columns (Miltenyi Biotech, CA, USA) according to the manufacturer’s protocol. On Day 0, isolated CD8+ T cells were incubated with nano-aAPCs  in the B media containing different cytokines mixes. On Day 6 of culture, OT-I T cells expanded in the media containing nano-aAPCs in the presence of mIL-2 (TEff) or mIL-2, mIL-15, and mTGF-β (TRM-like) were harvested for RNA extraction with quick-RNA miniprep kit (Zymo research, CA, USA). Then RNA samples were submitted to Psomagen to generate RNA-seq libraries using TruSeq Stranded Total RNA Library Prep Kit (Illumina, CA, USA). Libraries were sequenced by a Illumina NovaSeq X Plus platform.

first: Shuyi

last: Li

first: Jonathan

last: Ecker

first: Jaechan

last: Lee

first: Pilar

last: O'Neal

last: Schneck


title: Structure of an aberrant spliceosome intermediate on its way to disassembly

status: Public on Jun 01 2024

city: Melbourne

first: Attila

last: Horvath

state: Victoria

address: 305 Grattan St

country: Australia

institute: Peter MacCallum Cancer Centre

summary: Intron removal during pre-mRNA splicing is of extraordinary complexity and its disruption causes a vast number of genetic diseases in humans. While key steps of the canonical spliceosome cycle have been revealed by combined structure-function analyses, structural information on an aberrant spliceosome committed to discard is not available. Here, we report the cryo-EM structure of a B state spliceosome intermediate primed for disassembly. We identify the DEAH-box helicase – G patch protein pair (Gih35-Gpl1) to maintain catalytic dormancy with Gpl1 recognizing a remodeled active site of the spliceosome due to a single-nucleotide insertion at the 3’ end of the 5’ exon. Remodeling is communicated to the spliceosome surface and the Ntr1 complex is recruited. Our data pave the way for a targeted analysis of spliceosome-associated quality control.

overall_design:
Detailed analysis of Ctr1-deficient and WT strains using high throughput poly(A)+ RNA sequencing.

RNAseq of RNA-IP experiemnts of Nrl1-NTP_Prp43-6xFlag S. pombe strains (2 biological repliactes), RNA-IP of no-tag control strain (negative control) and total RNA sequencing

first: Tamas

last: Fischer

first: Komal

last: Soni

first: Olexandr

last: Dybkov

first: Merlin

last: Schwan

first: Sasanan

last: Trakansuebkul

first: Dirk

last: Flemming

first: Klemens

last: Wild

first: Henning

last: Urlaub

first: Irmgard

last: Sinning


title: Th17-Derived Th1 Cells Resemble Progenitor Exhausted CD4+ T Cells and Exhibit Augmented Anti-tumor Efficacy

city: Guangzhou

first: Xiao

last: Ruipei

address: Guangzhou North Stress

institute: Southern Medical University

summary: Th17 cells exhibit superior stemness and differentiation capabilities. Under specific conditions, they can be induced to differentiate into various subtypes. Here we reported that Th17-derived Th1 cells, when compared to conventional Th1 and Th17 cells, manifest less exhausted phenotype and enhanced anti-tumor activity.

overall_design: Naïve T cells were isolated from splenocytes of C57BL/6 mice and cultured with plated-bound anti-CD3 (5 mg/mL) and soluble anti-CD28 (1 mg/mL) for 3 days under different conditions (Th1 for 10 ng/mL IL-12, 5 mg/mL anti-IL-4; Th17 for 1 ng/mL TGF-b, 20 ng/mL IL-6, 10 ng/mL IL-1b, 20 mg/mL anti-IFN-g, 5 mg/mL anti-IL-12 and 5 mg/ml anti-IL-4). Then T cells were collected and further cultured for 2 days with cytokines only. On day 5, cells were cultured with IL-2 (100 U/mL) for 2 more days. Then Th cells were re-stimulated with soluble anti-CD3 (0.05ug/mL), anti-CD28 (0.05ug/mL), IL2 (100U/mL) on day 7 in the presence of Th1 or Th17 conditions for 3 days. Total RNA of T cells was prepared using RNAex Pro RNA Reagent (AG, AG21102) on day 10 for RNAseq.

first: Zhe

last: Chen

first: Ruipei

last: Xiao

first: Xiaoyi

last: Lei

first: Enguang

last: Bi


title: Single-cell transcriptomic analysis of skeletal muscle regeneration across mouse lifespan identifies altered stem cell states associated with senescence

status: Public on May 31 2024

city: Ithaca

first: Iwijn

last: De Vlaminck

state: New York

address: 237 Tower Road

department: Biomedical Engineering

institute: Cornell University

summary: Skeletal muscle regeneration is driven by the interaction of myogenic and non-myogenic cells. In aging, regeneration is impaired due to dysfunctions of myogenic and non-myogenic cells, but this is not understood comprehensively. We collected an integrated atlas of 273,923 single-cell transcriptomes from muscles of young, old, and geriatric mice (~5, 20, 26 months-old) at six time-points following myotoxin injury. We identified eight cell types, including T and NK cells and macrophage subtypes, that displayed accelerated or delayed response dynamics between ages. Through pseudotime analysis, we observed myogenic cell states and trajectories specific to old and geriatric ages. To explain these age differences, we assessed cellular senescence by scoring experimentally derived and curated gene-lists. This pointed to an elevation of senescent-like subsets specifically within muscle stem cells in aged muscles in both single-cell and spatial transcriptomics datasets. This resource provides a holistic portrait of the altered cellular states underlying skeletal muscle regenerative decline across mouse lifespan.

overall_design: Spatial transcriptomics

first: Ioannis

last: Ntekas

first: Lauren

last: Walter

first: Benjamin

last: Cosgrove


title: CXXC5 stabilizes DNA methylation patterns in mouse embryonic stem cells [Bisulfite-Seq]

first: Gerd

last: Pfeifer

type: Methylation profiling by high throughput sequencing

overall_design: To gain insight into a role of CXXC5 in mouse embryonic stem cells, we performed whole genome bisulfite sequencing (WGBS) on two wildtype mES smaples and two CXXC5-CXXC4 double knockout mES cell clones.

first: Seung-Gi

last: Jin


title: Samd7 preserves cell identity and enforces the ‘one neuron-one receptor’ rule in vertebrate photoreceptors [5dpf_whole_eye]

first: Joseph

last: Corbo

summary: The exclusive expression of single sensory receptors in individual neurons (the ‘one-neuron-one receptor’ rule) is essential for vision and other sensory systems. Here, we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7-/- retina, red cones are transformed to hybrid red/UV-sensitive cones, green cones are transfated to blue cones, and the number of rods is greatly reduced. In the mouse Samd7-/- retina, dorsal M-cones are transformed to hybrid M/S-cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S-opsin. Altogether, Samd7 acts to repress short-wavelength cone gene expression in long-wavelength-sensitive cones, thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision.

overall_design: To perform RNA-seq on 5-dpf larvae, samd7stl888/+ heterozygotes were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine, anterior halves of the body were collected in RNAlater (Invitrogen) and stored at 4°C, and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed, eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA-extracted using the RNeasy Micro Kit (Qiagen), then DNAse-treated and repurified using the Rneasy MinElute Cleanup Kit (Qiagen). RNA concentrations ranged from 1.5-2.2 ng/µl (~20 ng RNA total), with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces.


status: Public on May 29 2024

city: Shanghai

first: Xiaonan

last: Wang

address: 227 Chongqing South Road

department: School of Public Health

institute: Shanghai Jiao Tong University School of Medicine

summary:
Adult haematopoietic stem cells (HSCs) are responsible for the lifelong production of blood and immune cells and must respond to extracellular cues including cytokines. The JAK/STAT pathway is a highly conserved pathway, activated by many cytokines, in which tyrosine-phosphorylated STATs (pSTATs) function as transcription factors. STAT5 is a pivotal downstream mediator of several cytokines known to regulate haematopoiesis but its function in the HSC compartment remains poorly understood. Here we show that STAT5-deficient HSCs exhibited an unusual phenotype - reduced multi-lineage repopulation and self-renewal combined with reduced cell cycle entry and increased differentiation. This reflected not only loss of a canonical pSTAT5 transcriptional program but also loss of distinct functions mediated by tyrosine-unphosphorylated STAT5 (uSTAT5). Consistent with this concept expression of an unphosphorylatable STAT5B mutant constrained HSC differentiation, promoted HSC maintenance and upregulated transcriptional programs associated with quiescence and stemness.  Moreover, treatment with a JAK1/2 inhibitor (ruxolitinib) increased the uSTAT5:pSTAT5 ratio, constrained HSC differentiation and proliferation and promoted HSC maintenance, thus phenocopying uSTAT5B overexpression. Our results therefore reveal a previously unrecognized interplay between pSTAT5 and uSTAT5 in the control of HSC function. In addition the demonstration that JAK inhibition promotes HSC maintenance has implications for gene therapy using HSCs and may contribute to the failure of JAK inhibitors to eradicate myeloproliferative neoplasms.
To evaluate the long-term functional capacity of uSTAT5B overexpressing HSCs, WT CD45.2 ESLAM HSCs were sorted and transduced with lentivirus containing STAT5B-YF, STAT5B-WT and EV in SCF and IL-11 cultures. Five days after infection, cells were sorted for viability (DAPI-, Thermo Fisher) and green fluorescent protein (GFP) expression and processed according to the manufacturer’s protocol for 10x Chromium (10x Genomics, Pleasanton, CA) experiments .

overall_design: Matthew J Williams

first: Hugo

last: Bastos

first: Michelle

last: Wantoch

first: Hyun

middle: J

last: Park

first: Gabriela

last: Grondys-Kotarba

first: Dean

middle: C

last: Pask

first: Tina

last: Hamilton

first: Nicola

last: Wilson

first: Sarah

last: Kinston

first: Ryan

last: Asby

first: Rachel

last: Sneade

first: George

last: Vassiliou

first: Elisa

last: Laurenti

first: Juan

last: Li

first: Berthold

last: Göttgens

first: Anthony

last: Green


title: The effect of RVG-SCMH1-Evs treatment on photothrombotic (PT) stroke mice

status: Public on May 26 2024

city: Nanjing

first: Yu

state: Jiangsu

address: Dingjiaqiao 87

department: Department of Pharmacology

institute: Southeast University

summary: Metabolic dysfunction is considered a key event after ischemic stroke. However, the underlying mechanisms of metabolic disorders in cerebral ischemia is unknown. Circular RNA SCMH1 (circSCMH1), a circular RNA that has been reported to function in brain repair after stroke, was confirmed to be involved in metabolic disorders. circSCMH1 was delivered by rabies virus glycoprotein (RVG)-modified sxtracellular vesicle. Mechanistically, circSCMH1  decreased kynurenine 3-monooxygenase (KMO) expression, a key enzyme in kynurenine metabolism. Our findings suggested that circSCMH1 inhibited  KMO expression, providing insight into the mechanism by which circSCMH1 promotes stroke recovery.

overall_design: To investigate the mechanism of circular RNA SCMH1 in functional recovery of photothrombotic (PT) stroke mice.


title: Cytomegalovirus vaccine vector-induced effector memory CD4+ T cells protect cynomolgus macaques from lethal aerosolized heterologous avian influenza

status: Public on May 24 2024

city: Minneapolis

first: Michael

last: Gale

state: Minnesota

address: 689 23rd Ave SE

department: Microbiology and Immunology

institute: University of Minnesota

summary: To investigate the transcriptional response in whole blood from administration of the Cytomegolovirus vaccine vector against heterologous avian flu

overall_design: Comparative gene expression analysis cynomolgus macaque whole blood RNA-seq data of each time point in vaccination regimen of 68-1 CyCMV/flu or full length CyCMV/flu.  Regimen consisted of prime given at D0 and boost at D105. All animals were challenged after vaccination with avian influenza H5N1 virus

first: Leanne

last: Whitmore

first: Jennifer

last: Tisoncik-Go

first: Inah

last: Golez

first: Elise

last: Smith

last: Gale Jr

first: Jonah

middle: B

last: Sacha


title: Optimized reporters for multiplexed detection of transcription factor activity

city: Amsterdam

first: Max

last: Trauernicht

address: Plesmanlaan 121

country: Netherlands

institute: Netherlands Cancer Institute

summary: A library of 35,500 reporters was designed for 86 TFs in order to identify optimized TF reporters. The library was transfected into diverse cell types and probed upon almost 100 TF perturbations. To compare TF activities with TF abundances, RNA-seq data was used. For mNPCs, mESCs and HepG2 cells, RNA-seq data was generated in house. For thee TF knockdowns in HepG2 cells, RNA-seq data was generated to validate the experimental design.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: We quantified gene expression levels by RNA-seq in mNPCs derived from ES-E14tg2a (Peric-Hupkes et al., PMID: 20513434), ES-E14tg2a cells, and HepG2 cells. 1x10^6 cells were cultured and subjected to RNA-seq in two to three biological replicates.

first: Teodora

last: Filipovska

first: Bas

last: van Steensel

first: Chaitanya

last: Rastogi


title: Next Generation Sequencing and Quantitative Analysis of mRNAs in Caenorhabditis elegans Mutant Animals Exhibiting Impaired Hedgehog, mTORC2, or p38 Signaling

city: Chapel Hill

first: Robert

middle: Houston

last: Dowen

state: NC

address: 321 Fordham Hall, CB7100

department: Cell Biology and Physiology

institute: University of North Carolina at Chapel Hill

summary: To maximize fitness, organisms must appropriately allocate energetic resources between essential cellular processes to maintain homeostasis. The nutritional regulators of energy homeostasis have been studied in detail; however, how developmental signals might impinge on these pathways to govern cellular metabolism is poorly understood. We identified a non-canonical role for Hedgehog (Hh), a classic regulator of development, in maintaining intestinal lipid homeostasis in C. elegans. We find that expression of two Hh ligands, GRD-3 and GRD-4, is controlled by the LIN-29/EGR transcription factor in the hypodermis, where the Hh secretion factor CHE-14/Dispatched also facilitates non-cell autonomous Hh signaling. Using C. elegans, we demonstrate that Hh metabolic regulation does not occur through the canonical Hh transcription factor, TRA-1/GLI, but rather through non-canonical signaling that engages mTOR Complex 2 (mTORC2) in the intestine. Hh mutants display impaired lipid homeostasis, including reduced lipoprotein synthesis and fat accumulation, decreased growth, and upregulation of autophagy factors, mimicking loss of mTORC2. Additionally, we found that Hh inhibits p38 MAPK signaling in parallel to mTORC2 activation and that both pathways act together to modulate of lipid homeostasis. Our findings show a non-canonical role for Hedgehog signaling in lipid metabolism via regulation of core homeostatic pathways and reveal a new mechanism by which developmental timing events govern metabolic decisions.

overall_design: The mRNAs of wild-type and mutant day 1 adult animals were profiled by high-throughput sequencing, in triplicate.


title: The protein moonlighting dominates the phenotypic divergence of the Sef1 transcriptional regulatory networks in yeasts

status: Public on May 23 2024

city: Taipei

first: Po-Chen

last: Hsu

address: 128 Academia Road, Section 2, Nankang

department: Institute of Molecular Biology

country: Taiwan

institute: Academia Sinica

summary: We report the ChIP-seq profiling of a transcriptional factor Sef1 in Sccharomyces cerevisiae, and show that ScSef1 targets many TCA cycle and many others genes but has very limited regulatory effects to these target genes.

overall_design: ScSef1 ChIP-seq analysis was performed from three or four biological replicates of YPD- and YPGly-grown Sef1-TAP tagged cells, using Illumina NextSeq500 sequencer.

first: Jun-Yi

last: Leu


title: Teriflunomide/leflunomide synergize with chemotherapeutics by decreasing mitochondrial fragmentation via DRP1 in SCLC

status: Public on May 22 2024

city: Duarte

first: Haiqing

last: Li

state: CA

address: 1500 E Durate Rd

department: Shared Resources Department

institute: City of Hope National Medical Center

summary: Although up to 80% small-cell lung cancer (SCLC) patients response good for first-line chemotherapy regimen, most patients develop recurrence of the disease within weeks to months. Here, we report cytostatic effect of Leflu and Teri on SCLC cell proliferation through inhibition of DRP1 phosphorylation at Ser616 and decreased mitochondrial fragmentation. When administered together, Teri and carboplatin (Carbo) acted synergistically to significantly inhibit cell proliferation and DRP1 phosphorylation, reduce abundance of intermediates in pyrimidine de novo pathway, and increase apoptosis and DNA damage. Combination of Leflu&Carbo has anti-tumorigenic effect in vivo. Additionally, lurbinectedin (Lur) and Teri potently and synergistically inhibited spheroid growth, and depleted uridine and DRP1 phosphorylation in mouse tumors. Our results suggest combinations of Carbo and Lur with Teri or Leflu are efficacious and underscore how the relationship between DRP1/DHODH and mitochondrial plasticity serves as a potential therapeutic target to validate these treatment strategies in SCLC clinical trials.

overall_design: SCLC SBC3 cells Spheroids were treated with the appropriate drugs to study the new treatment strategy.

first: Tamara

last: Mirzapoiazova

first: Ravi

last: Salgia


title: Emx2 underlies the development and evolution of marsupial gliding membranes

city: Houston

first: Olga

last: Dudchenko

state: TX

address: 1 Baylor Plaza

institute: Baylor College of Medicine

summary: We study the genomic and developmental basis of the mammalian gliding membrane, or patagium, an adaptative trait that has repeatedly evolved in different lineages, including in closely related marsupial species. Through comparative genomic analysis of fifteen new marsupial genomes, both from gliding and non-gliding species, we find that the Emx2 locus experienced lineage-specific patterns of accelerated cis-regulatory evolution in gliding species. We confirm our finding via epigenomics, transcriptomics, and in vivo marsupial transgenics.

sample_organism:
Macropus fuliginosus
Macropus giganteus
Pseudocheirus peregrinus
Vombatus ursinus
Petaurus breviceps
Pseudochirops cupreus
Distoechurus pennatus
Dactylopsila trivirgata
Pseudochirops archeri
Tarsipes rostratus
Phalanger gymnotis
Pseudochirops corinnae
Petauroides volans
Acrobates pygmaeus
Pseudocheirus occidentalis

overall_design: 15 marsupial genomes, from closely related gliding and non-gliding species

first: Jorge

last: Moreno

first: Erez

middle: L

last: Aiden

first: Ricardo

last: Mallarino


title: Maintenance of haematopoietic stem cells by tyrosine-unphosphorylated STAT5 and JAK inhibition - STAT5 deficient HSPCs (LK)

city: Cambridge

first: Hugo

last: Bastos

address: Jeffrey Cheah Biomedical Centre, Puddicombe Way

department: Haematology

institute: Wellcome - MRC Cambridge Stem Cell Institute

summary: Adult haematopoietic stem cells (HSCs) are responsible for the lifelong production of blood and immune cells and must respond to extracellular cues including cytokines. The JAK/STAT pathway is a highly conserved pathway, activated by many cytokines, in which tyrosine-phosphorylated STATs (pSTATs) function as transcription factors. STAT5 is a pivotal downstream mediator of several cytokines known to regulate haematopoiesis but its function in the HSC compartment remains poorly understood. Here we show that STAT5-deficient HSCs exhibited an unusual phenotype - reduced multi-lineage repopulation and self-renewal combined with reduced cell cycle entry and increased differentiation. This reflected not only loss of a canonical pSTAT5 transcriptional program but also loss of distinct functions mediated by tyrosine-unphosphorylated STAT5 (uSTAT5). Consistent with this concept expression of an unphosphorylatable STAT5B mutant constrained HSC differentiation, promoted HSC maintenance and upregulated transcriptional programs associated with quiescence and stemness.  Moreover, treatment with a JAK1/2 inhibitor (ruxolitinib) increased the uSTAT5:pSTAT5 ratio, constrained HSC differentiation and proliferation and promoted HSC maintenance, thus phenocopying uSTAT5B overexpression. Our results therefore reveal a previously unrecognized interplay between pSTAT5 and uSTAT5 in the control of HSC function. In addition the demonstration that JAK inhibition promotes HSC maintenance has implications for gene therapy using HSCs and may contribute to the failure of JAK inhibitors to eradicate myeloproliferative neoplasms.

overall_design: Lineage negative, cKit positive bone marrow cells were sorted from WT and STAT5KO mice. The transcriptional profiles of these cells was analysed by 10X genomics single cell RNA sequencing.

first: Matthew

last: Williams

first: Juan

first: Xiaonan

last: Wang

first: Michelle

last: Wantoch

first: Hyun

last: Park

first: Gabriela

last: Grondys-Kotarba

first: Dean

last: Pask

first: Tina

last: Hamilton

first: Nicola

middle: K

last: Wilson

first: Ryan

last: Asby

first: Rachel

last: Sneade

first: Sarah

last: Kinston

first: Elisa

last: Laurenti

first: George

middle: S

last: Vassiliou

first: Berthold

last: Göttgens

first: Anthony

last: Green


title: Long-read Ribo-STAMP simultaneously measures transcription and translation at full length isoform resolution

status: Public on May 20 2024

city: La Jolla

first: Brian

last: Yee

address: 9500 Gilman Dr

department: Health

institute: UCSD

summary: Transcription and translation are intertwined processes where mRNA isoforms are crucial intermediaries. However, methodological limitations in analyzing translation at the mRNA isoform level have impaired our ability to comprehensively establish links between the full-length transcripts and the translatome. This has left gaps in our understanding of critical biological processes, regulatory mechanisms, and disease progression. To address this, we develop an integrated computational and experimental framework called long-read Ribo-STAMP (LR-Ribo-STAMP). LR-Ribo-STAMP capitalizes on advancements in long-read sequencing and RNA-base editing-mediated technologies to simultaneously and scalably profile translation and transcription at both gene and mRNA isoform levels for the first time. In this report, we show agreement between gene-level translation profiles obtained with LR-Ribo-STAMP and those from previously validated short-read Ribo-STAMP data in unperturbed cells. At the mRNA isoform level, we show that LR-Ribo-STAMP successfully profiles translation in unperturbed cells and links mRNA isoforms and regulatory features, such as upstream ORFs (uORFs) and regulatory sequences, to translation measurements. We further demonstrate the method’s effectiveness in profiling disease models by profiling translation at gene and isoform levels in a triple-negative breast cancer cell line under normoxia and hypoxia. Here, we find that LR-Ribo-STAMP effectively delineates orthogonal transcriptional and translation shifts between conditions at gene and isoform levels. At the isoform level, LR-Ribo-STAMP uniquely identifies key regulatory elements and shifts in mRNA isoform transcription that correlate with changes in translational, providing an example of insight that can inform the development of novel therapeutics. Overall, LR-Ribo-STAMP is a significant advancement in translation methods and can have profound implications for basic research and clinical applications.

overall_design: Three replicates each of unperturbed HEK293T cells expressing dox-inducible APOBEC1-only or Ribo-STAMP expression vectors to benchmark long-read Ribo-STAMP and profile translation at gene and mRNA isoform level in unperturbed cell state context. Two replicates each of MDA-MB-231 (triple-negative breast cancer cell line) expressing the Ribo-STAMP expression vector with and without 48h cobalt (II) chrolide treatment to induce hypoxia to apply long-read Ribo-STAMP for profiling disease models.

first: Pratibha

last: Jagannatha

first: Alex

middle: T

last: Tankka

first: Daniel

middle: A

last: Lorenz

first: Tao

last: Yu

first: Kristopher

middle: W

last: Brannan

first: Jason

last: Underwood

first: Gene

last: Yeo


title: Early transcriptomic response of innate immune cells  to subcutaneous BCG vaccination of mice

city: Moscow

first: Liya

last: Kondratyeva

address: Ulitsa Miklukho Maklaya, 16/10

country: Russia

institute: Institute of bioorganic chemistry of the Russian Academy of Sciences

summary: Bacille Calmette-Guerin (BCG) vaccination might contribute to nonspecific enhancement of resistance to various infections. Thus, BCG in addition to the formation of specific immunity against mycobacteria is also capable of inducing “trained immunity” - the ability of the innate immune system to maintain the memory of past infections and use it to develop immune defenses against new ones. In this study, we performed RNA sequencing of innate immune cells derived from mice bone marrow and spleen three day after subcutaneous BCG vaccination to evaluate early transcriptomic response of innate immune cells

overall_design: We performed RNA sequencing of innate immune cells derived from murine bone marrow and spleen three day after subcutaneous BCG vaccination. To do this, we obtained single-cell suspensions from the organs, stained the suspensions with conjugated anti-CD45, anti-CD11b, anti-NK1.1, anti-Ly6G, and anti-Ly6C antibodies, and subjected the suspen-sions to fluorescence-activated cell sorting (FACS). The prepared cell suspensions were used for FACS sorting and three cell populations were collected: monocytes [CD45+CD11b+Ly6C+], neutrophils [CD45+CD11b+Ly6G+] and NK-cells [CD45+CD11b-NK1.1+]. The samples were collected for each mouse from both control and BCG vaccinated groups. Monocytes and neutrophils were collected during sorting of bone marrow cell suspensions and NK-cells were collected from spleens. Then we have pre-pared double-indexed cDNA libraries for Illumina sequencing from the collected samples. The quality of the cDNA libraries was confirmed via Agilent Tape Station 2200 system, and the resulting cDNA library mix was subjected to NovaSeq 6000 sequencing. The RNA sequencing data was further processed with HISAT2 (GRCm38/mm10 was used as the reference genome), featureCount (GENCODE vM23, GRCm38.p6 was used as the reference transcriptome) and DESeq2 tools.

first: Alexey

last: Kuzmich

first: Irina

last: Linge

first: Victor

last: Pleshkan

first: Eugene

last: Snezhkov

first: Olga

last: Rakitina

first: Sofia

last: Kondratieva

last: Alekseenko


title: Transcriptional outcome of telomere elongation in human fibrosarcoma model

status: Public on May 18 2024

city: New Delhi

first: Shantanu

last: Chowdhury

state: Delhi

address: Mathura Road

department: Functional Genomics

country: India

institute: CSIR-Institute of Genomics and Integrative Biology

summary: The study focuses on understanding the transcriptional changes and the pathways altered when human cancer (fibrosarcoma ) cells  are subjected to telomere elongation.

overall_design: HT1080 human fibrosarcoma cell line was subjected to telomere elongation by constitutive overexpression of TERT and TERC components of the telomerase holoenzyme ( Lingner et al., 2006). The cells were characterized for telomere length and found to have about 2.5-fold longer telomeres and were designated as HT1080- long telomeres (HT1080-LT). Total RNA extracted from HT1080 cells (short telomeres) and HT1080-LT ( long telomeres) cells were sequenced using the Illumina Hiseq 2000 platform.

first: Ananda

last: Mukherjee

first: Shalu

last: Sharma

first: Mohammed

last: Faruq

first: Anshul

last: Budharaja

first: Ishaan

last: Gupta


title: Maintenance of ESLAM haematopoietic stem cells by tyrosine-unphosphorylated STAT5 and JAK inhibition

first: Hugo

last: Bastos

overall_design: Bone Marrow resident ESLAM HSCs were sorted from WT and STAT5-deficient mice and submitted for plate based single cell RNA sequencing by Smartseq2

first: Matthew

last: Williams

first: Juan

last: Li

first: Xiaonan

last: Wang

first: Michelle

last: Wantoch

first: Hyun

last: Park

first: Gabriela

last: Grondys-Kotarba

first: Dean

last: Pask

first: Tina

last: Hamilton

first: Nicola

last: Wilson

first: Ryan

last: Asby

first: Rachel

last: Sneade

first: Sarah

last: Kinston

first: Elisa

last: Laurenti

first: George

last: Vassiliou

first: Berthold

last: Göttgens

first: Anthony

last: Green


title: Single-cell profiling reveals a critical role for hypoxia-inducible factor-1a signaling in leukocytes during human craniotomy infection

status: Public on May 09 2024

city: White/Caucasian

first: Tammy

last: Kielian

state: NE

address: 985900 Nebraska Medical Center

institute: UNMC

summary: Neurosurgeries complicated by bacterial infection are often associated with poor clinical prognosis and prolonged treatment strategies involving multiple surgeries, placing additional morbidity on an already fragile patient population. Craniotomy is one of the most common neurosurgical procedures with an infection rate of 1-7%; however, the cellular and molecular signatures associated with craniotomy infection in human subjects have not yet been investigated. We performed a retrospective study of over 2,500 craniotomies, which revealed diversity in patient demographics, pathogen identity, and surgical landscapes associated with infection. Leukocyte profiling in tissues procured from craniotomy infection patients revealed a predominance of granulocytic myeloid-derived suppressor cells (G-MDSCs), which were absent from the blood. Single-cell transcriptomics identified immunometabolic shifts in leukocytes invading infected tissues, including a prominent hypoxia-inducible factor-1 (HIF-1) signature, whereas peripheral blood cells from the same patient were largely quiescent. The importance of HIF-1 signaling was validated using a mouse model of Staphylococcus aureus craniotomy infection, where the local delivery of HIF-1 inhibitor microparticles attenuated proinflammatory cytokine production concomitant with a significant increase in leukocyte infiltrates into the infected brain and galea. These findings establish G-MDSCs as a major infiltrate during human craniotomy infection and identify conserved transcriptional signatures across several leukocyte populations that may represent promising therapeutic targets for craniotomy infection in the face of increasing antimicrobial resistance.

overall_design:
To better understand the human immune response to craniotomy infection, we collected residual surgical tissue from patients with craniotomy infection. These specimens included blood, subgaleal, epidural, bone flap, and intracranial tissue samples. Samples were collected from 4 patients, designated patient 7, 11, 14, or 15.
We processed each sample into a single-cell solution and enriched each sample for CD45+ immune cells via fluorescence-activated cell sorting, before analysis via scRNA-seq.

first: Zachary

last: Van Roy

first: Gunjan

last: Kak

first: Lee

middle: E

last: Korshoj

first: Joe

last: Menousek

first: Cortney

last: Heim

first: James

last: Campbell

first: Carol

last: Geary

first: Bo

last: Liu

first: Scott

first: William

last: Thorell


title: TGFBR3 inhibits the aggressive of papillary thyroid cancer by inhibiting PI3K/AKT pathway and EMT

city: Guang Zhou

first: zhang

middle: han

last: rong

state: Guang dong Province

address: Zhong shan Second Road

institute: The First Affiliated Hospital of SunYat-sen University

summary: Transforming Growth Factor Beta Receptor III (TGFBR3) had been shown to play a tumor suppressor role in a variety of cancers. However, its role in papillary thyroid cancer (PTC) remains a mystery. The purpose of this study was to determine the molecular mechanism and biological behavior of TGFBR3 in PTC, to further explore the regulatory pathway involved in TGFBR3 on PTC and provide potential targets for the diagnosis and treatment of invasive PTC.

overall_design: To explore the expression of TGFBR3 in PTC. Construct TGFBR3 overexpression cell line, explore the influence of TGFBR3 overexpression on PTC cells. Explore the downstream pathway of TGFBR3 on PTC. Investigate how TGFBR3 regulates the biological behavior of PTC through PI3K/AKT and EMT.

first: Hanrong

last: Zhang

first: Junxin

last: Chen

first: Xin

first: Hai

last: Li