title: Transcriptional regulation in the absence of inositol trisphosphate receptor signaling

status: Public on Dec 03 2024

city: philadelphia

first: Suresh

last: Joseph

state: PA

address: 1020 Locust street

institute: Thomas Jefferson University

summary: The activation of IP3 receptor (IP3R) Ca2+ channels generates agonist-mediated Ca2+ signals that are critical for the regulation of a   wide range of biological processes. It is therefore surprising that CRISPR induced loss of all three IP3R isoforms (TKO) in HEK293 and HeLa cell lines yields cells that can survive, grow and divide, albeit more slowly than wild-type cells. In an effort to understand the adaptive mechanisms involved, we have examined the activity of key Ca2+ dependent transcription factors (NFAT, CREB and AP-1) and signaling pathways using luciferase-reporter assays, phosphoprotein immunoblots and whole genome transcriptomic studies. In addition, the diacylglycerol arm of the signaling pathway was investigated with protein kinase C (PKC) inhibitors and siRNA knockdown. The data showed that agonist-mediated NFAT activation was lost but CREB activation was maintained in IP3R TKO cells. Under base-line conditions transcriptome analysis indicated the differential expression of 828 and 311 genes in IP3R TKO HEK293 or HeLa cells, respectively, with only 18 genes being in common. Three main adaptations in TKO cells were identified in this study: 1) increased basal activity of NFAT, CREB and AP-1; 2) an increased reliance on Ca2+- insensitive PKC isoforms; and 3) increased production of reactive oxygen species and upregulation of antioxidant defense enzymes. We suggest that whereas wild-type cells rely on a Ca2+ and DAG signal to respond to stimuli, the TKO cells utilize the adaptations to allow key signaling pathways (e.g. PKC, Ras/MAPK, CREB) to transition to the activated state using a DAG signal alone.

overall_design:
RNAseq profiling of HEK293 cells wild-type cells (WT) and HEK293 cells in which all three IP3R isoforms have been deleted (TKO)

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first: Michael

last: Young

first: David

last: Booth

last: Smith

first: Marco

last: Tigano

first: Gyorgy

last: Hajnoczky


title: Gene expression profile at single cell level of YS-derived cells and monocyte-derived cells from brain parenchyma and leptomeninges of Ms4a3-Cre: R26-TdTomato: Cx3cr1-gfp mice

status: Public on Dec 02 2024

city: Rehovot

first: Jung-Seok

last: Kim

address: 234 Herzl Street, Wolfson Building 321

department: Immunology and Regenerative Biology

country: Israel

institute: Weizmann Institute Of Science

type: Expression profiling by high throughput sequencing

summary: Ms4a3-Cre: R26-TdTomato: Cx3cr1-gfp mice allow discrimination between YS-derivd  and monocyte-derived macrophages in the brain parenchyma and leptomeninges by color. We used single cell RNAseq for RNA profiling to compare YS-derived micrglia, monocyte-derived microglia, YS-derived leptomeningeal macrophages, and monocyte-derived leptomeningeal macrophages.

overall_design: Brain parenchymal and leptomenigeal cells were isolated seperately from the brain of  one-year-old Ms4a3-Cre: R26-TdTomato: Cx3cr1-gfp mice. Cells were sorted by color of reporters with staining to discriminate microglia and macrophages.

first: Sébastien

last: Trzebanski

first: Aryeh

last: Solomon

first: Steffen

last: Jung


title: Gene expression profile at sorted microglia and border-associated macrophage with Ms4a3-Cre: R26-TdTomato: Cx3cr1-gfp bone marrow transplantation

summary: After irradiation and bone-marrow transplantation, most of microglia and boarder-associated macrophages in the central nervous system is replaced by engranft cells. Taking advantages of Ms4a3-Cre: R26-TdTomato: Cx3cr1-gfp mice, we discriminated cells from monocyte precursors and hematopoietic stem cells with colors at 32 weeks following irradiation and bone-marrow transplantation. We sorted HSC-derived, monocyte-derived, and endogenouse microglia and leptomeningeal macrophages, and analyzed gene expression level between different cell populations using a bulk RNAseq method.

overall_design: Wild type C57BL6/C mice were lethally irradiated, and took Ms4a3-Cre: R26-TdTomato: Cx3cr1-gfp bone marrow transplantation. After 32 weeks of irradiation and bone morrow transplantation, host cells (no color), HSC-derived cells (GFP+), and monocyte-derived cells (GFP+ and TdTomato+) were sorted, and used for bulk RNAseq transcriptome analysis.


title: Synaptic decline precedes dopaminergic neuronal loss in human midbrain organoids harboring a triplication of the SNCA gene

city: Esch sur Alzette

first: Alise

last: Zagare

state: Belval

address: 7 Avenue des Hauts-Fourneaux

country: Luxembourg

institute: University of Luxembourg

summary: Increased levels of the protein alpha-synuclein (α-syn) are associated with the development of neurodegenerative diseases like Parkinson's disease (PD). In physiological conditions, α-syn modulates synaptic plasticity, neurogenesis and neuronal survival. Here, we used a PD patient specific midbrain organoid model derived from induced pluripotent stem cells harboring a triplication in the SNCA gene to study PD-associated phenotypes. The model recapitulates the two main hallmarks of PD, which are α-syn aggregation and loss of dopaminergic neurons. Additionally, impairments in astrocyte differentiation were detected. Transcriptomics data indicate that synaptic function is impaired in PD specific midbrain organoids. This is further confirmed by alterations in synapse number and electrophysiological activity. We found that synaptic decline precedes neurodegeneration. Finally, this study substantiates that patient specific midbrain organoids allow a personalized phenotyping, which make them an interesting tool for precision medicine and drug discovery. However, its pathogenic accumulation and aggregation results in toxicity and neurodegeneration.

overall_design: Comparative gene expression analysis between healthy control and Parkinson's disease patients with SNCA triplication

first: Jennifer

last: Modamio

first: Claudia

last: Saraiva

first: Gemma

last: Gomez-Giro

first: Sarah L

last: Nickels

first: Javier

last: Jarazo

first: Paul

last: Antony

first: Peter

last: Barbuti

first: Rashi

last: Halder

first: Christian

last: Jaeger

first: Reijko

last: Krueger

first: Enrico

last: Glaab

first: Jens

last: C Schwamborn


title: RNA seq analysis for impact of high glucose and salt on Zebrafish Embryo development

city: Anderson

first: Renfang

middle: Song

last: Taylor

state: IN

address: 1100 5th Street

department: Biology

country: USA

institute: Anderson University

summary:
Introduction
Zebrafish (Danio rerio) is an important model organism extensively used for studying developmental biology and genetics. The Wnt pathway plays a crucial role in embryonic development in vertebrates. High glucose and salinity can disrupt the normal Wnt signaling pathway and cause abnormal embryonic development. In this study, we aimed to investigate the effect of high glucose and salinity on embryological development of zebrafish through the Wnt pathway.
Methods
embryos were treated with normal media and high glucose for 5 days from 3 hours post-fertilization(hpf) to 96hpf separately as control and experimental groups. RNA is extracted from control and experimental group for replicates for RNAseq. Day 5 Larva is fixed in 10% formalin for H&E staining to detect organogenesis changes under high glucose and Saline conditions. Zebrafish eggs were treated with normal media and high salt (2%) for 3 days from 3 hpf to 72hpf separately as control and experimental groups. Day 1 and Day 2 larvae are fixed in 10% formalin for H&E staining.  We also analyzed the Wnt signaling pathway in these fish under different glucose and salinity conditions.
Results
Our results showed that exposure to high glucose and salinity caused abnormalities in embryological development of the fish. We observed poor hatching rate, reduced body length, deformities in various body parts, and mortality of the fish. Glucose delayed the zebrafish embryo development by slowing the hatch rate about 24 hours. The brain, heart, and tail started showing morphology smaller in the glucose group compared to the control group at 24 hpf. Heartbeat is slower in the glucose group compared to the control group on day 2 and 3 with a statistically significant difference. The brain, heart, and tail started showing morphology smaller in the salt group compared to the control group at 48 hpf. These conditions contribute to significant physiological differences that may provide insight on functionality post-embryological development. Testing to find consistent trends in morphology and genetic differences in high salt conditions, and genetic alterations for high glucose conditions are being further researched in this experiment. 1674 genes are regulated in high glucose treatment and 4323 genes changed expression in high saline condition. We also found that high glucose and salinity disrupted the normal Wnt signaling pathway by downregulating the expression of Wnt pathway genes and proteins. The expression of Wnt pathway inhibitors, such as Axin2 and DKK1, was upregulated, while the expression of Wnt pathway activators, such as β-catenin and Wnt3a, was downregulated in fish exposed to high glucose and salinity.
Conclusion
High glucose and salinity can have a detrimental effect on the embryological development of zebrafish through the Wnt signaling pathway. Our results suggest that exposure to high glucose and salinity can disrupt the normal development of zebrafish and lead to developmental abnormalities. Further studies are needed to investigate the underlying molecular mechanisms that regulate the Wnt pathway in response to high glucose and salinity in zebrafish.

overall_design:
Zebrafish embryos were cultured at control, high glucose and high salt conditions for 72 hours. RNA was extracted for RNA seq.
Control groups vs high glucose vs high salt groups.

first: Justin

last: Hensley

first: Ebony

last: Thompson

middle: S


title: Platelet thromboxane limits T cell immunity to cancer metastasis via ARHGEF1

city: Cambridge

first: Rahul

last: Roychoudhuri

address: Tennis Court Road

department: Department of Pathology

country: United Kingdom

institute: University of Cambridge

summary: Metastasis is the spread of cancer cells from primary tumours to distant organs and is the cause of 90% of cancer deaths globally.  Newly metastasising cancer cells are uniquely vulnerable to immune attack, deprived of the highly immunosuppressive microenvironment of established tumours.  There is interest in exploiting this vulnerability to prevent recurrence in early cancer patients at risk of metastasis.  Here, we show that inhibition of cyclooxygenase (COX)-1, including with aspirin, enhances immunity to cancer metastasis by releasing T cells from suppression by platelet-derived thromboxane A2 (TXA2). Platelet TXA2 triggers an immunosuppressive pathway within T cells dependent upon the guanine exchange factor ARHGEF1, suppressing T cell receptor (TCR)-driven kinase signalling, proliferation and effector functions. T cell-specific conditional deletion of Arhgef1 in mice increases T cell activation at the metastatic site, provoking immune-mediated rejection of lung and liver metastases.  Consequently, restricting the availability of TXA2 using aspirin, selective COX-1 inhibitors or through platelet-specific deletion of COX-1 reduces the rate of metastasis in a manner dependent upon T cell-intrinsic expression of ARHGEF1 and signalling by TXA2 in vivo.  These findings reveal a novel immunosuppressive pathway limiting T cell immunity to cancer metastasis, providing a mechanistic basis for the anti-metastatic activity of aspirin and paving the way for more effective anti-metastatic immunotherapies.

overall_design: FACS-sorted naïve CD8+ T cells from wild-type (WT) and Arhgef1-deficient mice were stimulated with anti-CD3/CD28 antibodies, rhIL-2 and thromboxane A2 analogue U46619 for 5 days, followed by bulk RNA sequencing analysis.

first: Jie

last: Yang

first: Alex

middle: C

last: Evans


overall_design: Whole lungs were harvested from wild-type (WT) and Arhgef1-knockout (KO) littermates 7 days after intravenous injection of B16 melanoma cells for bulk RNA-seq analysis.

first: Alexander


title: Lactate dehydrogenase B noncanonically promotes ferroptosis defense in KRAS-driven lung cancer

status: Public on Dec 04 2024

city: Bern

first: Liang

last: Zhao

address: Murtenstrasse 24

country: Switzerland

institute: DBMR

summary: To investigate SLC7A11 inhibition-induced ferroptosis in LDHB-deficient lung cancer cells requires glutaminolysis

first: Ren-wang

last: Peng


title: Absolute transcription-start nucleotide m6Am stoichiometry quantification by CROWN-Seq

city: New York

first: Samie

last: Jaffrey

state: New York

address: 1300 York Ave

department: Pharmacology

institute: Weill Cornell Medicine

summary: The 5’ end RNA molecules transcribed by RNA polymerase II are known to be modified in human cells. Specifically, A-initiated transcripts can be methylated at both the 2’-O position in the ribose (2’-O-methylation, or cap-1) and the N6-position in the base (N6-methylation). Almost all A-initiated transcripts have the cap-1 modification, however, the N6-methylation is optional. As a result, A-initiated transcripts in the cell can be Am (cap-1 only) or m6Am (cap-1 and N6-methylation) modified. To study m6Am, we developed CROWN-seq, which simultaneously obtains the transcription-start information and the stoichiometry of N6-methyl. With CROWN-seq, we found that the previous m6Am mapping studies highly underestimated the diversity of the transcription-start site (nucleotide) of the transcriptome. In this study, we used CROWN-seq to reveal m6Am sites and stoichiometry among different human cell lines. We demonstrated that m6Am stoichiometry in mRNA is in general high, however, m6Am stoichiometry in snRNA, snoRNA, and their pseudogenes is more variable.

overall_design: We performed both technical replicates and biological replicates of CROWN-seq in different cell lines. Technical replicates were performed by using the same purified mRNA as input.

first: Jianheng

last: Liu

middle: R

first: Nicholson

last: Luke


title: Single cell RNA-Seq of blood cells in irradiated patient

status: Public on Dec 06 2024

city: suzhou

first: zhang

middle: shu

last: yu

address: No. 199, Ren'ai Road

department: soochow university

country: China

institute: Soochow University

summary: This study aims to explore mRNA expression of  irradiated people’s blood  by single cell RNA-Seq. In this study, the blood was isolated from patients. The blood of irradiated patient was collected from a patient who exposed to irradiated.

overall_design: the bolld of irradiatd patient  was collected from patient who was exposed to irradiated.

first: Shuyu

last: Zhang

first: Yan

last: Tao

first: Daojiang

last: Yu


title: Macrophages protect against sensory axon degeneration in diabetic neuropathy

city: Los Angeles

first: Riki

last: Kawaguchi

state: CA

address: 760 Westwood Plaza, Room 37-420

department: Department of Psychiatry and Neurology Department of Psychiatry

institute: University of California, Los Angeles

summary: Diabetic peripheral neuropathy (DPN) is a common complication of diabetes, causing sensory loss and debilitating neuropathic pain1,2. Although the onset and progression of DPN have been linked with dyslipidemia and hyperglycemia3, the contribution of inflammation in the pathogenesis of DPN has not been investigated. Here, we use a High Fat High Fructose Diet (HFHFD) to model DPN and its metabolic syndrome in mice. Diabetic mice develop persistent heat hypoalgesia after three months, but a reduction in epidermal skin innervation only manifests at 6 months. Using single-cell sequencing, we find that CCR2+ macrophages infiltrate the sciatic nerves of diabetic mice well before axonal degeneration is detectable. We show that these infiltrating macrophages share gene expression similarities with nerve crush-induced macrophages4 and express neurodegeneration-associated microglia marker genes5 although there is no axon loss or demyelination yet. Inhibiting this macrophage recruitment in diabetic mice by genetically or pharmacologically blocking CCR2 signaling results in a more severe heat hypoalgesia and accelerated skin denervation. These findings reveal a novel neuroprotective recruitment of macrophages into peripheral nerves of diabetic mice that delays the onset of terminal axonal degeneration, thereby reducing sensory loss. Potentiating and sustaining this early neuroprotective immune response in patients represents, therefore, a potential means to reduce or prevent DPN.

overall_design: We fed male C57Bl6 mice on a high fat high fructose diet (HFHFD) or control diet (CD) for 12 weeks. Mice fed HFHFD become diabetic. We FACS sorted Live Cd45+ immune cells from hindpaw skin and sciatic nerves and performed single cell RNA sequencing. We identified infiltration of CCR2+ macrophages in the nerves but not the skin of diabetic mice.

first: Sara

last: Hakim

first: Clifford

last: Woolf


title: Impact of severe traumatic brain injury (TBI) on cerebral blood flow and the endothelial cell phenotype

city: Milwaukee

first: Ankan

last: Gupta

state: WI

address: 8701 W Watertown Plank Road

department: Pediatrics

institute: Medical College of Wisconsin

summary: TBI causes the disruption of blood vessels in brain leading to hemorrhage, edema, and changes in cerebral blood flow. How such altered vascular and flow condition affect endothelial cells, the cellular unit of blood vessels, is not known. We used single cell RNA sequencing (scRNAseq) to analyze the heterogenity in endothelial cell clusters in brain, and how key cellular and signaling signatures of these clusters are temporally changed post-TBI at the site of injury.

overall_design: We used closed skull cortical impact (CCI) mouse model. The brain cortex specimens were isolated from the site of injury at day 1, 7 and 28 post-TBI. Site-matched specimens were also harvested from uninjured control mice. Single cells were harvested from the collected tissue specimens. Myelin debris was removed from the cells and the specimens were immediately fixed in 4% paraformaldehyde. Fixed samples were subjected to scRNAseq as per manufacturer's protocol.

first: Zachary

last: Bice

first: Vaya

last: Chen

first: Yiliang

first: Anthony

middle: J

last: Veltri

first: Chien-Wei

last: Lin

first: Xialong

last: Ma

first: Amy

middle: Y

last: Pan

first: Rahima

last: Zennadi

first: Sean

middle: P

last: Palecek

first: Ashraf

middle: M

last: Mohieldin

first: Surya

last: Nauli

first: Ramani

last: Ramchandran

first: Kevin

last: Rarick


title: Origin of extra-embryonic mesenchyme points to evolutionary novelty in primate early development

city: Farmington

first: Paul

last: Robson

state: CT

address: 10 Discovery Dr

institute: The Jackson Laboratory For Genomic Medicine

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Functional characterization of a surface antigen suppressing ApiAP2 in Eimeria tenella

status: Public on Dec 01 2024

city: Nanning

first: Dandan

last: Hu

state: China

address: Daxue East Rd #100, College of Animal Science and Techenology, Guangxi University.

institute: Guangxi University

summary: Apicomplexa parasite Eimeria is the causative agent of coccidiosis, which leads to enteritis in animals and causes huge economic burden to the farm industry. The Apicomplexan Apetala2/ERF (ApiAP2) transcription factors have proved to play key roles in various processes in other Apicomplexa parasites. However, little is known about the function of ApiAP2s in Eimeria species. In this study, we functionally characterized an ApiAP2 by direct knockout mediated by CRISPR-Cas9. Our results showed that the invasion efficiency, total oocyst output and virulence to the host were significantly impaired after EtAP2-S1 depletion. RNA-Seq and CUT&Tag analyze showed that EtAP2-S1 targets on the promoters of numerous genes and its knockout results in upregulation of 59 sag genes. Additionally, the knockout strain exhibits significantly reduced virulence but provides excellent immune protection, which makes it a good vaccine candidate. This study demonstrates that EtAP2-S1 is a fitness coffering gene that suppress the expression of sag genes in E. tenella, and this is the first practice in developing gene knockout vaccine for the control of coccidiosis.

overall_design:
Total RNAs from the sporozoites of EtHCYA and AP2-S1-KO strains were isolated for RNA-SEQ. Three biological replicates were employed.
To further characterize the direct target genes of AP2-S1, CUT&Tag experiments were performed using the AP2-S1 overexpression strain.

first: Peiyao

last: Li

first: Rina

last: Su

first: Ganglin

last: Ren

first: Hongbin

last: Si

first: Xingju

last: Song


title: Origin of extra-embryonic mesenchyme points to evolutionary novelty in primate early development [RNA-seq]

summary: As ontogeny does not recapitulate phylogeny and little developmental restraint exists prior to gastrulation, early mammalian development differs significantly between species. A prime example is the temporal difference in the first emergence of extra-embryonic mesenchymal cells (ExMC) between mouse and human. Here, we report a fast and efficient in vitro cell model of human ExMC formation from induced pluripotent stem cells. This ExMC emerges from the apparent late blastocystic trophoblast. We define HAND1 as an essential regulator of ExMC specification, with HAND1 null cells reverting to the trophoblast lineage. Regulatory phenotyping defines primary target genes of, and primate-specific genomic regions bound by, HAND1. These data emphasize the nascent evolutionary innovation in early development relevant to human health but, through pleiotropy, also to later in life human biology. The novel findings that trophectoderm is a source of ExMC lineage and how recently evolved young transposable elements regulate this process emphasize the evolutionary novelties of our model to explore the primate embryogenesis.

overall_design: hiPSC cells (KOLF2) were culured in different media for the differentiation to different cell lineages. BMP4 and SU5402 with or without A-83 were used for trophoblast differentiation and annotated as BS or BSA-83. BMP4, SU5402 and ActivinA were used for extra-embryonic mesenchymal cell differentiation and annotated as BSA. Both WT and HAND1 Knock-out cell lines were used in this study.

first: Zukai

last: Liu


title: Enteric glia regulate Paneth cell secretion and intestinal microbial ecology

city: Boston

first: Meenakshi

last: Rao

state: MA

address: 300 Longwood Ave

department: Pediatrics-Division of Gastroenterology

institute: Boston Children's Hospital

summary: Glial cells of the enteric nervous system (ENS) interact closely with the intestinal epithelium and secrete signals that influence epithelial cell proliferation and barrier formation in vitro. Whether these interactions are important in vivo, however, is unclear because previous studies reached conflicting conclusions. To better define the roles of enteric glia in steady state regulation of the intestinal epithelium, we characterized the glia in closest proximity to epithelial cells and found that the majority express PLP1 in both mice and humans. To test their functions using an unbiased approach, we genetically depleted PLP1+ cells in mice and transcriptionally profiled the small and large intestines. Surprisingly, glial loss had minimal effects on transcriptional programs and the few identified changes varied along the gastrointestinal tract. In the ileum, where enteric glia had been considered most essential for epithelial integrity, glial depletion did not drastically alter epithelial gene expression but caused a modest enrichment in signatures of Paneth cells, a secretory cell type important for innate immunity. In the absence of PLP1+ glia, Paneth cell number was intact, but a subset appeared abnormal with irregular and heterogenous cytoplasmic granules, suggesting a secretory deficit. Consistent with this possibility, ileal explants from glial-depleted mice secreted less functional lysozyme than controls with corresponding effects on fecal microbial composition. Collectively, these data suggest that enteric glia do not exert broad effects on the intestinal epithelium but have an essential role in regulating Paneth cell function and gut microbial ecology.

overall_design: To investigate whether enteric glia are  necessary for epithelial cell function, we used a glial depletion model in which Plp1+ cells were inducibly depleted in 5-6 week old male mice. After 11 days, whole intestinal segments from duodenum, proximal ileum, and proximal colon were taken for bulk-RNA sequencing. For scraped epithelium, 6cm of the most distal ileum was collected. A total of 32 samples were submitted from Cre+ (glial depleted) and WT (glial sufficient) littermates, evenly distributed between groups.  n=4  colon, n=4 duodenum, n=4 ileum, n=4  scraped epithelium ileum.

first: Aleksandra

last: Prochera

first: Anoohya

middle: N

last: Muppirala

first: Gavin

middle: A

last: Kuziel

first: Salima

last: Soualhi

last: Shepherd

first: Liang

last: Sun

first: Biju

last: Issac

first: Harry

last: Rosenberg

first: Farah

last: Karim

first: Kristina

last: Perez

first: Kyle

middle: H

last: Smith

first: Tonora

last: Archibald

first: Seth

last: Rakoff-Nahoum

first: Susan

last: Hagen


title: Origin of extra-embryonic mesenchyme points to evolutionary novelty in primate early development [scRNA-seq]

first: Paul

last: Robson

overall_design: WT KOLF2.C1 cell line was passaged as single cell and then reated with BMP4 and SU5402. Cells were harvested at different time points for single cell RNA-seq on 10x genomics platform.

first: William

last: Flynn


title: Gene expression profile of microglia and border-associated macrophages carrying hDNMT3A mutation

first: Jung-Seok

last: Kim

summary: To probe for the impact of a age-related clonal hematopoiesis associated somatic mutation on monocyte-derived microglia and macrophages in the CNS, we took advantage of a mouse model that harbors a replacement of the murin Dnmt3a gene with a cDNA cassette encoding the human DNMT3A R882H variant. To seed the CNS with monocyte-derived cells from hDNMT3A mut hematopoietic stem cells, we engrafted Cx3cr1-gfp mice with bone marrow of 10-month-old Vav-Cre: hDNMT3A fl/fl mice. After 4 months following irradiation, endogenous microglia and leptomeningeal macrophages, and engrafted cells were isolated from both mutation and wild type groups for bulk RNAseq analysis.

overall_design: Cx3cr1 gfp/+ mice were lethaly irradiated, and took either Vav-Cre: hDNMT3A fl/fl or hDNMT3A fl/fl bone marrow. After 4 months of irradiation, both GFP+ and GFP- microglia and leptomeningeal macrophages were isolated from both Mut and WT groups for sorting.

first: Steffen

last: Jung


title: Origin of extra-embryonic mesenchyme points to evolutionary novelty in primate early development [ChIP-seq]

overall_design: WT and HAND1 KO cell lines were cultured in BSA condition for 4 days and then collected for ChIP-seq with both HAND1 and H3K4me3 antibodies.

first: Zukai

last: Liu


title: The Weight of Multiple Hits: How TBI and Infectious Encephalitis Co-Modulate Adverse Outcomes

city: Blacksburg

first: Alicia

last: Pickrell

address: 970 Washington St NW

institute: Virginia Tech

summary: Compared to patients with no TBI history, patients with a TBI history at least one month before infectious encephalitis have an increased risk of mortality, epilepsy, and dementia or delirium. Those with a history of TBI have an increased risk of various outcomes, including mortality, epilepsy, and dementia. Bulk RNA sequencing of the hippocampus from mice subjected to CCI injury and VEEV infection demonstrated that key pathways, specifically those involved in granzyme-mediated cell death, were enriched compared to VEEV infection alone.

overall_design: All experimental procedures were performed on male mice aged 8–10 weeks. In brief, mice received a controlled cortical impact 1.5 months before VEEV inoculation and were sacrificed one week later for hippocampal tissue extraction (Figure 5). Mice were split into three groups: mock, VEEV, and TBI/VEEV (n=4/group). All studies complied with the guidelines outlined in the NIH Guide for the Care and Use of Laboratory Animals. Controlled cortical impact. Surgeries were conducted following established procedures [38-42]. Mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg), and buprenorphine SR (0.5 mg/kg) was administered subcutaneously before surgery. A Φ = 4 mm craniotomy was made over the right parietal bone (− 2.5 mm A/P and 2.0 mm lateral from bregma). Cortical injury was induced using an electronic controlled cortical impactor (Custom design and fabrication; Φ = 3 mm round tip) at a velocity of 5.0 m/s, depth of 2.0 mm, and 150 ms dwell time. Mice were intranasally infected with 2x107 PFU of VEEV TC-83 (except for uninfected control mice).

first: Liliana

last: Ladner

first: Collin

middle: T

last: Ocampo

first: Colin

last: Kelly

first: Caitlin

last: Woodson

first: Eric

last: Marvin

first: Kylene

last: Kehn-Hall

first: Michelle

last: Theus


title: Assessing mechanisms driving phenol isopropylated phosphate (IPP)-induced larval photomotor response deficits in zebrafish

city: Clemson

first: Subham

last: Dasgupta

state: South Carolina

address: 142 Long Hall

department: Biological Sciences

institute: Clemson University

summary: Isopropylated phenyl phosphates (IPP) are an additive organophosphate flame retardant (OPFR) which has been extensively used in furniture, electronics, automobiles, plastics, and children’s products to slow down the spread of fire. The processing and distribution of IPP- containing products have been prohibited but its continuous leaching from end use products has retained the concern of its toxicity. The present study was designed to evaluate IPP-induced developmental toxicity using zebrafish embryos. We first performed  range finding experiments with embryonic zebrafish, exposed to 0 - 200 M IPP from 6 to 120 h post fertilization and found significant morphological impacts like pericardial edema, yolk sac edema and spinal curvature at higher concentrations. Following this, relying on secondary analyses of our whole embryo mRNA-seq data, we quantified neurotransmitters and found significant increase in the levels of dopamine and its metabolite, 3-methoxytyramine. We then conducted, in vitro retinoic acid receptor (RAR) signaling assay and noticed significant inhibition of RARα, but not RAR and RAR. For behavioral readouts, we performed larval photomotor response (LPR) assay at sublethal concentrations and observed hypoactive locomotory behavior in exposed larvae. Whole-mount immunohistochemistry for 5-methylcytosine and global DNA methylation assay showed significant IPP-induced hypermethylation in situ. Finally, based on whole embryo RNA-seq data, we hypothesized that IPP affects the development of brain and eyes. Firstly, we performed global DNA methylation in brain and eyes, but did not find significant effects. Then, we conducted mRNA sequencing on dissected brains and eyes, and found 2 and 135 differentially expressed genes, respectively. Gene ontology revealed that IPP affect voltage gated ion channel activity, synaptic transmission and neurotransmitter signaling. Collectively, our data shows that IPP induces morphological abnormalities and disrupts larval photo motor response, potentially through RA inhibition and methylomic regulation. Finally, we observed that IPP affects gene expression within the developing eye, establishing synaptic transmission, vision and muscle contraction as a potential causative factor for LPR responses.

overall_design: Zebrafish embryos were exposed to 0 or 0.2 uM IPP from 6 to 120 hpf, eyes and brains were dissected out, RNA extraction and RNA sequencing using a Novogene Library preparation Pipeline and sequencing on an Novaseq 600 (Illumina, San Diego, CA) (2x150 bp, ~40m reads/sample).

first: Sunil

last: Sharma

first: Alfredo

last: Rojas

first: Abhishek

last: Gour

first: Rosemaria

last: Serradimigni

first: Connor

last: Leong

first: Abhisheak


title: Restriction mechanism of peri-centromeric DNA loop expansion necessary to ensure accurate chromosome segregation [Micro-C]

city: Tokyo

first: Sao

middle: Anh

last: Nguyen

address: 1-1-1 Yayoi, Bunkyo-ku

department: Institute for Quantitative Biosciences

country: Japan

institute: University of Tokyo

summary: Centromeres in budding yeast are surrounded by ~10-kb DNA loops generated by the cohesin complex. These peri-centromeric loops are shown to play a role in faithful chromosome segregation. It remains not fully understood how the dynamics of the peri-centromeric loops are controlled. Here we found that gene deletion of two cohesin regulators, Wpl1 and Eco1, resulted in larger peri-centromeric loops that connected the centromeres and genome regions up to ~ 300 kb distant in G2/M-arrested cells. Deletion of Wpl1 and Eco1 synergistically contributed to the loop size enlargement. ChIP-seq revealed that cohesin and its ATPase activator, Scc2, were colocalized at the anchor sites of the extended peri-centromeric loops, specifically in the double-deletion mutant Δwpl1 Δeco1. Consistently, acute depletion of Scc2 in the G2/M phase resulted in the disappearance of the extended loops. Given that Scc2 is bound prominently only at the centromeres in wild-type cells, our results suggest that Wpl1 and Eco1 jointly promote the dissociation of Scc2 from cohesin and inhibit excessive expansion of peri-centromeric DNA loop size. Notably, we found that nuclear division after benomyl block and release was significantly delayed in the Δwpl1 Δeco1 double-deletion mutant, and this delay was rescued by Scc2 depletion in the G2/M phase. This indicates that cohesin regulators cooperatively regulate the size of the peri-centromeric DNA loops to ensure faithful chromosome segregation.

overall_design: Micro-C experiment in budding yeast Saccharomyces cerevisiae to study chromosome structure in various mutant.

first: Takashi

last: Sutani

first: Yukata

last: Suzuki

first: Katsuhiko

last: Shirahige


title: NVP-BHG712 alleviates ovariectomy-induced osteoporosis by modulating osteoclastogenesis

city: Jinan

first: Yueran

last: Zhao

state: Shandong

address: 44 Wenhua West Road

department: Center for Reproductive Medicine

institute: Shandong University

summary: To investigate the effect of NVP-BHG712 on osteoclast differentiation, BMMs were removed from femorae of C57BL/6 mice and treated with NVP-BHG712 for RNA-seq

overall_design: Comparative gene expression profiling analysis of RNA-seq data for BMMs of mice treated with or without NVP-BHG712 after stimulation with RANKL.

first: Xin

last: Liu


title: Glucocorticoid- and Pioglitazone-Induced Proteinuria Reduction in Experimental Nephrotic Syndrome Both Correlate with Glomerular ECM Modulation

city: Columbus

first: Julie

last: Dougherty

state: OH

address: 700 Children's Dr, Research Building 2

department: Research

institute: Nationwide Children's Hospital, Columbus, Ohio

summary: Idiopathic nephrotic Syndrome (NS) is a common glomerular disease. While glucocorticoids (GC) are the primary treatment, the PPARγ agonist pioglitazone (Pio) also reduces proteinuria in patients with NS and directly protects podocytes from injury. Since both drugs reduce proteinuria, we hypothesized these effects result from overlapping transcriptional patterns. Systems biology approaches compared glomerular transcriptomes from rats with PAN-induced NS treated with GC vs. Pio and identified 29 commonly regulated genes-of-interest, primarily involved in extracellular matrix (ECM) remodeling. Correlation with clinical idiopathic NS patient datasets confirmed glomerular ECM dysregulation as a potential mechanism of injury. Cellular deconvolution in silico revealed GC- and Pio-induced amelioration of altered genes primarily within podocytes and mesangial cells. While validation studies are indicated, these analyses identified molecular pathways involved in the early stages of NS (prior to scarring) suggesting that targeting glomerular ECM dysregulation may enable a future non-immunosuppressive approach for proteinuria reduction in idiopathic NS.

overall_design: Day 11 glomeruli were isolated from rats with PAN-induced NS treated with methylprednisolone or with pioglitazone. Sham treated health controls were included as reference. N=4 rats were used for RNAseq. Data were analyzed for PAN-induced changes in gene expression and amelioration of said alterations by either drug. This study focused on the overlapping amerliorations of disease-induced gene expression between methylprednisolone and pioglitazone.

first: Sagar

last: Bhayan

first: Yu

last: Kamigaki

first: Shipra

last: Agrawal

first: Saranga

last: Wijeratne

first: James

last: Fitch

first: Amanda

last: Waller

first: Katelyn

last: Wolfgang

first: Peter

last: White

first: Bryce

last: Kerlin

first: William

middle: E

last: Smoyer


title: Restriction mechanism of peri-centromeric DNA loop expansion necessary to ensure accurate chromosome segregation

first: Sao

last: Nguyen


title: LncRNA AC025580.2 mediates the senescence of periodontal ligament stem cells in inflammatory environments via the alternative splicing of TP53BP1

city: Xi‘an

first: Mei

last: Xu

address: 145th West Changle Road

institute: The Fourth Military Medical University

type: Non-coding RNA profiling by high throughput sequencing

summary: Periodontal ligament stem cells (PDLSCs) are key cells that suppress periodontal damage during both the progression and recovery stages of periodontitis. Although substantial evidence has demonstrated that incubation under an inflammatory condition may accelerate senescence of PDLSCs, whether such cellular senescence contributes to inflammation-induced cell changes however remains unexplored. In this study, we first observed PDLSC senescence in periodontitis based on comparisons of matched patients, and this cellular senescence was demonstrated in healthy cells that were subjected to inflammatory conditions. We subsequently designed further experiments to investigate the possible mechanism underlying inflammation-induced PDLSC senescence with a particular focus on the role of long noncoding RNAs (lncRNAs). LncRNA microarray analysis and functional gain/loss studies revealed AC025580.2 as a regulator of inflammation-mediated PDLSC senescence. By full-length transcriptome sequencing, we found that overexpressed AC025580.2 interacted with SRSF3 to promote the alternative splicing (AS) of TP53BP1, leading to the upregulation of the TP53BP1-204 transcript. Further functional studies showed that decreased expression of TP53BP1-204 reversed PDLSC senescence, and AC025580.2 overexpression-induced PDLSC senescence was abolished by TP53BP1-204 knockdown. Our data suggest for the first time that LncRNA AC025580.2 plays a key role in regulating PDLSC senescence in inflammatory environments by modulating the AS of TP53BP1.

overall_design:
PDLSCs were cultured in normal medium (H-PDLSCs) or in medium containing inflammatory cytokines (I-PDLSCs) .
lncRNA expression data from PDLSC

first: Dian

last: Gan

first: Xi-Yu

last: Zhang

first: Xiao-Tao

last: He

first: Rui

middle: X

last: Wu

first: Yuan

last: Yin

last: Jin

first: Lin

last: Li

first: Yu-Jie

last: Tan

first: Bei-Min

last: Tian

first: Xuan

first: Fa-Ming

last: Chen


title: Preclinical efficacy of a live vesicular stomatitis virus-based Lassa virus vaccine candidate advanced for human clinical trials

status: Public on Nov 30 2024

city: Seattle

first: Maxwell

last: Neal

state: NY

address: 307 Westlake Ave N

institute: Seattle Children's Research Institute

summary: We advanced a promising experimental vaccine for immunizing against the Lassa virus surface glycoprotein as a candidate for human trials. Preclinical evaluation of the vaccine candidate based on a live vesicular stomatitis virus (VSV) vector showed that it was highly efficacious in cynomolgus macaques consistent with earlier research studies, and that the animals developed serum antibodies that could mediate antiviral effector functions including direct neutralization of virus infectivity. As part of this evaluation, we analyzed whole-blood transcriptomes from each study subject prior to vaccination and at 1 day and 3 days post-vaccination.

overall_design: Ten Chinese-origin cynomolgus macaques (Macaca fascicularis) were assigned to two vaccine groups of five animals each. Each group of vaccinated animals was composed of two males and three females. One group was administered a single dose of 2x10^7 PFU VSV∆G-LASV-GPC, the other group received a lower dose of 2x10^5 PFU, both by intramuscular (IM) injection. Animals were vaccinated once with 1 ml of virus injected into the caudal thigh. A control group of three animals (one female and two males) received an equivalent volume of vaccine diluent (PBS, 5% trehalose) delivered IM.

first: Gavin

last: Morrow

first: Leslie

last: Miller

first: John

last: Aitchison

first: Christopher

last: Parks


title: next generation sequencing of icariin protection against contrast induced acute kidney injury

status: Public on Nov 29 2024

city: Guangzhou

first: Yan

last: Lin

state: Alabama

address: 63th Duobao Road

institute: Nanfang College Guangzhou

summary: This study aims to investigate the underlying mechanism of protective effect of icariin in contrast medium induced acute kidney injury.

overall_design: Next generation sequencing was applied for rat kidneys in three groups: control group, CIAKI group and CIAKI+icariin group. There're three rats in each group.

first: Zena

last: Huang


title: Effect of Vitamin D Modulation on the TH2 Cell Transcriptomic Landscape

status: Public on Nov 27 2024

first: Vrushali

middle: Dipak

last: Fangal

address: 181 Longwood Avenue

department: Brigham and Women's Hospital

institute: Harvard Medical School

summary: This dataset examines the transcriptional effects of vitamin D deficiency in TH2 cells isolated from Mus musculus (C57BL/6 strain). Vitamin D-deficient mice were bred and maintained under UV-free conditions with a vitamin D-depleted diet. TH2 cells from these mice (N=7) were compared to TH2 cells from wild-type mice (N=3) to assess the impact of chronic vitamin D deficiency on gene expression. RNA-seq was used to generate raw gene-level counts.

overall_design: This study explores transcriptional changes in TH2 cells from vitamin D-deficient versus wild-type mice. Naïve CD4+ T cells were isolated, polarized into TH2 cells, and analyzed using RNA-seq. Differential expression analysis was performed to identify genes and pathways affected by vitamin D deficiency.

middle: D

first: Ayşe

last: Kiliç

first: Hooman

last: Mirzakani

first: Augusto

last: Litonjua

first: Marie

middle: B

last: Damey

first: Bruce

last: Levy

first: Scott

last: Weiss


summary: This dataset profiles the transcriptional response of in vitro polarized TH2 cells treated with calcitriol (active vitamin D) and vehicle controls. RNA-seq was used to identify differentially expressed genes influenced by vitamin D signaling. This study provides insight into the molecular mechanisms through which vitamin D modulates immune responses in TH2 cells.

overall_design: The experimental design investigates the transcriptional impact of calcitriol (active vitamin D) treatment in in vitro polarized TH2 cells.TH2 cells were treated with 100 nM calcitriol for 24 hours. Vehicle controls were exposed to 2% ethanol (v/v). Differential expression analysis was performed between the calcitriol-treated group (N=11) and the vehicle control group (N=11). RNA-seq was performed to quantify gene expression, followed by downstream differential expression and enrichment analyses to identify genes and pathways impacted by calcitriol treatment.


title: Spatial Transcriptomics and Network-Based Bioinformatics Differentiate Intestinal Phenotypes of Cardiac and Classical Necrotizing Enterocolitis

status: Public on Nov 28 2024

city: Oklahoma City

first: Jonathan

last: Wren

state: OK

address: 825 N.E. 13th Street

department: Genes & Human Disease Research Program

institute: OMRF

summary: Classical necrotizing enterocolitis (classical NEC) is a devastating gastrointestinal disease of the neonatal period, and cardiac NEC is a subset of the disease occurring in infants with comorbid congenital heart disease. Despite nearly identical symptomatology, the two NEC subtypes may represent etiologically distinct diseases. We used spatial transcriptomics and network analyses to compare tissue regions of interest derived from surgically resected ileum of patients with cardiac  and classical NEC, finding evidence classical and cardiac NEC may arise from differing etiologies.

overall_design: Surgically resected ileum from 2 patients with cardiac NEC (14 total regions of interest) and 5 patients with classical NEC (42 total regions of interest) were used for spatial transcriptomics analysis. Roughly equal numbers of immune-rich (CD45+) and epithelial-rich (PanCK+) regions of interests were selected per patient.

first: Kathryn

last: Burge

first: Hala

last: Chaaban


title: Normal and Oropharyngeal-carcinoma derived monocyte responses to OPC-cell line conditioned media.

status: Public on Nov 25 2024

city: Manhasset

first: Vincent

last: Bonagura

address: 350 Community Drive

department: Pediatric Immunology

institute: Northwell Health

summary: Tumor Associated Macrophages (TAMs) represent a critical immunological barrier to effective anti-tumor mechanisms of developing carcinomas of the oropharynx. Tumor infiltrating monocytes become activated and differentiate into TAMs which contribute to the immunological landscape of tumors as either containing or devoid of effector T-cells. We first compared transcriptional responses of normal and OPC derived monocytes from single individuals, at the single cell level, after exposure to media conditioned by SCC-25 and SCC-154 OPC cell lines. Since media conditioned by both SCC-154 and SCC-25 contain secreted IL1α, stimulation of these monocytes with recombinant IL1α was included as a control. We showed that to these conditioned media, CXCL1, CXCL5 and CCL2 transcripts reached higher levels in a greater proportion of OPC-patient monocytes compared to control monocytes. These early monocyte responses were validated in a cohort of isolated monocytes exposed to the same media. These findings represent that monocytes may be secreting TME influencing cytokines, prior to their own differentiation, which in turn recruit detrimental cell populations including T-regs, MDSCs and other monocytes.

overall_design: Primary monocytes from a healthy and OPC-diagnosed individual were stimulated with culture media conditioned by SCC-25 or SCC-154 OPC cell lines, or recombinant human IL-1α.

last: Papayannakos

first: James

last: DeVoti

first: Mohd

last: Israr

first: Arnon

last: Arazi

first: Fung

last: Lam

first: Bettie M.

last: Steinberg


title: Early life cisplatin exposure induces nerve growth factor mediated neuroinflammation and chemotherapy induced neuropathic pain

city: Nottingham

first: Mark

middle: John

last: Paul-Clark

state: Nottinghamshire

address: Clifton Campus

department: Bioscience

institute: Nottingham Trent University

summary: Chemotherapy-induced neuropathic pain (CINP) is a common adverse health related comorbidity that manifests later in life in paediatric cancer patients. Current analgesia is ineffective, aligning closely with our lack of understanding of CINP. The aim of this study was to investigate how cisplatin induces nerve growth factor mediated neuroinflammation and nociceptor sensitisation. In a rodent model of cisplatin induced survivorship pain, cisplatin induced a neuroinflammatory environment in the dorsal root ganglia (DRG) demonstrated by nerve growth factor (NGF) positive macrophages infiltrating into the DRG. Cisplatin treated CD11b/F480 positive macrophages expressed more NGF compared to vehicle treated. Primary DRG sensory neuronal cultures demonstrated enhanced NGF-dependent TRPV1 mediated nociceptor activity after cisplatin treatment. Increased nociceptor activity was also observed when cultured DRG neurons were treated with conditioned media from cisplatin activated macrophages. Elevated nociceptor activity was dose-dependently inhibited by a neutralising NGF antibody. Intraperitoneal administration of a NGF neutralising antibody reduced cisplatin-induced mechanical hypersensitivity and aberrant nociceptor intraepidermal nerve fibre density. These findings identify that a monocyte/macrophage driven NGF/TrkA pathway is a novel analgesic target for adult survivors of childhood cancer.

overall_design:
To investigate the inflammatory response in dorsal root ganglia (DRG) after treatment with cisplatin and vehicle with the intention of dissecting the inflammatory processes influence on chemotherapy-induced peripheral neuropathies
We performed gene expression profiling analysis of RNA-seq data obtained from 2 neonatal Wistar rats treated with vehicle (PBS) and 2 neonatal Wistar rats treated with cisplatin (Sigma-Aldrich; 0.3mg/kg). DRG's were isolated 16 days post-treatment.
Differential gene expression was performed between DRGs isolated from vehicle compared with cisplatin treated rodents.

first: Mark John

first: Richard Philip

last: Hulse

first: Benjamine James Alexander

last: Dickins

first: Marlene

last: Da Vitoria Lobo

first: Lydia

last: Hardowar

first: Tameille

last: Valentine

first: Lucy

last: Tomblin

first: Charlotte

last: Guest

first: Dhyana

last: Sharma


title: Anti-leukemic effect of TGN-1062, a novel CDK7 inhibitor, on AML-leukemic stem cells by targeting RNA modification and Mitochondrial metabolism

status: Public on Nov 24 2024

city: Duarte

first: Hsun

middle: Teresa

last: Ku

address: 1500 E. Duarte Road

department: Translational Research and Cellular Therapeutics

institute: City of Hope

summary: High expression of CDK7, a cell cycle regulator and transcriptional modulator, correlates with poor outcomes in acute myeloid leukemia (AML). We report that TGN-1062, a novel CDK7 inhibitor, exhibits significant antileukemic effects by inhibiting cell growth and inducing apoptosis in AML cell lines and primary CD34+CD38- blasts (enriched for leukemia stem cells; LSCs), while sparing normal CD34+CD38- cells (enriched for hematopoietic stem cells; HSCs). Our study revealed that TGN-1062 treatment in primary CD34+CD38- AML blasts suppress RNA m6A modification by downregulating METTL3/14 proteins, key components of the m6A methyltransferase complex that regulate RNA stability and translation. Decrease in METTL proteins reduced BCL-2 m6A RNA modification and led to increased BCL-2 mRNA decay and protein levels. BCL-2 downregulation resulted in disruption of mitochondrial metabolism and mitofusion in LSCs through decrease of HMGB1/HSPB1 and NRF2/DRP1 signaling, respectively. In vivo, TGN-1062 significantly reduced leukemia burden and prolonged survival in a murine MllPTD/WT/Flt3ITD/ITD AML model and in FLT3-WT and inv(16) AML patient-derived xenografts (PDXs). Increased survival in secondary transplant experiments supported TGN-1062 activity on LSCs. Of note, we proved that TGN-1062 synergized with the BCL-2 inhibitor, venetoclax in eliminating LSCs in both murine and PDX AML models, underscoring its potential as a promising therapeutic approach for AML patients.

overall_design: Transcriptome profiling of patient-drived primary AML cells trreated with vehicle vs TGN-1062.

first: Truong

last: Nguyen


title: Fate plasticity of interneuron specification

city: Santa Cruz

first: Mohammed

middle: Andres

last: Mostajo-Radji

address: 2300 Delaware Ave

department: Genomics Institute

institute: University of California Santa Cruz

summary: The generation of neuronal subtypes in the mammalian central nervous system is driven by competing genetic programs. The medial ganglionic eminence (MGE) gives rise to two major cortical interneuron (cIN) populations, marked by Somatostatin (Sst) and Parvalbumin (Pvalb), which develop on different timelines. The extent to which external signals influence these identities remains poorly understood. Pvalb-positive cINs are particularly important for regulating cortical circuits through strong perisomatic inhibition, yet they have been difficult to model in vitro. Here we investigated the role of the environment in shaping and maintaining Pvalb cINs. We grafted mouse MGE progenitors into a variety of 2D and 3D coculture models, including mouse and human cortical, MGE, and thalamic systems with dissociated cells, organoids, organotypic cultures, and conditioned media. Across models, we observed distinct proportions of Sst- and Pvalb-positive cIN descendants. Strikingly, grafting MGE progenitors into 3D human, but not mouse, corticogenesis models led to efficient, non-autonomous differentiation of Pvalb-positive cINs. This differentiation was characterized by upregulation of Pvalb maturation markers, downregulation of Sst-specific markers, and the formation of perineuronal nets. Furthermore, lineage-traced postmitotic Sst-positive cINs, when grafted, also upregulated Pvalb expression. These results reveal an unexpected level of fate plasticity in MGE-derived cINs, demonstrating that their identities can be dynamically shaped by the surrounding environment.

overall_design: Genetically-labeled mouse cortical interneurons were grafted onto FBS-depleted human cortical organoids. 14 days post graft, the cells were isolated through flourescent-activated cell sorting to construct single cell sequencing libraries.

first: Walter

middle: R

last: Mancia Leon

first: Arnar

last: Breevoort

first: Jesus

last: Gonzalez-Ferrer

first: Hunter

last: Schweiger

first: Julian

last: Lehrer

first: Li

last: Zhou

first: Matthew

last: Schmitz

first: Yonatan

last: Perez

first: Tanzila

last: Mukhtar

first: Julia

last: Chu

first: Madeline

middle: G

last: Andrews

first: Frederika

last: Sullivan

first: Dario

last: Tejera

first: Eric

middle: C

last: Choy

first: Mercedes

middle: F

last: Paredes

first: Mircea

last: Teodorescu

first: Arnold

last: Kriegstein

first: Arturo

last: Alvarez-Buylla

first: Alex

last: Pollen


title: Genetic properties underlying transcriptional plasticity and evolvability in E. coli

city: Suita

first: Chiara

last: Furusawa

state: Osaka

address: Furuedai

institute: RIKEN Quantitative Biology Center

summary: The rate and direction of phenotypic evolution depends on the availability of phenotypic variants induced either genetically or environmentally for selection act upon. Theoretical suggest that genetic interactions between genes governing phenotypes could explain how the bias in phenotypic variability arises. However, it remains unknown whether the phenotypic variability is explained by real known genetic interactions as expected. To address this question, we analyzed transcriptional variability in a model microorganism, Escherichia coli, in response to different environmental and genetic perturbations. Thanks to the rich knowledge of the genetic interactions of E. coli, we identified common genetic properties that potentially affect transcriptional variability in both environmental and genetic causes. These empirical evidences support the relevance of the genetic interactions to shape phenotypic variability that is shared by different perturbations. Our results provide a mechanistic insight into how evolution learns from past environments to generalize to new environments.

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: Transcriptomic profiles of the MA lineages  (12 isolates and two founders) of E. coli were analized by DNA microarrays. Two biological replicates of each founder were also analyzed. A total of 16 samples were analyzed. All samples were cultured in LB and total RNA was extracted from the cells in the exponential growth phase.

first: Saburo

last: Tsuru

first: Chikara


title: N6-methyladenosine in DNA promotes genome stability

status: Public on Nov 22 2024

city: New York

first: Mariano

last: Oppikofer

address: 450 East 29th Street

summary: DNA base lesions, such as incorporation of uracil into DNA or base mismatches, can be mutagenic and toxic to replicating cells. To discover factors in repair of genomic uracil, we performed a CRISPR knockout screen in the presence of floxuridine, a chemotherapeutic agent that incorporates uracil and fluoro-uracil into DNA. We identified known factors, such as uracil DNA N-glycosylase (UNG), but also unknown factors, such as the N6-adenosine methyltransferase, METTL3, as required to overcome floxuridine-driven cytotoxicity. Visualized with immunofluorescence, the product of METTL3 activity, N6-methyladenosine, formed nuclear foci in cells treated with floxuridine. The observed N6-methyladenosine was embedded in DNA, called 6mA, which was confirmed using mass spectrometry. METTL3 and 6mA were required for repair of lesions driven by additional base damaging agents, including raltitrexed, gemcitabine, and hydroxyurea. Our results establish a role for METTL3 and 6mA to promote genome stability in mammalian cells, specially in response to base damage.

overall_design: Cas9-expressing HT-29 cells were transduced with a lentiviral sgRNA library, split into four pools. Each pool was transduced at a MOI of 0.3 and 1 µg/mL puromycin-containing medium was added the next day. Selection was continued until 4 days post transduction, which was considered the initial time point, t0. At this point the transduced cells were divided into two populations and split into technical triplicates. One population was untreated and 2.2 nM floxuridine was added to the other. Cells were grown with or without floxuridine until t11 or t15, subculturing every three to four days. Cell pellets were frozen at each time point for genomic DNA (gDNA) isolation. A library coverage of ≥500 cells/sgRNA was maintained throughout the screen. gDNA from cell pellets was isolated using QIAGEN Gentra Puregene kit and genome-integrated sgRNA sequences were amplified by PCR using KOD Hot Start Polymerases. i5 and i7 multiplexing barcodes (Ilumina) were added in a second round of PCR and final gel-purified products were sequenced on Illumina HiSeq2500 or NextSeq500 systems to determine sgRNA representation in each sample. Gene knockouts enriched at t11 or t15 as compared to t0 were identified using Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK) analysis.

first: Conti

last: Brooke A

first: Tong

last: Deyan

first: Xiang

last: Qing

first: Arroyo

last: Jason D

first: Shang

last: Lei


title: Cellular signatures in human blood track bone mineral density in postmenopausal women

first: Takayuki

last: Fujii

address: 515 E 71st St

institute: Hospital for Special Surgery

summary: Osteoclasts are sole bone-resorbing cells and are formed by the fusion of osteoclast precursor cells (OCPs) derived from myeloid lineage cells. Animal studies reveal that circulating osteoclast precursor cells (cOCPs) in blood travel to bone and fuse with boneresident osteoclasts. However, the characteristics of human cOCPs and their association with bone diseases remain elusive. We have identified and characterized human cOCPs and found a positive association between cOCPs and osteoporosis. Sorted cOCPs have a higher osteoclastogenic potential than other myeloid cells and effectively differentiate into osteoclasts. cOCPs exhibit distinct morphology and transcriptomic signatures. The frequency of cOCPs in the blood varies among treatment-naïve postmenopausal women and has an inverse correlation with lumbar spine bone density and a positive correlation with serum CTX, a bone resorption marker. The increased cOCPs in treatment-naïve osteoporosis patients were significantly diminished by denosumab, a widely used antiresorptive therapy. Our study reveals the distinctive identity of human cOCPs and the potential link between the dynamic regulation of cOCPs and osteoporosis and its treatment. Taken together, our study enhances our understanding of human cOCPs and highlights a potential opportunity to measure cOCPs through a simple blood test, which could potentially identify high-risk individuals.

overall_design: Human CD14+ cells were harvested from peripheral blood mononuclear cells of a healthy donor using magnetic cell isolation kit. Cells were treated with MCSF and RANKL and  sincle-cell RNA-seq was performed.

first: Kaichi

last: Kaneko

first: Jefferson

last: Tsai

first: Deniece

last: Meñez

first: Oh

last: Brian

first: Andrew

last: Junwoo Suh

first: Masataka

last: Mizuno

first: Akio

last: Umemoto

first: Eugenia

last: Giannopoulou

first: Emily

last: Stein

first: Richard

last: Bockman

first: Kyung-Hyun

last: Park-Min


title: Transcriptomic analysis of coxsackievirus B3 infection in induced pluripotent stem cell-derived brain-like endothelial cells

status: Public on Nov 21 2024

city: Tuscaloosa

first: Daryl

last: Lam

institute: University of Alabama

summary:
ABSTRACT: Viral aseptic meningitis is a neuroinflammatory condition that occurs when viruses gain access to the central nervous system (CNS) and induce inflammation. The blood-brain barrier (BBB) is comprised of brain endothelial cells (BECs) that stringently regulate the passage of molecules, toxins, and pathogens from the circulation into the CNS. Through their unique properties, such as complex tight junctions, reduced rates of endocytosis, expression of efflux transporters, and restricted expression of leukocyte adhesion molecules, the BBB is often able to limit pathogen entry into the brain; however, certain neurotropic pathogens, such as coxsackievirus B3 (CVB3) are able to infect the CNS. We have previously demonstrated that CVB3 can infect and disrupt induced pluripotent stem cell-derived brain-like endothelial cells (iBECs), but the host response to this infection remains unknown. Here, we investigate global host transcriptional changes during CVB3 infection of iBECs using RNA sequencing. We validated our data set by exploring pathways altered by CVB3 using quantitative real-time PCR (qPCR) and enzyme-linked immunosorbent assay of upregulated cytokines and interferon signaling molecules.

IMPORTANCE: Coxsackievirus B3 (CVB3) is a leading cause of viral aseptic meningitis that can cause severe disease in susceptible individuals. To gain access to the central nervous system, CVB3 must cross central nervous system barriers, such as the blood-brain barrier. Previously, we have shown that CVB3 infects a human stem cell-derived brain-like endothelial cell model. Here, we report the global transcriptome of stem cell-derived brain-like endothelial cells to CVB3 infection and provide proof-of-concept validation of the dataset using molecular biology techniques. These data could inform novel mechanisms of CVB3-mediated blood-brain barrier dysfunction.

KEYWORDS: Coxsackievirus B3, blood-brain barrier, brain endothelial cells, RNA sequencing, induced pluripotent stem cells

overall_design: To investigate Coxsackievirus B3 infection (after 2 days and 5 days) on human pluripotent stem-cell derived brain-like endothelial cells using transcriptomics (RNA seq) with differential expression analyses, qPCR, and ELISA.

first: Sarah

last: Hathcock

first: Julia

last: Mamana

first: Taryn

last: Keyzer

first: Nadine

last: Vollmuth

first: Mohammad-Reza

last: Shokri

first: Henry

last: Mauser

first: Robert

middle: N

last: Correll

middle: W

first: Brandon

middle: J

last: Kim

first: Jon

last: Sin


title: lncRNA/mRNA expression profiling for 4 Human RNA samples

city: Bethesda

first: Sichuan

last: Xi

state: MD

address: Building 10

institute: NCI/NIH

type:
Expression profiling by array
Non-coding RNA profiling by array

summary:
We have completed the Human lncRNA microarray analysis of the 4 samples that you submitted. Total RNA from each sample was quantified using the Nanodrop 1000 and the RNA integrity was assessed by Agilent 2100 Bioanalyzer. About 5 μg total RNA of each sample was used for labeling and array hybridization as the following steps: 1) Reverse transcription with invitrogen Superscript ds-cDNA synthesis kit; 2) ds-cDNA labeling with Nimblegen one-color DNA labeling kit; 3) Array hybridization using the NimbleGen Hybridization System and followed by washing with the Nimblegen wash buffer kit. 4) Array scanning using the Axon GenePix 4000B microarray scanner (Molecular Devices Corporation).  Scanned images (TIFF format) were then imported into NimbleScan software (version 2.5) for grid alignment and expression data analysis. Expression data were normalized through quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software. The Probe level (*_norm_RMA.pair) files and mRNA level (*_RMA.calls) files were generated after normalization. The 4 mRNA level files were imported into Agilent GeneSpring Software (version 11.0) for further analysis. lncRNAs and mRNAs that at least 2 out of 4 samples have values greater than or equal to lower cut-off: 50.0 (“All Targets Value”) were chosen for data analysis. Differentially expressed lncRNAs and mRNAs were identified through Fold Change filtering. Pathway Analysis and GO analysis were applied to determine the roles of these differentially expressed mRNAs played in these biological pathways or GO terms. Finally, Hierarchical Clustering was performed to show distinguishable lncRNA and mRNA expression profiling among samples.   Array Information: Human 4x44K lncRNA expression array information.
Data Analysis for lncRNAs. Data Analysis for lncRNAs.  Data Analysis for mRNAs.  1. Raw mRNA data normalization and low intensity filtering: Raw signal intensities were normalized in RMA method by NimbleScan v2.5, and low intensity mRNAs were filtered. (mRNAs that at least 2 out of 4 samples have values greater than or equal to lower cut-off: 50.0 were chosen for further analysis)  2. Quality assessment of mRNA data after filtering: Contains Box Plot and Scatter Plot for mRNAs after filtering.  3. Differentially expressed mRNAs screening: Contains significant differentially expressed mRNAs that passed Fold Change filtering (Fold Change >= 2.0).  4. Heat Map and Hierarchical Clustering: Hierarchical Clustering of the mRNAs after filtering.  5. Pathway analysis: Pathway analysis of the differentially expressed mRNAs.  6. GO analysis: GO term analysis of the differentially expressed mRNAs.  1. Raw lncRNA data normalization and low intensity filtering: Raw signal intensities were normalized in RMA method by NimbleScan v2.5, and low intensity lncRNAs were filtered. (lncRNAs that at least 2 out of 4 samples have values greater than or equal to lower cut-off: 50.0 were chosen for further analysis).  2. Quality assessment of lncRNA data after filtering: Contains Box Plot and Scatter Plot for lncRNAs after filtering.  3. Differentially expressed lncRNAs screening: Contains significant differentially expressed lncRNAs that passed Fold Change filtering (Fold Change >= 2.0).  4. Heat Map and Hierarchical Clustering: Hierarchical Clustering of the lncRNAs after filtering.   Data Analysis for mRNAs  1. Raw mRNA data normalization and low intensity filtering: Raw signal intensities were normalized in RMA method by NimbleScan v2.5, and low intensity mRNAs were filtered. (mRNAs that at least 2 out of 4 samples have values greater than or equal to lower cut-off: 50.0 were chosen for further analysis)  2. Quality assessment of mRNA data after filtering: Contains Box Plot and Scatter Plot for mRNAs after filtering.  3. Differentially expressed mRNAs screening: Contains significant differentially expressed mRNAs that passed Fold Change filtering (Fold Change >= 2.0).  4. Heat Map and Hierarchical Clustering: Hierarchical Clustering of the mRNAs after filtering.  5. Pathway analysis: Pathway analysis of the differentially expressed mRNAs.  6. GO analysis: GO term analysis of the differentially expressed mRNAs.  1. Raw lncRNA data normalization and low intensity filtering: Raw signal intensities were normalized in RMA method by NimbleScan v2.5, and low intensity lncRNAs were filtered. (lncRNAs that at least 2 out of 4 samples have values greater than or equal to lower cut-off: 50.0 were chosen for further analysis).  2. Quality assessment of lncRNA data after filtering: Contains Box Plot and Scatter Plot for lncRNAs after filtering.  3. Differentially expressed lncRNAs screening: Contains significant differentially expressed lncRNAs that passed Fold Change filtering (Fold Change >= 2.0).  4. Heat Map and Hierarchical Clustering: Hierarchical Clustering of the lncRNAs after filtering.   Data Analysis for mRNAs  1. Raw mRNA data normalization and low intensity filtering: Raw signal intensities were normalized in RMA method by NimbleScan v2.5, and low intensity mRNAs were filtered. (mRNAs that at least 2 out of 4 samples have values greater than or equal to lower cut-off: 50.0 were chosen for further analysis)  2. Quality assessment of mRNA data after filtering: Contains Box Plot and Scatter Plot for mRNAs after filtering.  3. Differentially expressed mRNAs screening: Contains significant differentially expressed mRNAs that passed Fold Change filtering (Fold Change >= 2.0).  4. Heat Map and Hierarchical Clustering: Hierarchical Clustering of the mRNAs after filtering.  5. Pathway analysis: Pathway analysis of the differentially expressed mRNAs.  6. GO analysis: GO term analysis of the differentially expressed mRNAs.

first: Jigui

last: Shan


title: The Circadian Clock in the Ageing Plant

city: York

first: Ethan

middle: James

last: Redmond

address: Wentworth Way

department: Department of Biology

institute: University of York

summary: The plant circadian clock is linked to development on every scale, from control of the cell cycle to determination of flowering time. Of particular importance is the photoperiodic pathway, through which changes of day length (photoperiod) are interpreted. Circadian regulation of this pathway is generally studied in seedlings. This allows researchers to test the effect of different environmental conditions, such as extreme photoperiods or temperature, more rapidly than in adult plants.  However, this approach fails to identify links between the photoperiodic pathway and other ageing-related processes, such as senescence. To close this knowledge gap, we collected data from Arabidopsis thaliana at two different timescales: the ‘flowering’ timescale and the ‘daily’ timescale. By shifting the photoperiod from short days (SD) to long days (LD), we induced flowering, via the photoperiodic pathway. We then sampled across 24h at 2, 7, and 12 days after this transition. We find that the regulation of senescence-associated genes begins soon after the transition from SD to LD. We explore how downstream targets of the circadian clock have changes in expression across the flowering timescale. Surprisingly, many genes shift from their time of peak expression, suggesting a widespread rewiring of the circadian clock’s targets during the flowering transition.

overall_design:
We first grew Arabidopsis Ws-2 wild-type plants under short-day (8h light; 16h dark) conditions, for a total of 22 days. We then transitioned the plants to long-day (16h light; 8h dark) conditions.
We collected samples across three days (the 2nd, 7th, and 12th days after the transition), at seven timepoints across each day (ZT 0, 1, 4, 8, 12, 16, 20).
We performed RNA-seq on each sample to produce a 'timeseries of timeseries' of gene expression.

first: James

last: Ronald

first: Seth

last: Davis

first: Daphne

last: Ezer


title: Neuronal polyunsaturated fatty acids are protective in FTD/ALS

status: Public on Nov 15 2024

city: London

first: Adrian

last: Isaacs

address: Cruciform Building, Gower Street

department: Neurodegenerative Diseases

institute: University College London

summary: We report a conserved transcriptomic signature of reduced fatty acid and lipid metabolism gene expression in human post-mortem ALS spinal cord and a Drosophila model of the most common genetic cause of FTD/ALS, a repeat expansion in C9orf72. To investigate lipid alterations, we performed lipidomics on C9FTD/ALS iPSC-neurons and post-mortem FTLD brain tissue. This revealed a common and specific reduction in phospholipid species containing polyunsaturated fatty acids (PUFAs). To determine whether this PUFA deficit contributes to neurodegeneration, we fed C9FTD/ALS flies PUFAs, which yielded a modest increase in survival. However, increasing PUFA levels specifically in neurons of the C9orf72 flies, by overexpressing fatty acid desaturase enzymes, led to a substantial extension of lifespan. Neuronal overexpression of fatty acid desaturases also suppressed stressor induced neuronal death in C9FTD/ALS patient iPSC-neurons. These data implicate neuronal fatty acid saturation in the pathogenesis of FTD/ALS and suggest that interventions to increase PUFA levels specifically within neurons will be beneficial.

overall_design: mRNA profiles from heads of 7 day old female (G4C2)36; elavGS flies induced with RU486 or ethanol for 5 days

first: Ashling

last: Giblin

first: Alla

last: Mikheenko

first: Linda

last: Partridge


title: RNA-seq profiles of C. elegans with various dietary conditions and gene perturbations related to vitamin B12 metabolism

status: Public on Nov 20 2024

city: Worcester

first: Xuhang

last: Li

state: Massachusetts

address: 55 N Lake Ave

department: Department of Systems Biology

institute: University of Massachusetts Medical School

summary: Genome-scale metabolic network models (MNMs) can be used with a mathematical modeling tool called flux balance analysis to gain insights into metabolism at a systems level. Such models represent metabolism of an organism as a whole and do not automatically reveal how the flux overall is wired, i.e., which reactions carry flux, and which do not, and measuring flux at a large scale is challenging. Enzyme levels, as proxied by mRNA levels, can be used to predict reaction fluxes. However, integrating gene expression data with MNMs only partly constrains flux models. Here, we derive how metabolism is wired in unperturbed animals using a large-scale Worm Perturb-Seq dataset. We present a highly constrained, semi-quantitative optimal flux distribution that reflects how metabolism is wired in the animal at the adult stage. We discover several features about C. elegans metabolism that were heretofore unknown, including cyclic flux through the pentose phosphate pathway, and the primary use of amino acids and bacterial RNA as an energy source, all of which we validated by isotope tracing. Our strategy for inferring metabolic wiring based solely on gene expression should be applicable to a variety of systems, including human cells. This dataset includes the RNA-seq profiles collected during the proof-of-concept studies on the metrics used for predicting flux wiring from Worm Perturb-Seq dataset. It includes three sets of data: the profiles of animals fed live versus paraformaldehyde-killed (PFA killed) bacteria; the profiles of gene knockdowns in propionate degradation metabolism under a gradient of vitamin B12 supplementation; and the profiles of gene knockdowns in Met/SAM cycle under vitamin B12 and choline supplementations.

overall_design: This GEO record includes three independent datasets used for investigating different topics in this study.  The first includes RNA-seq profiles of C. elegans animals fed live versus paraformaldehyde-killed (PFA-killed) HT115 bacteria collected in biological triplicates. The second involves profiles of RNAi knockdowns of two genes in propionate canonical (pccb-1 and mmcm-1) and two genes in shunt (acdh-1 and hphd-1) degradation pathways, under a gradient of vitamin B12 supplementation (0-16nM). The third involves RNAi knockdowns of genes related to Met/SAM cycle (sams-1, ahcy-1, gcst-1, mel-32, pmt-1, cbs-1, metr-1, mthf-1, gcst-1/mel-32 double knockdown, metr-1/cbs-1 double knockdown) with and without the supplementation of vitamin B12 or choline. All data were collected using the multiplexed RNA-seq method developed in Worm Perturb-seq (WPS), a massively parallel RNAi and RNA-seq technology. This method generates whole-animal, bulk, 3'end RNA-seq profiles. Specifically, samples were multiplexed to form pooled sequencing libraries with around 50 samples per library. The read 1 is used to encode barcode information and read 2 maps to the 3' end region of C. elegans transcripts. Barcode information can be found from the Supplementary Table "barcode_and_metadata_one_carbon_cycle_vignette.csv" and "barcode_and_metadata_propionate_degradation_vignette.csv". These libraries can be directly processed by the WPS data processing pipeline (https://github.com/XuhangLi/WPS) providing the barcode information attached. A custom processing of the data is feasible following the typical demultiplexing methods for single-cell RNA-seq data that are able to process CEL-seq2 library. Notably, the first dataset, profiles for animals fed live versus dead bacteria, was also generated with WPS method, but readily demultiplexed into single fastq files per sample. This is because other samples pooled in the corresponding library were not included in this study, thus were excluded to avoid confusions.

first: Hefei

last: Zhang


title: Systems-level design principles of metabolic rewiring in an animal

summary: The regulation of metabolism is vital to any organism and can be achieved by transcriptionally activating or repressing metabolic genes. Although many examples of transcriptional metabolic rewiring have been reported, a systems-level study of how metabolism is rewired in response to metabolic perturbations is lacking in any animal. Here we apply Worm Perturb-Seq (WPS)-a high-throughput method combining whole-animal RNA-interference and RNA-sequencing to around 900 metabolic genes in the nematode Caenorhabditis elegans. We derive a metabolic gene regulatory network (mGRN) in which 385 perturbations are connected to 9,414 genes by more than 110,000 interactions. The mGRN has a highly modular structure in which 22 perturbation clusters connect to 44 gene expression programs. The mGRN reveals different modes of transcriptional rewiring from simple reaction and pathway compensation to rerouting and more complex network coordination. Using metabolic network modelling, we identify a design principle of transcriptional rewiring that we name the compensation-repression (CR) model. The CR model explains most transcriptional responses in metabolic genes and reveals a high level of compensation and repression in five core metabolic functions related to energy and biomass. We provide preliminary evidence that the CR model may also explain transcriptional metabolic rewiring in human cells.

overall_design: In this study, we applied Worm Perturb-seq (WPS), a massively parallel RNAi and RNA-seq technology, to perturb around 900 metabolic genes in nematode C. elegans, followed by a RNA-seq readout. The technology generates multiplexed RNA-seq libraries, each of which contains the triplicate samples of around 16 single gene perturbations and six empty vector controls (total ~50 samples per library). The RNA-seq library construction follows an adapted CEL-seq2 protocol, giving a multiplexed bulk RNA-seq library for 3'-end sequencing of C. elegans genes. The read 1 is used to encode barcode information and read 2 maps to the 3' end region of C. elegans transcripts. Barcode information can be found from the Supplementary Table "barcode_and_metadata_full_dataset.csv". These libraries can be directly processed by the WPS data processing pipeline (https://github.com/XuhangLi/WPS) providing the barcode information attached. A custom processing of the data is feasible following the typical demultiplexing methods for single-cell RNA-seq data that are able to process CEL-seq2 library. Please refer to our manuscript for decription of the design of read 1.


title: Connexin 43 dephosphorylation mediates the Dchs1/YAP/TEAD signaling pathway to impact  cardiac fibrosis

city: Suzhou

first: min

last: wang

address: 242 Guangji Road

institute: Affiliated Suzhou Hospital of Nanjing Medical University

summary: The gap junction protein connexin 43 (Cx43) has been implicated in the development of cardiac fibrosis. We found that Cx43 dephosphorylation at serine 282 (S282) is related to cardiomyocyte apoptosis and arrhythmias in hearts damaged by ischemia/reperfusion. Here, we investigated the effect of Cx43 S282 phosphorylation on fibrosis. We found a decrease in Cx43 S282 phosphorylation in transforming growth factor beta-1 (TGF-β)-induced fibrosis in fibroblasts and an angiotensin II-induced rat model. We transferred a lentivirus with S282A (alanine) into cardiac fibroblasts and an adenovirus with S282A into the hearts of rats. We found that the Cx43 S282A mutation caused fibrosis in cardiac fibroblasts and in the hearts of rats, which suggested that the phosphorylation of Cx43 S282 is important in the development of fibrosis. Furthermore, mRNA sequencing showed that Cx43 dephosphorylation at S282 increased the expression of Dchs1, and Dchs1 inhibited Yes-associated protein (YAP) phosphorylation, subsequently activating the YAP/TEAD signaling pathway and increasing the development of fibrosis. This study suggests that the phosphorylation of Cx43 S282 could be an effective antifibrotic target in cardiac fibroblasts, indicating a novel mechanism and a molecular target that might be promising for the treatment of cardiac fibrosis.

overall_design: we sequenced mRNA from injected mouse hearts (Cx43-wt and S282A) using gene set enrichment analysis (GSEA) and the standardized enrichment score (NES). After data analysis, the Hippo signalling pathway was identified as the main pathway involved in p282-Cx43-mediated regulation of MF.

first: Xinxin

last: Yan

first: Zhongshan

last: Gou

first: Min

last: Wang

first: Wanning

first: Yaqing

last: Shao

first: Ying

last: Huang

first: Chenchen

last: Wei

first: Ping

first: Kangyun

last: Sun


title: Study on the Analgesic and Compatibility Mechanism of Yuan-Hu Zhi Tong Prescription for Alleviating CCI-induced Neuropathic Pain

status: Public on Nov 11 2024

city: Beijing

first: Wu

last: Dan

address: Dongzhimennei south steet 16

institute: Institute of Chinese Materia Medica，China Academy of Chinese Medical Sciences

summary: Neuropathic pain (NP) is a complex chronic pain due to the nervous system damage or diseases.Yuan-Hu Zhi Tong Prescription (YZP) is a well-known Traditional Chinese Medicine (TCM) formula for pain relief, which is composed of Corydalis Rhizoma (Yuanhu, YH) and Angelicae Dahuricae Radix (Baizhi, BZ). However, understanding of the analgesic and compatibility mechanisms of YZP remains limited. The present study found that YZP was more effective in relieving mechanical allodynia and thermal hypersensitivity than YH and BZ administered alone. To understand the potential analgesic and compatibility mechanisms of YZP on neuropathic pain (NP), RNA-seq and bioinformatics analyses were performed. The results revealed that YZP exerts analgesic effects mainly by relieving inflammation in the spinal cord through various mechanisms, such as suppressing leukocyte activation and cytokine-mediated signaling pathways. Moreover, YZP can relieve NP by modulating the neuropeptide signaling pathway and promoting damaged tissue regeneration. In terms of compatibility, YH and BZ demonstrated synergistic effects on the inflammatory response and neuropeptide signaling pathway. Furthermore, BZ was found to regulate calcium ion transport and promote skeletal muscle tissue regeneration, thereby alleviating concomitant symptoms of NP. Hub genes such as Ucn, Ptgs2, Gal, and others may play a pivotal role in YZP analgesia and the synergistic effects of YH-BZ compatibility.

overall_design: The CCI rats were randomly assigned to four groups: the CCI group, the CCI+YZP group, the CCI+YH group, and the CCI+BZ group, with 7 rats in each group. YZP (10.8 g crude drug/kg), YH (7.2 g crude drug/kg,) and BZ (3.6 g crude drug/kg) were administered respectively and continuously for 5 days from the postoperative day (POD) 6 to 10. The ipsilateral L4-L6 SC of rats were collected on POD 10, immersed in TRIzol (Invitrogen, CA, USA), and immediately frozen in liquid nitrogen for the RNA-seq (n=3 per group).

first: Dan

last: Wu

first: Jin

last: Su

first: Haiyu

last: Xu

first: Yanqiong


title: Restriction mechanism of peri-centromeric DNA loop expansion necessary to ensure accurate chromosome segregation [ChIP-seq]

city: Tokyo

first: Sao

middle: Anh

last: Nguyen

address: 1-1-1 Yayoi, Bunkyo-ku

institute: University of Tokyo

summary: Centromeres in budding yeast are surrounded by ~10-kb DNA loops generated by the cohesin complex. These peri-centromeric loops are shown to play a role in faithful chromosome segregation. It remains not fully understood how the dynamics of the peri-centromeric loops are controlled. Here we found that gene deletion of two cohesin regulators, Wpl1 and Eco1, resulted in larger peri-centromeric loops that connected the centromeres and genome regions up to ~ 300 kb distant in G2/M-arrested cells. Deletion of Wpl1 and Eco1 synergistically contributed to the loop size enlargement. ChIP-seq revealed that cohesin and its ATPase activator, Scc2, were colocalized at the anchor sites of the extended peri-centromeric loops, specifically in the double-deletion mutant Δwpl1 Δeco1. Consistently, acute depletion of Scc2 in the G2/M phase resulted in the disappearance of the extended loops. Given that Scc2 is bound prominently only at the centromeres in wild-type cells, our results suggest that Wpl1 and Eco1 jointly promote the dissociation of Scc2 from cohesin and inhibit excessive expansion of peri-centromeric DNA loop size. Notably, we found that nuclear division after benomyl block and release was significantly delayed in the Δwpl1 Δeco1 double-deletion mutant, and this delay was rescued by Scc2 depletion in the G2/M phase. This indicates that cohesin regulators cooperatively regulate the size of the peri-centromeric DNA loops to ensure faithful chromosome segregation.

overall_design: Chromatin immunoprecipitation-sequencing (ChIP-seq) targeting Scc1, Scc2 and Rpo21 in budding yeast S.cerevisiae in several conditions (wt, ∆eco1_∆wpl1,  ∆wpl1, Gal-Ub-Eco1, Pds5-AID, etc.)

middle: A

first: Takashi

last: Sutani

first: Yukata

last: Suzuki

first: Katsuhiko

last: Shirahige


title: Integration of transcriptional and epigenetic regulation of TFEB reveals dual functional roles in Pan-cancer [ChIP-seq]

status: Public on Nov 10 2024

city: Avenida da Universidade Taipa

first: Chen

last: Ming

address: Macau

department: Faculty of Health Sciences

country: Macau

institute: the University of Macau

summary: Transcription factor EB (TFEB) mainly regulates the autophagy-lysosomal pathway, associated with many diseases, including cancer. However, the role of TFEB in pan-cancer has not been investigated systematically. In this study, we comprehensively analyzed TFEB targets under three stresses in Hela cells by cross-validation of RNA-seq and ChIP-seq. 1,712 novel TFEB targets have not been reported in the Gene Set Enrichment Analysis and ChIP Enrichment Analysis databases. We further investigated their distributions and roles among the pan-cancer co-expression networks across 32 cancers constructed by multiscale embedded gene co-expression network analysis (MEGENA) based on the Cancer Genome Atlas (TCGA) cohort. Specifically, TFEB might serve as a hidden player with multifaceted functions in regulating pan-cancer risk factors, e.g., CXCL2, PKMYT1, and BUB1, associated with cell cycle and immunosuppression. TFEB might also regulate protective factors, e.g., CD79A, related to immune promotion in the tumour microenvironment. We further developed a Shiny app website to present the comprehensive regulatory targets of TFEB under various stimuli, intending to support further research on TFEB functions. Summarily, we provided references for the TFEB downstream targets responding to three stresses and the dual roles of TFEB and its targets in pan-cancer, which are promising anticancer targets that warrant further exploration.

overall_design: Chromatin immunoprecipitation sequencing of one input sample and two replicates of Hela cells expressing 3×Flag-TFEB without treatment or with treatment of CCCP, sucrose and Torin1. To investigate  regulatory targets of TFEB under three different stresses , we did ChIP-seq for Hela cells expressing 3×Flag-TFEB without treatment or with treatment of CCCP, sucrose and Torin1. Each group has three replicates. We then performed TFEB binding site profiling analysis using ChIP-seq data from the four groups, i.e., Ctrl, CCCP-stimuli, Sucrose-stimuli, and Torin-1 stimuli. By comparing the differential binding sites of TFEB between three stimuli groups and the Ctrl group, we identified the potential TFEB regulatory target genes.

first: Jing-Fang

last: Luo

first: Shijia

first: Jiajing

last: Fu

first: Peng

first: Ningyi

first: Jia-Hong

last: Lu


title: Targeting CD206+ macrophages disrupts the establishment of a key anti-tumor immune axis [Bulk RNA-seq]

status: Public on Nov 08 2024

city: San Francisco

first: Bushra

last: Samad

address: 505 Parnassus Ave

institute: USCF Immunoprofiler

summary: The direct and indirect effects of targeting specific tumor-associated macrophage (TAM) subsets in cancer are not well-defined. TAM targeting strategies are frequently based on the ‘M1’ or ‘M2’ polarization categories, even as substantial data challenges this binary modeling of macrophage cell state. One molecule consistently referenced as a delineator of a putative immunosuppressive ‘M2’ state is the surface protein CD206. We thus made a novel conditional CD206 (Mrc1) knock-in mouse to specifically visualize and/or deplete CD206+ TAMs and assess their correspondence with pro-tumoral immunity. Early, but not late depletion of CD206+ macrophages and monocytes (here, ‘Mono/Macs’) led to an indirect loss of a key anti-tumor network of NK cells, conventional type I dendritic cells (cDC1) and CD8 T cells. Among myeloid cells, we found that the CD206+ TAMs are the primary producers of CXCL9, and able to differentially attract activated CD8 T cells. In contrast, a population of stress-responsive TAMs (“Hypoxic” or Spp1+) and immature monocytes, which lack CD206 expression and become prominent following early depletion, expressed markedly diminished levels of CXCL9. Those NK and CD8 T cells which enter CD206-depleted tumors express vastly reduced levels of the corresponding receptor Cxcr3, the cDC1-attracting chemokine Xcl1 and cDC1 growth factor Flt3l transcripts. Consistent with the loss of this critical network, early CD206+ TAM depletion decreased tumor control by antigen specific CD8 T cells in mice. Likewise, in humans, the CD206Replete, but not the CD206Depleted Mono/Mac gene signature correlated robustly with CD8 T cell, NK cell and stimulatory cDC1 gene signatures and transcriptomic signatures skewed towards CD206Replete Mono/Macs associated with better survival. Together, these findings negate the unqualified classification of CD206+ ‘M2-like’ macrophages as immunosuppressive and provide further evidence of the context-dependence of macrophage function in tumors.

overall_design:
To identify patterns of gene expression within six broadly defined cell populations, we also performed cell sorting for bulk RNA-sequencing from freshly resected primary human tumors including compartments denoted: 1. “Live”: All viable cells at the time of sorting, 2. “Tconv”: sorted conventional CD4+ and CD8+ T cells, 3. “Treg”: CD25+ CD4+ (enriched for regulatory) T cells, 4. “Myeloid”: Lymphocyte-negative HLA-DR+ (enriched for myeloid) cells, 5. “Stromal”: CD45- CD44+Thy1+ cells, and 6. “Tumor”: all other CD45- cells. We use the bulk RNASeq data from the 'Tconv', 'Myeloid' and 'Live' compartments for this study from both previously published data and new data

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The table below lists GEO accessions reused/reanalyzed for this study.
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first: Arja

last: Ray

first: Alexis

last: Combes


title: SUV39H1 Maintains Cancer Stem Cells in Glioblastoma [ATAC-seq]

status: Public on Nov 06 2024

city: Bloomington

first: QIQI

last: XIE

state: IN

address: 3209 E 10th St Apt E6

department: Biology

institute: Indiana University Bloomington

summary: Glioblastoma (GBM) is the deadliest brain cancer, driven in part by GBM stem cells (GSCs) that contribute to therapeutic resistance and tumor recurrence. Effective targeting and elimination of GSCs hold promise for preventing GBM recurrence and achieving potential cures. In this study, we explored the role of the epigenetic regulator SUV39H1 in GSC maintenance and GBM progression. We observed that SUV39H1 is upregulated in GBM samples compared to normal brain tissues. Single-cell RNA-seq data indicated that SUV39H1 is preferentially expressed in GSCs relative to non-stem GBM cells, possibly due to super-enhancer-mediated transcriptional activation. Knockdown of SUV39H1 in patient-derived GSCs impaired their proliferation and stemness. RNA-seq analysis revealed that SUV39H1 regulates G2/M cell cycle progression, stem cell maintenance, and cell death pathways in GSCs. Integrated ATAC-seq (assay for transposase-accessible chromatin followed by sequencing) and RNA-seq analyses demonstrated that targeting SUV39H1 altered chromatin accessibility in key genes associated with these pathways. Treatment with chaetocin, a SUV39H1 inhibitor, mimicked the effects of SUV39H1 knockdown in GSCs and sensitized them to the GBM chemotherapy drug temozolomide (TMZ). In vivo studies using an intracranial patient-derived xenograft model showed that targeting SUV39H1 inhibited GSC-driven tumor formation in mice. Our findings identify SUV39H1 as a critical regulator of GSC maintenance and suggest that targeting SUV39H1 could disrupt GSCs and enhance the efficacy of existing chemotherapy, offering a promising strategy for improving GBM treatment outcomes.

overall_design: The submission includes samples of GSC3565 cells. The experimental conditions involve treatment with either vehicle or 50 nM chaetocin for 48 hours. The study includes biological replicates for each condition to ensure data reliability. Control samples treated with vehicle are included for comparison with chaetocin-treated samples to investigate the effect of chaetocin on chromatin accessibility in GSC3565 cells.

first: Chunying

last: Li

first: Qiqi

last: Xie

first: Bihui

last: Cao

first: Jia

last: Shen


title: A New Role for Huntingtin-Interacting Protein 1-Related (HIP1R) in Rheumatoid Arthritis Synovial Fibroblast Transcriptomic, Biological Processes and Invasiveness

first: Teresina

last: Laragione

address: 1468 Madison Ave

department: Medicine

institute: Icahn School of Medicine at Mount Sinai

summary: Huntingtin-interacting protein 1 Related (HIP1R) protein, shares some function similarities with HIP1, and HIP1 regulates arthritis and RA fibroblast-like synoviocytes (FLS) invasiveness. Therefore, we hypothesized that HIP1R might be involved in the regulation of FLS phenotypes and molecular processes relevant to RA. siRNA was used to knockdwon HIP1R, HIP1 or control in RA FLS followed by cell studies for invasion in Matrigel, migration, proliferation and adhesion. RNA was sequenced and analyzed. HIP1R knockdown significantly reduced RA FLS invasiveness and migration (P<0.05). The DEGs in siRNA HIP1R had an enrichment for GO processes “astrocyte and glial cell projection”, “small GTPase signaling” and “PDGFR signaling”. The most significantly DEGs had decreased expression in siRNA HIP1R and included AKT1S1, GABBR2, GPR56, and TXNDC12. siRNA HIP1 RA FLS had an enrichment for the “Rap1 signaling pathway” and “Growth factor receptor binding”. The most significantly DEGs in HIP1 siRNA included FGF2, PGF, and SLC39A8. HIP1R and HIP1 DEG lists had a greater than expected number of similar genes (P=0.0015), suggesting that, despite the major differences detected, both have partially overlapping functions in RA FLS. The most significant DEGs in both HIP1R and HIP1 analyses are involved in cancer cell behaviors and outcomes. HIP1R is a new gene implicated in RA FLS invasiveness and migration, and regulates unique pathways and cell processes relevant to both RA as well as cancer biology. Our study provides new insight into processes implicated in FLS invasiveness, which is relevant for joint damage in RA, and identify new potential gene targets for FLS-specific treatments.

overall_design: siRNA was used to knockdown HIP1r and HIP1 in RA FLS.  Cells were then studied in invasion, migration, proliferation and adhesion assays, and RNA was used for RNA sequencing and pathway analyses.

first: Carolyn

last: Harris

first: Percio

last: Gulko


title: Conserved DNA Methylation Signatures in the Prefrontal Cortex of Newborn and Juvenile Guinea Pigs Following Antenatal Betamethasone Exposure

city: Toronto

first: Stephen

last: Matthews

address: 1 King's College Circle, Rm 3302

country: Canada

institute: University of Toronto

type: Methylation profiling by high throughput sequencing

summary: We investigated the effect of antenatal corticosteroid exposure (ACS) on the DNA methylation patterns in the prefrontal cortex (PFC) of female guinea pig offspring. Guinea pig dams were treated with saline or betamethasone on gestational days 50/51. PFC was collected from newborn (post-natal day 1) and juvenile (PND 14) offspring. Methylation assessment using genomic DNA was performed with reduced representation bisulfite sequencing techniques.

overall_design: Differential DNA methylation in the prefrontal cortex of newborn female guinea pig offspring was assessed to compare profiles between Betamethasone and saline control exposures. The PND1 cohort has n=7 per treatment condition, total of 14. The PND14 cohort has n=7 in the saline group and n=6 in the Betamethasone group, to a total of 13.

first: Bona

last: Kim

first: Alice

last: Kostaki

first: Stephen G