title: Human Enteroid Monolayers: A Novel, Functionally-Stable Model for Investigating Oral Drug Disposition

first: James

middle: William

last: MacDonald

address: 4225 Roosevelt Way NE

department: Environmental and Occupational Health Sciences

institute: University of Washington

summary: To further the development of an in vitro model that can more faithfully recapitulate drug disposition of orally administered drugs, we investigated the utility of human enteroid monolayers to simultaneously assess intestinal drug absorption and first-pass metabolism processes. We cultured human enteroid monolayers from three donors, derived via biopsies containing duodenal stem cells that were propagated and then differentiated atop permeable Transwell® inserts, and confirmed transformation into a largely enterocyte population via RNA-seq analysis and immunocytochemical (ICC) assays. Proper cell morphology was assessed and confirmed via bright field microscopy and ICC imaging of tight junction proteins and other apically and basolaterally localized proteins. Enteroid monolayer barrier integrity was demonstrated by elevated transepithelial electrical resistance (TEER) that stabilized after 10 days in culture and persisted for 42 days. These results were corroborated by low paracellular transport probe permeability at 7 and 21 days in culture. The activity of a prominent drug metabolizing enzyme, CYP3A, was confirmed at 7, 21, and 42 days culture under basal, 1?,25(OH)2 vitamin D3-induced, and 6',7'-dihydroxybergamottin-inhibited conditions. The duration of these experiments is particularly noteworthy, as this is the first study assessing DMET function for enteroids cultured for greater than 12 days. The sum of these results suggests enteroid monolayers are a promising in vitro model to investigate and quantitatively predict an orally administered drug's intestinal absorption and/or metabolism and associated drug-drug/natural product-drug interactions.

overall_design: Matched enteroid monolayer and undifferentiated spheroids from three donors were compared to characterize differences and infer suitability of enteroid monolayers as a model for oral drug disposition.

first: Christopher

last: Arian

first: Eimear

last: O'Mahony

middle: W

first: Theo

last: Bammler

first: Mark

last: Donowithz

first: Ed

last: Kelly

first: Kenneth

last: Thummel


title: Histone H3 E50K remodels chromatin to confer oncogenic activity  and support EMT phenotype [Cut & Tag]

status: Public on Jan 15 2025

city: Atlanta

first: Jennifer

middle: Marie

last: Spangle

state: Georgia

address: 1365C Clifton Rd NE

country: USA

institute: Emory University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Sequencing of human patient tumors has identified recurrent missense mutations in genes encoding core histones. We report that mutations that convert histone H3 amino acid 50 from a glutamate to a lysine (H3E50K) support an oncogenic phenotype. Expression of H3E50K is sufficient to transform human cells as evidenced by an increase in cell migration and invasion, and an increase in proliferation and clonogenicity. H3E50K also increases the invasive phenotype in the context of co-occurring BRAF mutations, which are present in patient tumors characterized by H3E50K. H3E50 lies on the globular domain surface in a region that contacts H4 within the nucleosome. We find that H3E50K selectively increases chromatin accessibility and perturbs proximal H3 post-translational modifications including H3K27me3; together these changes to chromatin dynamics dysregulate gene expression to support the epithelial to mesenchymal transition. Functional studies using S. cerevisiae reveal that, while yeast cells that express H3E50K as the sole copy of histone H3 show sensitivity to cellular stressors, including caffeine, H3E50K cells display some genetic interactions that are distinct from the characterized H3K36M oncohistone yeast model. Taken together, these data suggest that additional H3 mutations have the potential to support oncogenic activity and function through distinct mechanisms that dysregulate gene expression.

overall_design: H3K27me3 CUT&Tag was performed in histone H3.3 E50K and H3.3 WT cells to see transcription repression based on the low H3K27me3 in H3E50K cells. We are using immortalized but untransformed HMEC DD cells with dominant negative p53 cells that stably overexpressed a C-terminal TY1 epitope-tagged H3.3 WT and E50K.

first: Kirti

last: Sad

first: Dorelle

middle: V

last: Fawwal

first: Celina

middle: Y

last: Jones

first: Emily

middle: J

last: Hill

first: Katie

middle: T

last: Skinner

first: Miranda

last: Adams

first: Severin

last: Lustenberger

first: Richard

middle: S

last: Lee

first: Sandhya

last: Lohano

first: Satvik

middle: R

last: Elayavalli

first: Jonathan

last: Farhi

first: Christina

middle: C

last: Mehta

first: Laramie

middle: D

last: Lemon

first: Milo

middle: B

last: Fasken

first: Andrew

middle: L

last: Hong

first: Steven

middle: A

last: Sloan

first: Anita

middle: H

last: Corbett

middle: M


title: Discrete vulnerability to pharmacological CDK2 inhibition is governed by heterogeneity of the cancer cell cycle [CRISPR]

city: Buffalo

first: Jianxin

last: Wang

state: NY

address: 665 Elm Street

department: Molecular and Cellular Biology

institute: Roswell Park Comprehensive Cancer Center

summary: Cyclin dependent kinase 2 (CDK2) regulate cell cycle and is an emerging target for cancer therapy.  There are relatively small numbers of tumor models that exhibit strong dependence on CDK2 and undergo G1 cell cycle arrest following CDK2 inhibition. The expression of P16INK4A and cyclin E1 determines this sensitivity to CDK2 inhibition. The co-expression of these genes occurs in breast cancer patients highlighting their clinical significance as predictive biomarkers for CDK2-targeted therapies.  In cancer models that are genetically independent of CDK2; pharmacological inhibitors suppress cell proliferation by inducing 4N cell cycle arrest and increasing the expressions of phospho-CDK1 (Y15) and cyclin B1.   CRISPR screens identifiy CDK2 loss as a mediator of resistance to INX-315. Furthermore, CDK2 deletion reverses the G2/M block induced by CDK2 inhibitors and restores cell proliferation. Complementary drug screens define multiple means to cooperate with CDK2 inhibition beyond G1/S. These include the depletion of mitotic regulators as well as CDK4/6 inhibitors cooperate with CDK2 inhibition in multiple phases of the cell cycle.  Overall, this study underscores two fundamentally distinct features of response to CDK2 inhibitors that are conditioned by tumor context and could serve as the basis for differential therapeutic strategies in a wide range of cancers.

overall_design: HCC1806 and MiaPaCa-2 cells were infected with the TKOv3 lentiviral particles at an MOI ~0.3 and the positive clones were selected using puromycin to achieve a mutant pool comprising at least 200-fold coverage of the library. The resulting mutant pool was grown in the absence and presence of INX-315 (500 nM) and palbociclib (250 nM)   for at least 5 passages. Genomic DNA was extracted and subjected to sequencing library construction by amplifying the gRNA inserts

first: Vishnu

last: Kumarasamy

first: Michelle

last: Roti

first: Yin

last: Wan

first: Adam

last: Dommer

first: Hanna

last: Rosenheck

first: Sivasankar

last: Putta

first: Alec

last: Trub

first: John

last: Bisi

first: Jay

last: Strum

first: Patrick

last: Roberts

first: Seth

last: Rubin

first: Costakis

last: Frangou

first: Karen

last: McLean

first: Erik

last: Knudsen

first: Agnieszka

middle: K

last: Witkiewicz


title: A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis [mpra_bc_mapping]

city: Boston

first: James

middle: Christopher

last: Taggart

state: MA

address: 200 Longwood Avenue

department: Systems Biology

institute: Harvard Medical School

summary: RNA endonucleases are the rate-limiting initiator of decay for many bacterial mRNAs. However, the positions of cleavage and their sequence determinants remain elusive even for the well-studied Bacillus subtilis. Here we present two complementary approaches – transcriptome-wide mapping of endoribonucleolytic activity and deep mutational scanning of RNA cleavage sites – that reveal distinct rules governing the specificity among B. subtilis endoribonucleases. Detection of RNA terminal nucleotides in both 5′- and 3′-exonuclease-deficient cells revealed >103 putative endonucleolytic cleavage sites with single-nucleotide resolution. We found a surprisingly weak consensus for RNase Y targets, a contrastingly strong primary sequence motif for EndoA targets, and long-range intramolecular secondary structures for RNase III targets. Deep mutational analysis of RNase Y cleavage sites showed that the specificity is governed by many disjointed sequence features, each with mild contributions. Our results highlight the delocalized nature of mRNA stability determinants and provide a strategy for elucidating endoribonuclease specificity in vivo.

overall_design: A library of plasmids encoding barcoded RNAs with mutated variants of RNase Y cleavage sites was constructed. This library was introduced to B. subtilis and both RNA and genomic DNA was harvested. To determine the efficiency of cleavage at each cleavage site variant, barcodes were PCR amplified and quantified by sequencing. A separate amplicon was sequenced to map barcodes to variant sequences.

first: Julia

last: Dierksheide

first: Hannah

last: LeBlanc

first: Jean-Benoît

last: Lalanne

first: Sylvain

last: Durand

first: Frédérique

last: Braun

first: Ciarán

last: Condon

first: Gene-Wei

last: Li


title: A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis [threeprime_end_seq]

overall_design: RNA 5′/3′-end sequencing and Rend-seq were performed on B. subtilis strains deficient in exonucleolytic activity. These datasets, combined with similar measurements performed on strains with an additional knockouts of individual endoribonucleases, were used to determine the locations of endoribonucleolytic cleavage transcriptome-wide.


title: A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis [mpra_quant]


title: A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis [fiveprime_end_seq]

first: Taggart

last: James

first: Dierksheide

last: Julia

first: LeBlanc

last: Hannah

first: Lalanne

last: Jean-Benoît

first: Durand

last: Sylvain

first: Braun

last: Frédérique

first: Condon

last: Ciarán

first: Li

last: Gene-Wei


title: Transcriptome and DNA methylation analyses provide insight into the heterosis of growth-related traits in hybrid yellow croaker

status: Public on Jan 14 2025

city: TianJin

first: Lu

last: chuang

address: BinHai

country: China

institute: Northwest Normal University

type: Expression profiling by high throughput sequencing

summary: Background: Interspecific hybrid combinations of Larimichthys crocea × Larimichthys polyactis exhibit heterosis in terms of growth traits; however, the molecular regulatory mechanism underlying this phenomenon remains unclear. DNA methylation plays a pivotal role in regulating gene expression and is involved in growth and development processes. In this study, we comprehensively investigated intricate regulatory processes by integrating transcriptome and methylome datasets from brain, liver, and muscle tissues. Results: We analyzed a total of 72 sequence datasets, including transcriptome and genome-wide DNA methylome data, from 36 tissue samples using LC, LP, LPC and LCP. We elucidated the distinct expression patterns of these four populations and examined their interactions with DNA methylation. Our findings revealed diverse DNA methylation profiles and demonstrated a greater number of hypo-DMRs in hybrid yellow croakers than in their parental lines. The majority (86~92%) of these DMRs were observed within the CG context. Moreover, we found that most DMRs were located within promoter regions as well as exons and introns. A total of 1288 DMEGs were identified through correlation analysis between DNA methylation and transcriptional activity. Functional enrichment analysis revealed that most of the DMEGs were significantly enriched in pathways related to the protein export pathway, proteasome, terpenoid backbone biosynthesis, ubiquitin-mediated proteolysis, autophagy-other pathway. Furthermore, we screened candidate growth-related genes, such as stat2, capn2, akt1, mTOR, and mef2aa. Among these, the expression levels of capn2, mTOR, and akt1 exhibited a positive correlation with DNA methylation levels, whereas the expression levels of stat2 and mef2aa showed a negative correlation. These findings suggest that alterations in DNA methylation patterns may promote growth advantages in hybrid yellow croaker by modulating the expression of these genes. Conclusions: Epigenetic changes exert distinct influences on genes related to growth heterosis. The presented data establish a foundation for comprehending the epigenetic and transcriptomic alterations underlying the growth of hybrid yellow croaker, thereby providing preliminary insights into the molecular mechanisms of growth heterosis. These findings have significant implications for breeding programs aimed at enhancing yellow croaker production.

sample_organism:
Larimichthys
Larimichthys crocea
Larimichthys polyactis

overall_design: RNA-seq analysis of brain, liver, and muscle tissues from large yellow croaker, small yellow croaker, and their reciprocal hybrids.


title: MI-883 Constitutes a Novel Dual-Action Ligand Targeting Constitutive Androstane Receptor and Pregnane X Receptor for Hypercholesterolemia Intervention

city: Hradec Kralove

first: Alena

last: Mrkvicova

address: Simkova

country: Czech Republic

institute: LFHK

summary: We describe the effect of a novel combined human CAR agonist/PXR antagonist MI-883 on the liver transcriptome. RNA-seq transcriptomic data in control (n=5) and MI-883-treated (n=5) humanized PXR-CAR-CYP3A4/3A7 male mice fed with high-fat diet demonstrate that MI-883 significantly regulates genes involved in xenobiotic metabolism, and fatty acid and cholesterol homeostasis. This compound thus reduces plasma cholesterol levels and enhances fecal bile acid excretion.

overall_design:
Humanized PXR-CAR-CYP3A4/3A7 mice were fed with high-fat diet. Then animals were treated with either MI-883 treatment or vehicle in p.o. gavage. Liver tissue was used dor transcriptomic analysis.
Differential expression analysis of liver transcriptome of humanized PXR-CAR-CYP3A4/3A7 mice fed with high fat diet after treatment with MI-883.
GO pathway analysis of metabolic processes in the liver of of humanized PXR-CAR-CYP3A4/3A7 mice after treatment with MI-883 (n=5) and control  mice (vehicle-treated, n=5).

first: Petr

last: Pavek

first: Radim

last: Nencka

first: Jan

last: Dusek

first: Ivana

last: Mejdrova


title: Oligodendrocyte precursor cells facilitate neuronal lysosome release

city: Homburg

first: Lipao

last: Fang

address: Kirrbergerstrasse 100

department: Molecular Physiology, CIPMM

country: Germany

institute: University of Saarland

summary: Oligodendrocyte precursor cells (OPCs) shape brain function through complex mechanisms. We found that OPCs facilitate neuronal lysosome exocytosis by contacting neuronal somata. Reduced number or process branching of OPCs decreased these contacts, accompanied with lysosome accumulation and altered metabolism in neurons, and more senescent neurons with age. In Alzheimer's mouse models, OPC-neuron contacts were fewer, and lysosomes accumulated. Our findings are relevant for preventing aging-related pathologies and treatment of neurodegenerative diseases.

overall_design: To investigate the role of OPC-CAV1.2/CAV1.3 in neuronal function, we generated inducible OPC-CAV1.2/CAV1.3 conditional knockout (cKO) mice by cross-breeding NG2-CreERT2 mice, which allow temporal control of OPC-specific gene recombination, with floxed CAV1.2/CAV1.3 mice. mRNA was isolated from OPCs and neurons of the cKO mice and subsequently sent for sequencing.

first: Li-Pao

first: Frank

last: Kirchhoff

first: Xianshu

last: Bai


title: Genome-wide absolute quantification of chromatin looping

status: Public on Jan 13 2025

city: Cambridge

first: James

middle: Martin

last: Jusuf

address: Building 56 Room 787B, 32 Vassar Street

department: Biological Engineering

institute: Massachusetts Institute of Technology

summary: We developed a method to estimate the 3D interaction probabilities of chromatin loops across the genome on an absolute scale from Micro-C maps. To calibrate the method, we performed Micro-C on two engineered mouse embryonic stem cell (mESC) lines, each containing a fluorescently labeled chromatin loop that was quantified in previous live imaging studies. One loop is an endogenous loop containing the Fbn2 gene, and the other is a synthetic loop near the Npr3 gene. We performed two replicates of Micro-C per cell line. Using our absolute quantification method, we find that loops generally form with low probabilities. We also provide an ultra-deep merged Micro-C map for mESCs that combines all existing mESC Micro-C datasets to date, containing a total of 15.6 billion unique interactions.

overall_design: Genome-wide Micro-C was performed on two replicates of the engineered cell line containing the fluorescently tagged Fbn2 loop ("Fbn2 cell line"), as well as two replicates of the engineered cell line containing the fluorescently tagged synthetic Npr3 loop ("Npr3 cell line"); all cells were in untreated condition.

first: Simon

last: Grosse-Holz

first: Michele

last: Gabriele

first: Pia

last: Mach

first: Ilya

last: Flyamer

first: Christoph

last: Zechner

first: Luca

last: Giorgetti

first: Leonid

last: Mirny

first: Anders

last: Hansen


title: The cell polarity protein MPP5/PALS1 controls the subcellular localiza-tion of the oncogenes YAP and TAZ in hepatocellular carcinoma

city: Mannheim

first: Carolina

last: Delatorre

address: Theodor-Kutzer-Ufer 1-3

institute: University Heidelberg

summary:
The oncogenes yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are potent liver oncogenes. Because gene mutations cannot fully explain their nuclear enrichment, we aim to understand which mechanisms cause YAP/TAZ activation in liver cancer cells. The combination of proteomics and functional screening identified numerous apical cell polarity complex proteins interacting with both proteins. Co-immunoprecipitation (Co-IP) ex-periments confirmed that MPP5 (synonym: PALS1) physically interacts with YAP and TAZ. After removing different MPP5 protein domains, Co-IP analyses revealed that the PDZ domain plays a crucial role in YAP binding. The interaction between YAP and MPP5 in the cytoplasm of cancer cells was demonstrated by proximity ligation assays (PLAs). In human hepatocellular carcinoma (HCC) tissues, a reduction of apical MPP5 expression was observed, positively correlating with the nuclear accumulation of YAP and TAZ. Expression data analysis illustrated that MPP5 is in-versely associated with YAP/TAZ target gene signatures in human HCCs. Low MPP5 levels de-fine an HCC patient group with poor clinical outcome. In summary, MPP5 facilitates the nuclear exclusion of YAP and TAZ in HCC cells. This qualifies MPP5 as a tumor-suppressor gene and ex-plains how changes in cell polarity can foster tumorigenesis.
.

overall_design: MPP5 was inhibited by gene-specific siRNAs for 24 hours. Two out of six siRNAs were used, which caused the strongest inhibition effects (MMP5_3 and MPP5_5). Untreated cells (UTC) and nonsense-siRNA treated cells (NTC) were used as controls. Each biological sample consits or 3 replicates.

first: Kai

last: Breuhahn


title: A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis [rend_seq]

last: Taggart

first: Julia

last: Dierksheide

first: Hannah

last: LeBlanc

first: Jean-Benoît

last: Lalanne

first: Sylvain

last: Durand

first: Frédérique

last: Braun

first: Ciarán

last: Condon

first: Gene-Wei

last: Li


title: The transcriptional response of cortical neurons to concussion reveals divergent fates after injury

city: Bethesda

first: Claire

middle: E

last: LE PICHON

state: MD

address: 35A Convent Drive, 2D933

department: NICHD

institute: National Institutes of Health

summary: Traumatic brain injury (TBI) is a risk factor for neurodegeneration, however little is known about how different neuron types respond to this kind of injury. In this study, we follow neuronal populations over several months after a single mild TBI (mTBI) to assess long ranging consequences of injury at the level of single, transcriptionally defined neuronal classes. We find that the stress responsive Activating Transcription Factor 3 (ATF3) defines a population of cortical neurons after mTBI. We show that neurons that activate ATF3 upregulate stress-related genes while repressing many genes, including commonly used markers for these cell types. Using an inducible reporter linked to ATF3, we genetically mark damaged cells to track them over time. Notably, we find that a population in layer V undergoes cell death acutely after injury, while another in layer II/III survives long term and retains the ability to fire action potentials. To investigate the mechanism controlling layer V neuron death, we genetically silenced candidate stress response pathways. We found that the axon injury responsive kinase MAP3K12, also known as dual leucine zipper kinase (DLK), is required for the layer V neuron death. This work provides a rationale for targeting the DLK signaling pathway as a therapeutic intervention for traumatic brain injury. Beyond this, our novel approach to track neurons after a mild, subclinical injury can inform our understanding of neuronal susceptibility to repeated impacts.

overall_design: Atf3-CreER mice were crossed to a nuclear envelope protein reporter and injected with tamoxifen at 4 and 5 days post mTBI. The ipsilateral cortex was subdissected at 7 days post injury. GFP+ nuclei were FACS sorted and barcoded using 10X Genomics Chromium Single Cell Processor. Samples were run on an Illumina HiSeq2500.

first: Mor

last: Alkaslasi

first: Eliza

last: Lloyd

first: Austin

last: Gable

first: Hanna

last: Silberberg

first: Hector

last: Yarur

first: Valerie

last: Tsai

first: Hugo

last: Tejeda

last: Le Pichon


title: The DYT6 dystonia causative protein THAP1 is responsible for proteasome activity via PSMB5 transcriptional regulation

city: Bunkyo-ku

first: Hiroki

last: Sugishita

state: Tokyo

country: Japan

institute: The University of Tokyo

summary: The proteasome plays a pivotal role in protein degradation, and its impairment is associated with various pathological conditions, including neurodegenerative diseases. It is well understood that Nrf1 coordinates the induction of all proteasome genes in response to proteasome dysfunction. However, the molecular mechanism regulating the basal expression of the proteasome remains unclear. Here we identified the transcription factor THAP1, the causative gene of DYT6 dystonia, as a regulator of proteasome activity through genome-wide genetic screens. We demonstrated that THAP1 directly regulates the expression of the PSMB5 gene, which encodes the central protease subunit β5. Depletion of THAP1 disrupted proteasome assembly, leading to reduced proteasome activity and the accumulation of ubiquitinated proteins. These findings reveal a new regulatory mechanism for the proteasome and suggest a potential role for proteasome dysfunction in the pathogenesis of dystonia.

overall_design: RNA-Seq for THAP1C54Y/C54Y embryo and control embryo at e10.5

first: Yan

last: Wang

first: Jun

last: Hamazaki

first: Sigeo

last: Murata


title: Genome-Wide Profiling of Circulating MicroRNAs in Adolescent Idiopathic Scoliosis

status: Public on Jan 12 2025

city: Montreal

first: Alain

last: Moreau

state: QC

address: 3175 Côte-Ste-Catherine

country: Canada

institute: CHU Sainte-Justine

summary: Adolescent Idiopathic Scoliosis (AIS) is the most common orthopedic condition requiring surgery, affecting 4% of adolescents. There is currently no proven method or prognostic test to identify symptomatic patients at risk of developing severe scoliosis who could benefit from growth-guided devices or minimally invasive non-fusion instrumentation surgeries. These innovative treatments must be performed at an early disease stage in younger patients to benefit from their growth potential. In this prospective cross-sectional study, we investigated the clinical utility of circulating microRNAs (miRNAs), an important class of small non-coding RNA, as biomarkers to predict the risk of developing severe scoliosis in AIS. Blood samples and clinical data were collected from 116 AIS patients who were followed until skeletal maturity and stratified according to their clinical outcome. Genome-wide expression profiling of miRNAs was performed with plasma obtained at the time of diagnosis of AIS (mean age of 13.3 ± 1.7 years with a mean Cobb angle of 24.4° ± 12.4°). This approach led to the identification of 15 circulating miRNAs that are upregulated in AIS patients who developed a severe scoliosis (Cobb angle ≥45°) at skeletal maturity compared to moderate and mild scoliosis groups (Cobb angle between 25°-44° and <25° respectively). After optimization and the application of Random Forest Models a panel of six miRNAs (miR-1-3p, miR-19a-3p, miR-19b-3p, miR-133b, miR-143-3p, and miR-148b-3p) out of 15 led us to develop an algorithm predicting the risk of developing a severe scoliosis with great accuracy (100%), sensitivity (100%) and specificity (100%).  Having a scoliosis predictive bioassay and decision-making tools to predict curve progression in order to find the best treatment plan will undoubtedly transform the orthopedic care system in the field of pediatric scoliosis by integrating innovative precision medicine approaches. In addition, investigation of genes targeted by these miRNAs could fill our gaps in our understanding of AIS pathogenesis and reveal new actionable targets.

overall_design:
Study population: This cross-sectional study is part of a larger longitudinal prospective study conducted in three pediatric spine centers. Healthy controls (HC) without a family antecedent of scoliosis were recruited during the same period from primary and high schools from the Greater Montreal’s area. Stratification by scoliosis severity was determined only in the participants who have completed their longitudinal follow-up and reached their skeletal maturity. This discovery cohort consisted of 34 AIS patients (21F/13M) classified as spinal deformity progressors (P), 35 AIS patients (20F/15M) classified as non-progressors (NP), and 14 matched healthy controls (9F/5M). A second subgroup was established to assess the capacity of our qPCR probes to amplify our candidate miRNAs, composed of six female AIS patients classified as spinal deformity progressors (P), six female AIS patients classified as non-progressors (NP), and six matched healthy controls (5F/ 1M). Finally a replication cohort, which was composed of 15 AIS patients (11F/4M) classified as spinal deformity progressors (P), 20 AIS patients (11F/9M) classified as non-progressors, and 10 matched healthy controls (4F/6M) was used.
Identification of circulating miRNAs associated with AIS: We conducted a comprehensive genome-wide expression profiling of circulating miRNAs using plasma samples from our discovery cohort. We employed the Agilent expression array-Human miRNA 8x60K chips to identify candidate circulating miRNAs differentially expressed in AIS patients compared to HC group.
Validation and replication assays of identified circulating miRNAs: Selected candidate miRNAs were validated initially by RT-qPCR using a distinct small cohort of AIS patients (n=12) and HC (n=6). Of note, we were able to amplify only 70 miRNAs out of 90 initially discovered using the Agilent microarray. Subsequently, these 70 miRNAs were validated in a larger cohort comprising 35 AIS patients and 10 matched HC.
Study population: This cross-sectional study is part of a larger longitudinal prospective study conducted in three pediatric spine centers. Healthy controls (HC) without a family antecedent of scoliosis were recruited during the same period from primary and high schools from the Greater Montreal’s area. Stratification by scoliosis severity was determined only in the participants who have completed their longitudinal follow-up and reached their skeletal maturity. This discovery cohort consisted of 34 AIS patients (21F/13M) classified as spinal deformity progressors (P), 35 AIS patients (20F/15M) classified as non-progressors (NP), and 14 matched healthy controls (9F/5M). A second subgroup was established to assess the capacity of our qPCR probes to amplify our candidate miRNAs, composed of six female AIS patients classified as spinal deformity progressors (P), six female AIS patients classified as non-progressors (NP), and six matched healthy controls (5F/ 1M). Finally a replication cohort, which was composed of 15 AIS patients (11F/4M) classified as spinal deformity progressors (P), 20 AIS patients (11F/9M) classified as non-progressors, and 10 matched healthy controls (4F/6M) was used.
Identification of circulating miRNAs associated with AIS: We conducted a comprehensive genome-wide expression profiling of circulating miRNAs using plasma samples from our discovery cohort. We employed the Agilent expression array-Human miRNA 8x60K chips to identify candidate circulating miRNAs differentially expressed in AIS patients compared to HC group.
Validation and replication assays of identified circulating miRNAs: Selected candidate miRNAs were validated initially by RT-qPCR using a distinct small cohort of AIS patients (n=12) and HC (n=6). Of note, we were able to amplify only 70 miRNAs out of 90 initially discovered using the Agilent microarray. Subsequently, these 70 miRNAs were validated in a larger cohort comprising 35 AIS patients and 10 matched HC.

first: Nasrin

last: Khatami

first: Iurie

last: Caraus

first: Mahamuda

last: Rahaman

first: Evguenia

last: Nepotchatykh

first: Mohamed

last: Elbakry

first: Wesam

last: Elremaly

first: Anita

last: Franco

first: Marie

last: Beauséjour

first: Anne-Marie

last: Laberge

first: Stefan

last: Parent

first: Hubert

last: Labelle

first: Carl-Éric

last: Aubin

first: Jean

last: Lachaine


title: Transcriptomic Profiling of Myeloid Cells in Acetaminophen-Induced Acute Liver Failure

city: Seonbuk-gu

first: Yoon Kyoung

last: Kim

state: Seoul

address: Hwarang-ro 76, Unit102

country: South Korea

institute: SHIFTBIO

summary: In the recent study, we found that mesenchymal stem cell (MSC)-derived extracellular vesicles expressing the SIRPα protein (SIRP-EVs) were significantly distributed within myeloid cells (CD11b+ cells) and kupffer cells (CD11b+/F4/80+ cells) in acetaminophen (APAP)-induced mouse acute liver failure (ALF) model. Furthermore, SIRP-EVs enhanced the phagocytic activity of macrophages by blocking CD47 on necroptotic hepatocytes and promoted liver regeneration. Therefore, we investigated the therapeutic effects of SIRP-EVs on CD11b+ cells in APAP-induced ALF conditions. CD11b+ cells in the liver, including resident and recruited CD11b+ cells, were harvested and analyzed through bulk RNA sequencing.

overall_design: APAP was administered intraperitoneally to induce ALF, and EVs were administered intravenously 8 hours after APAP induction. 48 hours after APAP induction, livers were harvested and dissociated into single cells. CD11b+ cells were then immediately isolated from liver single-cell suspensions and analyzed via bulk RNA-sequenceing.

first: Seohyun

first: Hyemin

last: Joo

first: Yoonjeong

last: Choi

first: Gi Beom

first: Gi-Hoon

last: Nam

first: In-San


title: A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis

first: James

last: Taggart

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

overall_design: Refer to individual Series


title: Role of Hepatocyte RIPK1 in Maintaining Liver Homeostasis during Metabolic Challenges [RNA-seq]

status: Public on Jan 11 2025

city: Nanjing

first: Dan

last: Weng

address: 200 Xiaolingwei Street

institute: Nanjing University of Science and Technology

summary: In this study, we identified Receptor interacting protein kinase 1 (RIPK1) in hepatocytes as a critical regulator in preserving hepatic homeostasis during metabolic challenges, such as short-term fasting or high-fat dieting. Our results demonstrated that hepatocyte-specific deficiency of RIPK1 sensitized the liver to short-term fasting-induced liver injury and hepatocyte apoptosis in both male and female mice. Despite being a common physiological stressor that typically does not induce liver inflammation, short-term fasting triggered hepatic inflammation and compensatory proliferation in hepatocyte-specific RIPK1-deficient (Ripk1Δhep) mice. Transcriptomic analysis revealed that short-term fasting oriented the hepatic microenvironment into an inflammatory state in Ripk1Δhep mice, with upregulated expression of inflammation and immune cell recruitment-associated genes. Single-cell RNA sequencing further confirmed the altered cellular composition in the liver of Ripk1Δhep mice during fasting, highlighting the increased recruitment of macrophages to the liver.

overall_design: We performed transcriptome sequencing on liver tissues of fed and fasted Ripk1fl/fl and Ripk1Δhep mice, respectively. A total of 10 samples were collected in 4 groups, including 2 replicates per group of fed mice and 3 replicates per group of fasting mice.

first: Weigao

last: Zhang

first: Hu

last: Liu

first: Danyang

first: Yuguo

last: Yi

first: Liang

last: Tao

first: Haibing

first: Yuxin

last: Chen


title: Nuclear ANLN regulates transcription initiation related Pol II clustering and target gene expression [RNA-Seq]

city: Shantou

first: yufei

last: Cao

address: No. 22, Xinling Road

institute: Shantou University Medical College

summary: Anillin (ANLN), a mitotic protein that regulates contractile ring assembly, has been reported as an oncoprotein. However, the function of ANLN in cancer cells, especially in the nucleus, has not been fully understood. Here, we report a previously unknown role of nuclear ANLN in gene transcriptional regulation. We found that nuclear ANLN directly interacts with the RNA polymerase II (Pol II) large subunit. ANLN enhances initiated Pol II clustering and promotes Pol II CTD phase separation. Short-term depletion of ANLN alters the chromatin binding and enhancer-mediated transcriptional activity of Pol II. The target genes of ANLN-Pol II axis are involved in oxidoreductase activity, Wnt signaling and cell differentiation. Our results reveal the function of nuclear ANLN in transcriptional regulation, providing a theoretical basis for ESCC treatment.

overall_design: After knockdown of ANLN in esophageal squamous cell carcinoma cells (KYSE150 cells) for 24 or 48 hours, total RNA was extracted, and gene expression was measured by RNA-Seq.

first: Yu-Fei

first: Hui

last: Wang

first: Yong

last: Sun

first: Bei-Bei

last: Tong

first: Wen-Qi

last: Shi

first: Liu

last: Peng

first: Yi-Meng

first: Yu-Qiu

last: Wu

first: Teng

last: Fu

first: Hua-Yan

last: Zou

first: Kai

first: Li-Yan

last: Xu

first: En-Min

last: Li


title: Nuclear ANLN regulates transcription initiation related Pol II clustering and target gene expression [Cut & Tag]

overall_design:
CUT&Tag analysis of ANLN and Pol II in esophageal squamous cell carcinoma cells (KYSE150 cells).
After knockdown of ANLN in KYSE150 cells for 24 hours, Pol II and H3K27ac were analyzed by CUT&Tag.


title: Droplet-based high-throughput 3D genome structure mapping of single cells with simultaneous transcriptomics (b23-mouse-brain)

status: Public on Jan 10 2025

city: Beijing

first: Xiaoliang

middle: Sunney

last: Xie

state: Beijing

address: No.5 Yiheyuan Road, Haidian District

institute: Peking University

summary: Single-cell three-dimensional (3D) genome techniques have advanced our understanding of cell-type-specific chromatin structures in complex tissues, yet current methodologies are limited in cell throughput. Here we introduce a high-throughput single-cell Hi-C approach (dscHi-C) and its transcriptome co-assay (dscHi-C-multiome) using droplet microfluidics. Using dicHi-C, we investigated chromatin structural changes during mouse brain aging by profiling 32,777 single cells across three developmental stages (3 month, 12 month and 23 month), yielding a median of 78,220 unique contacts. Our results show that genes with significant structural changes are enriched in pathways related to metabolic process and morphology change in neurons, and innate immune response in glial cells, highlighting the role of 3D genome organization in physiological brain ageing. Furthermore, our multi-omics joint assay, dscHi-C-multiome, enables precise cell type identification in the adult mouse brain and uncovers the intricate relationship between genome architecture and gene expression. Collectively, we developed the sensitive, high-throughput dscHi-C and its multi-omics derivative, dscHi-C-multiome, demonstrating their potential for large-scale cell atlas studies in development and disease.

overall_design: In this study, we performed droplet-based single-cell Hi-C (dscHi-C) and its transcriptome co-assay (dscHi-C-multiome) on human and mouse cell line, and mouse brain cortex tissues.

first: Honggui

first: Maoxu


title: Role of Hepatocyte RIPK1 in Maintaining Liver Homeostasis during Metabolic Challenges [scRNA-seq]

first: Dan

last: Weng

overall_design: We performed single-cell RNA sequencing of liver non-parenchymal cells isolated from Ripk1Δhep or Ripk1fl/fl mice upon fasting. A total of 22274 single-cell transcriptomes (10374 Ripk1fl/fl; 11900 Ripk1Δhep) were obtained.

first: Weigao

last: Zhang

first: Hu

first: Danyang

first: Yuguo

first: Liang

first: Haibing

first: Yuxin


title: Transcriptomic profilings of CT45A1-expressing colorectal cacner cells.

city: Taipei

first: Wei-Lun

last: Hwang

address: No.155, Sec.2, Linong Street

department: Department of Biotechnology and Laboratory Science in Medicine

country: Taiwan

institute: National Yang Ming Chiao Tung University

summary: CT45 family members are increased in several tumor types. However, molecules of action of CT45A1 in colorectal cancer (CRC) are elusive. In an attempt to understand the biological and clincal significance of CT45A1 in CRC, we generated the CT45A1-expressing CRC lines for identifying CT45A1-regulatory circuit.

overall_design: Two colorectal cancer cell lines (DLD-1 and HCT-15) with stable CT45A1 expression were generated by lentivirus transduction and puromycine selection. The total RNAs of CT45A1-expressing CRC cells and corresponding controls were isolated and subjected for RNA-sequencing.

first: Hsiang-Yueh

last: Huang

first: Hao-Wei

last: Teng


title: Droplet-based high-throughput 3D genome structure mapping of single cells with simultaneous transcriptomics (cell-line)

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Droplet-based high-throughput 3D genome structure mapping of single cells with simultaneous transcriptomics (b12-mouse-brain)


title: Intergenerational transport of double-stranded RNA in C. elegans can limit heritable epigenetic changes

city: College Park

first: Antony

middle: Merlin

last: Jose

address: Rm 2136, Bioscience Research Building

department: Cell Biology and Molecular Genetics

institute: University of Maryland College Park

summary: RNAs in circulation carry sequence-specific regulatory information between cells in plant, animal, and host-pathogen systems. Such RNA can cross generational boundaries, as evidenced by somatic double-stranded RNA (dsRNA) in the nematode C. elegans silencing genes of matching sequence in progeny. Here we dissect the intergenerational path taken by dsRNA from parental circulation and discover that cytosolic import through the dsRNA importer SID-1 in the parental germline and/or developing progeny varies with developmental time and dsRNA substrates. Loss of SID-1 enhances initiation of heritable RNA silencing within the germline and causes changes in the expression of the sid-1-dependent gene sdg-1 that last for more than 100 generations after restoration of SID-1. The SDG-1 protein is enriched in perinuclear germ granules required for heritable RNA silencing but is expressed from a retrotransposon targeted by such silencing. This auto-inhibitory loop suggests how retrotransposons could persist by hosting genes that regulate their own silencing.

overall_design: Differential expression analyses between sid-1 mutant and wild-type mixed-stage animals.

first: Nathan

last: Shugarts

first: Andrew

last: Yi

first: Winnie

last: Chan

first: Julia

last: Marré

first: Aishwarya

last: Sathya


title: Molecular control of PDPNhi macrophage subset induction by ADAP as a host defense in sepsis III

city: Suzhou

first: Pengchao

address: 199 Ren'ai Road

institute: Soochow University

summary: The macrophage response to infected microbes is crucial for the development of life-threatening sepsis. However, the mechanisms underlying pathophysiological regulation of macrophage subsets during sepsis remain poorly understood. Here, we report a novel mechanism in which the adhesion and degranulation-promoting adapter protein (ADAP) is essential for priming macrophages to induce podoplanin (PDPN) in response to bacterial infection during sepsis. We showed that ADAP expression was robustly induced in macrophages following LPS stimulation and was associated with sepsis severity. Furthermore, PDPN failed to be upregulated in TLR4 stimulated macrophages deficient in ADAP. A distinct PDPNhi peritoneal macrophage (PM) subset, which exhibited an M2-like phenotype and enhanced phagocytic activity, was generated in WT but not in ADAP-deficient septic mice. The blockade of PDPNhi PMs mimicked the effect of ADAP deficiency, which exacerbated sepsis. Mechanistically, BTK-mediated ADAP Y571 phosphorylation worked together with mTOR to converge on STAT3 activation for the transactivation of the PDPN promoter. Moreover, agonist activation of STAT3 profoundly potentiated the PDPNhi PM subset generation and alleviated sepsis severity in mice. Together, our findings reveal a novel mechanism whereby ADAP resets macrophage function by controlling the TLR4-induced upregulation of PDPN as a host innate immune defense during sepsis.

overall_design: PDPNhi and PDPNlo peritoneal macrophages from WT septic mice were sorted and subjected to RNA-seq.

first: Xinning

last: Wang

first: Xiaodong

last: Yang

first: Hebin

last: Liu


title: Exogenous bacterial cellulose induces plant tissue regeneration through the regulation of cytokinin and defense networks

city: Pozuelo de Alarcon

first: Miguel

middle: Angel

last: Moreno-Risueno

state: Madrid

department: Biotechnology

country: Spain

institute: Center for Plant Biotechnology and Genomics

summary: Regeneration is a unique feature of postembryonic development extensively observed in plants. Several hormonal signaling networks govern plant regeneration responses, and while these networks can potentially integrate external cues, the capacity to induce regeneration exogenously is limited and usually confined to meristematic tissues. As bacterial cellulose (BC) contains plant hormones and shows unique physicochemical properties, it provides a valuable tool to investigate the relationships between exogenous cues, hormonal responses and plant regeneration. We demonstrate that BC induces post-wounding regeneration in non-meristematic plant tissues. Furthermore, our results reveal that BC activates an alternative route to standard regenerative programs, with inconsistent activation of previously proposed core regeneration gene regulatory networks, including WOUND INDUCED DEDIFFERENTIATION1 (WIND1), and despite several transcriptional signatures for wounding- and callus-induced regeneration being observed. The BC-specific transcriptional program involves cytokinin signaling, although not exclusively, to promote plant regeneration. Intriguingly, we found a strong induction of plant biotic responses. We identified the WRKY8 transcription factor, known for regulating defense and abiotic stress responses, as a regulator of BC-mediated plant regeneration. WRKY8 activates redox homeostasis genes, which, in turn regulate reactive oxygen accumulation at the wound sites. Particularly, BC increases the superoxide anion content through the WRKY8 network to potentially promote cell proliferation following wounding. Collectively, our results demonstrate that the temporal concurrent activation of the cytokinin and defense responses can be targeted by BC application to promote plant wound regeneration through an alternative route to callus formation.

overall_design: To comprehensively address the endogenous regulation and determinants of BC-mediated regeneration we performed transcriptomic analyses through RNA sequencing (RNA-seq) of the wound region. Arabidopsis leaf cuts were covered with BC or left uncovered, and a 1 mm area surrounding the wound was collected at 0, 3, 6 and 24 hpw and analyzed through RNA-seq. Differential gene expression (DEG) analysis was performed to identify transcriptional changes specifically occurring over time in wounded tissue covered with BC

first: Núria

last: Coll

first: Alvaro

last: Sanchez Corrionero

first: Alejandro

last: Alonso-Díaz


title: Molecular control of PDPNhi macrophage subset induction by ADAP as a host defense in sepsis II

last: Zhang

overall_design: CD11b+F4/80- and CD11b+F4/80+ peritoneal cells from wild-type and Adap-KO septic mice were sorted using fluorescence-activated cell sorting (FACS) and subjected to RNA-seq.


title: Hepatic Lpcat3 Accelerates Nonalcoholic Fatty Liver Disease by slut1e1-related promoting fatty acid oxidation and reducing fatty acid synthesis [MCD RNA-seq]

city: 上海

first: 艳群

last: 罗

state: --- Select One ---

address: Cailun Road 1200

institute: shanghai university of traditional Chinese medicine

summary: Non-alcoholic steatohepatitis (NASH) is a common chronic liver disease associated with metabolic disorders such as obesity, diabetes, and high cholesterol. It results in inflammation and fibrosis of liver tissue, eventually leading to cirrhosis and liver cancer. While LPCAT3 has been linked to the formation of fat cells, its role in the development of NASH is not yet fully understood. The purpose of this study was to gain insights into the mechanisms that accelerate NASH induced by LPCAT3. We utilized a genetic engineering rodent model and transcriptomics sequencing approach to explore the role and regulatory mechanism of LPCAT3 in the development of NAFLD. Approach & Results: We studied the impact of Lpcat3 deficiency on NASH progression using three distinct Lpcat3 liver-specific knockout mouse (LKO) models and conducted RNA sequencing, lipidomics, and metabolomics studies on liver samples. Human samples were obtained and analyzed for LPCAT3 expression in patients with NASH to determine its correlation with disease severity. The HepG2 and Huh-7 cell lines were utilized for in vitro analyses. We discovered that LPCAT3 expression was elevated in human NASH livers, and its expression correlated with NAFLD activity score and fibrosis stage. Lpcat3 deficiency in the mouse liver slows the development of diet-induced NASH. Lpcat3 deficiency mechanistically reduces lipid production by inhibiting lipid metabolic pathwaysDeletion of Lpcat3 significantly improved lipid accumulation in NASH mice, possibly due to the involvement of sult1e1 in lipogenesis and oxidation regulation. Moreover, the hepatic overexpression of Lpcat3 significantly intensified lipid buildup in steatotic cells, indicating that LPCAT3 plays a crucial role in the progression of NASH.  Conclusions: The findings indicate that LPCAT3 regulates the participation of sult1e1 in both adipogenesis and oxidation, leading to enhanced NASH outcomes.

overall_design: To elucidate the role and underlying mechanisms of LPCAT3 in non-alcoholic fatty liver disease (NAFLD), we employed a methionine-choline deficient (MCD) diet to induce the pathology in C57BL/6 mice.

first: Tao

last: Wu

first: Yanqun

last: Luo

first: Caiyun

first: Guang

last: Ji


title: single-cell miRNA and mRNA cosequencing using PSCSR-seq V2

last: Cai

address: No. 7 Science Park Road, Zhongguancun Life Science Park

institute: National Institute Of Biological Sciences, Beijing (NIBS)

type: Non-coding RNA profiling by high throughput sequencing

summary: We incorporated mRNA profiling into a previously reported parallel single-cell small RNA profiling method (PSCSR-seq) and called this new approach PSCSR-seq V2. PSCSR-seq V2 can overcome the limitations of existing methods and enable single-cell miRNA and mRNA coexpression profiling in various biological samples.

overall_design: miRNA and mRNA coprofiling in cancer cells and mouse lung tissue

first: Jia

last: Li


title: Molecular control of PDPNhi macrophage subset induction by ADAP as a host defense in sepsis

overall_design: Peritoneal macrophages from wild-type and Adap-KO mice exposed to LPS in vitro were subjected to RNA-seq.


title: Ifi27l2a regulates the microglial inflammation in the context of aging and stroke

status: Public on Jan 09 2025

city: Houston,

first: Gab Seok

last: Kim

state: TX

address: 6431 Fannin St.

department: Department of Neurology

institute: The University of Texas Health Science Center at Houston

summary: Microglia are key mediators of inflammatory responses within the brain, as they regulate pro-inflammatory responses while also limiting neuroinflammation via reparative phagocytosis. To facilitate identification of potential mediators of inflammation, we performed single-cell RNA sequencing of aged mouse brains following stroke and found that Ifi27l2a was significantly up-regulated, particularly in microglia.

overall_design: Sham and PDMCAO surgeries were performed on young and aged mice. At 14 days after surgery, brains were isolated. Both sexes were used for scRNA-seq. Fourteen days post PDMCAO (or sham surgery), anesthetized mice were transcardially perfused with heparinized PBS (10 U/mL). Brains were removed from the skull and sliced coronally into 3 mm-thick blocks (from a region spanning +1 to -2 mm from bregma), covering the cortical infarction and secondary thalamic injury site. The brain slice was then minced with a razor blade and subjected to the brain tissue dissociation protocol.The 10X Genomics Chromium™, Single-Cell RNA-SeqSystem (10X Genomics, Pleasanton, CA) was used to prepare cells for scRNA-seq.

first: Gab

first: Sean

last: Marrelli

first: Joshua

last: Wythe

first: Manuel

last: Gutierrez


title: The N-terminal domain of gasdermin D induces liver fibrosis by reprogrammed lipid metabolism

city: Chengdu

first: Wang

last: Xue

address: South Section of Yangliu West Rd.1880#

institute: GemPharmatech Chengdu Co.,Ltd.,

summary: Background: The increasing prevalence of chronic liver disease has become a significant global health concern. Pyroptosis leads to cell rupture, releasing large amounts of pro-inflammatory cellular contents and promoting the progression of liver fibrosis. Therefore, targeting pyroptosis may serve as a new strategy for the treatment of chronic liver diseases.  Methods: We constructed GSDMD-NTki/wt & Alb-creki/wt mice using CRISPR/Cas9 gene editing technology and induced conditional cell death by doxycycline to construct a mouse model of liver fibrosis. We analyzed differentially expressed genes by RNA sequencing and explored their biological functions. Obeticholic acid was used to assess the modeling effects of fibrosis. Results: Doxycycline-treated GSDMD-NTki/wt & Alb-creki/wt mice showed severe liver damage, vacuolation of hepatocytes, increased collagen fibers, accumulation of lipid droplets, and much stronger expression of genes involved in liver fibrosis development in the liver compared with untreated mice. RNA-sequencing showed that upregulated differentially expressed genes in the GSDMD-NTki/wt & Alb-creki/wt doxycycline-treated group were involved in inflammatory responses, cell activation, and metabolic processes. Obeticholic acid was used to assess the modeling effects of fibrosis. Obeticholic acid treatment of GSDMD-NTki/wt & Alb-creki/wt mice reduced ALT and AST levels, and enhanced anti-fibrotic effects were observed. Conclusions: Our results demonstrated that we successfully constructed a mouse model of liver fibrosis, and GSDMD-NT induced fibrosis by mediating lipid metabolism.


title: Single-cell profiling of penta- and tetradactyl mouse limb buds identifies early mesenchymal progenitors required to specify digit numbers and asymmetry

city: Basel

first: Aimee

last: Zuniga

address: Mattenstrasse, 28 4058

department: Department of Biomedicine

country: Switzerland

institute: University of Basel

summary: Developing limb cells are heterogeneous. We used single cell RNA sequencing to compare a wildtype limb to two different Grem1 tetradactyl mutants at E10.75 and found differences in populations.

overall_design: Limbs were chopped from E10.75 mouse embryos. Cells were dissociated and used to prepare libraries

first: Victorio

last: Palacio

first: Anna

last: Pancho

first: Angela

last: Morabito

first: Jonas

last: Malkmus

first: Zhisong

last: He

first: Geoffrey

last: Soussi

first: Rolf

last: Zeller

first: Barbara

last: Treutlein

first: Aimée


title: An integrated approach for the accurate detection of HERV-K HML-2 transcription and protein synthesis.

status: Public on Jan 08 2025

city: New York

first: Lubbertus

last: Mulder

address: 1468 Madison Avenue

department: Microbiology

institute: Icahn School of Medicine at Mount Sinai

summary: Human endogenous retroviruses (HERVs) occupy a large portion of the human genome. Most HERVs are transcriptionally silent, but can be reactivated during pathological states such as viral infection and certain cancers. The HERV-K HML-2 clade includes elements that recently have integrated in the human germ line and often contain intact open reading frames (ORFs) that possibly support peptide and protein expression. Understanding HERV-K-host interactions and their potential as biomarkers is problematic due to the high similarity among different elements.  Previously we described a long-read single molecule real-time sequencing (PacBio) strategy to analyze HERV-K RNA expression profiles in different cell types. However, identifying HERV-K HML-2 proteins accurately is difficult without robust and reliable methods and reagents. Here we present a new approach to characterize the HML-2 elements that (a) are being translated and (b) produce enough protein to be detected and identified by mass spectrometry. Our data reveal that RNA expression profiling alone cannot accurately predict which HML-2 elements are responsible for protein production, as we observe several differences between the highest expressed RNAs and the elements that are the predominant source of HERV-K HML-2 protein synthesis. These studies represent an important advance towards untangling the complexity of HERV-K-host interactions.

overall_design: RNA-seq profiling of untreated NCCIT cells with respect to endogenous retrovirus expression in 3 biological replicates.

first: Charles

last: Gleason

first: Sandra

middle: N

last: Terry

first: Matthew

last: Hernandez

first: Gintaras

last: Deikus

first: Nancy

last: Francoeur

first: Aana

last: Hahn

first: Viviana

last: Simon


title: Histone H3 E50K remodels chromatin to confer oncogenic activity  and support EMT phenotype [ATAC-Seq]

city: Atlanta

first: Jennifer

middle: Marie

last: Spangle

address: 1365C Clifton Rd NE

institute: Emory University

summary: Sequencing of human patient tumors has identified recurrent missense mutations in genes encoding core histones. We report that mutations that convert histone H3 amino acid 50 from a glutamate to a lysine (H3E50K) support an oncogenic phenotype. Expression of H3E50K is sufficient to transform human cells as evidenced by an increase in cell migration and invasion, and an increase in proliferation and clonogenicity. H3E50K also increases the invasive phenotype in the context of co-occurring BRAF mutations, which are present in patient tumors characterized by H3E50K. H3E50 lies on the globular domain surface in a region that contacts H4 within the nucleosome. We find that H3E50K selectively increases chromatin accessibility and perturbs proximal H3 post-translational modifications including H3K27me3; together these changes to chromatin dynamics dysregulate gene expression to support the epithelial to mesenchymal transition. Functional studies using S. cerevisiae reveal that, while yeast cells that express H3E50K as the sole copy of histone H3 show sensitivity to cellular stressors, including caffeine, H3E50K cells display some genetic interactions that are distinct from the characterized H3K36M oncohistone yeast model. Taken together, these data suggest that additional H3 mutations have the potential to support oncogenic activity and function through distinct mechanisms that dysregulate gene expression.

overall_design: ATACseq was performed with H3.3E50K and H3.3WT HMECDD cells to see whether H3E50K could alter chormatin accessiblity. Further we examined whether EMT-associated genes and regulatory regions are differentially accessible via ATAC-seq. We are using immortalized but untransformed HMEC DD cells with dominant negative p53 cells that stably overexpressed a C-terminal TY1 epitope-tagged H3.3 WT and E50K.

first: Kirti

last: Sad

first: Dorelle

middle: V

last: Fawwal

first: Celina

middle: Y

last: Jones

first: Emily

middle: J

last: Hill

first: Katie

middle: T

last: Skinner

first: Miranda

last: Adams

first: Severin

last: Lustenberger

first: Richard

middle: S

last: Lee

first: Sandhya

last: Lohano

first: Satvik

last: Elayavalli

first: Jonathan

last: Farhi

first: Christina

last: Mehta

first: Laramie

last: Lemon

first: Milo

last: Fasken

first: Andrew

last: Hong

first: Steven

last: Sloan

first: Anita

last: Corbett


title: Cut&Tag of 15 week-old HD R6/1 mice in neuronal cell populations.

city: strasbourg

first: Charles

last: DECRAENE

address: 12 rue Goethe

department: UMR 7364 CNRS

country: France

institute: CNRS

summary: To characterize the epigenome of HD R6/1 mice in striatal cells using CUT&TAG on neuronal nuclei

overall_design: NeuN+ nuclei from mixed males and females WT and R6/1 mice striatum were used (75000 nuclei). Two different histone marks were evaluated: H2AK119ub and H3K27me3 coming from the same sample, in order to perform correlation analysis (n=3 replicates). Rabbit IgGs were used as control.

first: Karine

last: Merienne

first: N

last: Penaud

first: S

last: Le Gras

last: Decraene


title: Engineering synthetic signaling receptors to enable erythropoietin-free erythropoiesis

city: San Francisco

first: Prathamesh

last: Chati

state: CA

address: 1975 4th Street

institute: University of California, San Francisco

summary: Blood transfusion plays a vital role in modern medicine. However, frequent shortages pose a significant healthcare challenge. Ex vivo manufacturing of red blood cells (RBCs) derived from universal donor pluripotent stem cells is limited by the high cost of recombinant cytokines required for ex vivo erythroid differentiation. Erythropoietin (EPO) signaling through the EPO receptor is indispensable to RBC development, and EPO is one of the most expensive components in erythroid-promoting media. Here, we use design-build-test cycles to develop highly optimized small molecule-inducible synthetic EPO receptors (synEPORs) which are integrated at a variety of genomic loci using homology-directed repair genome editing. We find that integration of synEPOR at the endogenous EPOR locus in an induced pluripotent stem cell producer line cultured with small molecule yields equivalent erythroid differentiation, transcriptomic changes, and hemoglobin production compared to cells cultured with EPO. Due to the dramatically lower cost of small molecules vs. recombinant cytokines, these efforts eliminate one of the most expensive elements of ex vivo culture media—EPO cytokine. As cytokines dependence is a common barrier to ex vivo cell production, these strategies could improve scalable manufacturing of multiple clinically relevant cell types.

overall_design: RNA-seq profiling of PB005-derived hematopoietic stem and progenitor cells (HSPCs) and erythroid cells, including wild-type (WT) and genetically modified derivatives (PGK(synEPOR), HBA1(synEPOR), and EPOR(synEPOR)), at undifferentiated (Day 0) and differentiated (Day 14) stages of erythroid development.

first: Aadit

middle: P

last: Shah

first: Kyle

last: Cromer


title: Comparative framework and adaptation of ACME HS approach to single cell isolation from fresh-frozen endocrine tissue [dataset 2]

city: Moscow

first: Valentin

last: Trofimov

address: Ulyanova str.11

country: Russia

institute: Endocrinology Research Center

summary: Current dissociation methods for scRNA-seq studies of solid tissues do not guarantee intact single-cell isolation from fresh frozen samples, especially for sensitive and heterogeneous endocrine tissues. Here, we adapted the acetic-methanol dissociation method – ACME High Salt (ACME HS) to isolate intact single cells from fresh-frozen endocrine tumor samples. We compared enzymatic, ACME HS, and nuclear isolation methods ability to preserve major cell types and gene expression. We demonstrated that ACME HS dissociates and fixes cells, preserving cell morphology and high RNA integrity. This renders ACME HS a valuable alternative in the scRNA-seq protocols for challenging tissues.

overall_design: Single cells were isolated from tissue samples using ACME HS followed by cryopreservation in 3xSSC*10% DMSO and methanol cell fixation. Key adaptations of the ACME HS method were the supplement of solution composition with 0.1M NAC and the introduction of additional washing steps in cold high salt 3xSSC* buffer. ACME dissociation was conducted in ~ 1 hours on a rotator at room temperature, with periodic pipetting of the solution. Afterward, we removed the ACME solution and washed the pellet with a two-step washing in cold 3xSSC. Single cells or nuclei were captured and barcoded, and cDNA libraries were generated using the Chromium Next GEM Single Cell 3ʹGEM, Library & Gel Bead Kit v3.1 (10X Genomics) according to the manufacturer’s instructions. Final libraries were multiplexed and sequenced on an Illumina Novaseq 6000 platform, using the S4 Reagent Kit v1.5 and Illumina HiSeq2500 platform.

first: Marina

last: Utkina

first: Anastasia

last: Shcherbakova

first: Ruslan

last: Deviatiiarov


title: Comparative framework and adaptation of ACME HS approach to single cell isolation from fresh-frozen endocrine tissue [dataset 1]

overall_design: Single cells were isolated from tissue samples using ACME HS and enzymatic dissociation methods, freshly or followed by cryopreservation in 3xSSC*10% DMSO and methanol cell fixation. Key adaptations of the ACME HS method were the supplement of solution composition with 0.1M NAC and the introduction of additional washing steps in cold high salt 3xSSC* buffer. ACME dissociation was conducted in ~ 1 hours on a rotator at room temperature, with periodic pipetting of the solution. Afterward, we removed the ACME solution and washed the pellet with a two-step washing in cold 3xSSC. Single cells or nuclei were captured and barcoded, and cDNA libraries were generated using the Chromium Next GEM Single Cell 3ʹGEM, Library & Gel Bead Kit v3.1 (10X Genomics) according to the manufacturer’s instructions. Final libraries were multiplexed and sequenced on an Illumina Novaseq 6000 platform, using the S4 Reagent Kit v1.5 and Illumina HiSeq2500 platform.


title: Comparative framework and adaptation of ACME HS approach to single cell isolation from fresh-frozen endocrine tissue.

summary: Current dissociation methods for scRNA-seq studies of solid tissues do not guarantee intact single-cell isolation from fresh frozen samples, especially for sensitive and heterogeneous endocrine tissues. Here, we adapted the acetic-methanol dissociation method – ACME High Soult (ACME HS) to isolate intact single cells from fresh-frozen endocrine tumor samples. We compared enzymatic, ACME HS, and nuclear isolation methods ability to preserve major cell types and gene expression. We demonstrated that ACME HS dissociates and fixes cells, preserving cell morphology and high RNA integrity. This renders ACME HS a valuable alternative in the scRNA-seq protocols for challenging tissues.

overall_design: Single cells were isolated from tissue samples using enzymatic dissociation method. Approximately 5-250 mg of fresh tissue samples were washed in HBSS, thoroughly minced on ice, and placed in dissociating solution at 37°C with gentle pipetting every 5 minutes.  Tissue samples were dissociated with 25-30 ul enzyme D Multi Tissue Dissociation Kit (MTDK) (Miltenyi Biotec) or enzyme A Neural Tissue Dissociation Kit P (NTDK) (Miltenyi Biotec) or 2 mg/ml collagenase IV (Gibco, Thermo Fisher Scientific) or collagenase I (Gibco, Thermo Fisher Scientific) in 870 mM HBSS, 10% FBS, 20 mM HEPES for 20-30 minutes. Obtained homogenate was filtered in 3-5ml of Wash Buffer (1x DPBS, containing 10% FBS, 20mM HEPES, 6mM glucose) through a pre-wetted 70μm cell culture filter (Miltenyi Biotec) and centrifuged for 5 minutes at 300xg (4 °C).  After all, the pellet was resuspended in a Wash Buffer volume of 100-400 ul, depending on the pellet size. Single cells or nuclei were captured and barcoded, and cDNA libraries were generated using the Chromium Next GEM Single Cell 3ʹGEM, Library & Gel Bead Kit v3.1 (10X Genomics) according to the manufacturer’s instructions. Final libraries were multiplexed and sequenced on an Illumina Novaseq 6000 / Hiseq 2500 platform, using the S4 Reagent Kit v1.5.


title: Sir2 and Fun30 regulate ribosomal DNA replication timing via MCM helicase positioning and nucleosome occupancy

city: Seattle

first: Antonio

last: Bedalov

state: WA

address: 1100 Fairview Ave N

department: Translational Science and Therapeutics Division

institute: Fred Hutch Cancer Center

summary: The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well-known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA arrays (rDNA). We have previously reported that in the absence of SIR2, a derepressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both, activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30. Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.

overall_design: Yeast strain  Strains used in the study were derived from BY4741 and W303 and are provided in Suplemental Table 1. Cells were grown in standard yeast peptone 2% dextrose media except for hydroxyurea time course study which employed synthetic media.  Strains containing point mutations in FUN30 were made using a plasmid that contains CAS9 and gRNA targeting the PAM sites adjacent to the desired mutation sites in FUN30 as described. Oligos containing FUN30-targeting gRNAs were cloned into pML104. pML104 was a gift from John Wyrick (Addgene plasmid # 67638)(Laughery et al. 2015). The plasmids, which induce double strand breaks within FUN30, were co-transformed with DNA blocks containing the repair templates with the desired mutations and altered PAM sites, synthetically synthesized by IDT. FUN30 DNA was PCR amplified from the resulting transformants and sequenced to verify that the desired mutations have been introduced.  Edu-incorporating yeast strains were derived from W303 and contain four Brdu vectors that express HSV-TK and hENT1 ((Viggiani and Aparicio 2006). Plasmids containing BrdU cassete obtained from Addgene (Addgene plasmid # 71789-71792), were integrated at four different auxotrophic loci (HIS3, TRP1, LEU2, and URA3). Single integration of each Brdu cassette was confirmed via PCR using plasmid-specific primer sets as described ((Viggiani and Aparicio 2006), which assured that the strains contain exactly four copies of the BrdU cassette  Measurements of rDNA replication timing using S-seq  S-seq experiments were carried out as previously described (Foss et al. 2017). Cells were grown to log phase in YEPD media, fixed using 70% (vol/vol) ethanol, subjected to proteinase K digestion, and their DNA stained with Sytox Green as described (Foss 2001). Cells were sorted according to DNA content on a BD Biosciences FACSAria II cell sorter into G1 and S fractions. DNA from a minimum of 1 e-6 cells from each fraction was isolated using the YeaStar Genomic DNA Kit (Zymo Research). DNA was fragmented by sonication and sequenced.  MNase seq  We carried out MNase-Seq as previously described (Foss 2019). Briefly, cells grown to log phase in rich medium, Yeast Peptone Agar with 2% glucose (YEPD), from an overnight 25 mL culture were synchronized with 3 μM alpha-factor for 1.5 hrs. at 30 ° C. Arrested cells were crosslinked with 1% formaldehyde for 30 min at room temperature water bath with shaking. Formaldehyde was quenched with 125 mM glycine and cells were centrifuged at 3000 rpm for 5 min. Cells were washed twice with water and resuspended in 1.5 mL Buffer Z (1 M sorbitol, 50 mM Tris-HCl pH 7.4) with 1 mM beta-mercaptoethanol (1.1 μL of 14.3 M beta-mercaptoethanol diluted 1:10 in Buffer Z) per 25 mL culture. Cells were treated with 100 μL 20 mg/ mL zymolyase at 30 ° C for 20–30 min. Spheroplasts were centrifuged at 5000 rpm for 10 min and resuspended in 5 mL NP buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris pH 7.4, 5 mM MgCl2, 1 mM CaCl2) supplemented with 500 μM spermidine, 1 mM beta-mercaptoethanol and 0.075% NP-40. Nuclei were aliquoted in tubes with varying concentrations of micrococcal nuclease (Worthington), mixed via tube inversion, and incubated at room temperature for 20 mins. Chromatin digested with 1.9 U– 7.5 U micrococcal nuclease per 1/5th of spheroplasts from a 25 mL culture yielded appropriate mono-, di-, tri- nucleosome protected fragments for next-generation sequencing. Digestion was stopped with freshly made 5x stop buffer (5% SDS, 50 mM EDTA) and proteinase K was added (0.2 mg/ml final concentration) for an overnight incubation at 65 ° C to reverse crosslinking. DNA was extracted with phenol/chloroform and precipitated with ethanol. Micrococcal nuclease digestion was analyzed via gel electrophoresis prior to proceeding to library preparation. Sequencing libraries were prepared as described below for ChEC.  Chromatin Endogenous Cleavage (ChEC)  ChEC-seq was carried out as previously described (Foss et al. 2019; Foss et al. 2021). Briefly, MNase was activated by addition of CaCl2 to cells that were permeabilized with digitonin. DNA was extracted from cells using phenol and chloroform, precipitated using salt and ethanol and used to construct sequencing libraries without size fractionation. Cells were centrifuged at 1,500 x g for 2 mins, and washed twice in cold Buffer A (15 mM Tris pH 7.5, 80 mM KCl, 0.1 mM EGTA) without additives. Washed cells were carefully resuspended in 570 μL Buffer A with additives (0.2 mM spermidine, 0.5 mM spermine, 1 mM PMSF, ½ cOmplete ULTRA protease inhibitors tablet, Roche, per 5 mL Buffer A) and permeabilized with 0.1% digitonin in 30 ° C water bath for 5 min. Permeabilized cells were cooled at room temperature for 1 min and 1/5th of cells were transferred in a tube with freshly made 2x stop buffer (400 mM NaCl, 20 mM EDTA, 4 mM EGTA)/1% SDS solution for undigested control. Micrococcal nuclease was activated with 5.5 μL of 200 mM CaCl2 at various times (5 mins, and 10 mins) and the reaction stopped with 2x stop buffer/1% SDS. Once all time points were collected, proteinase K was added to each of the collected time points and incubated at 55 ° C water bath for 30 mins. DNA was extracted using phenol/chloroform and precipitated with ethanol. Micrococcal nuclease digestion was analyzed via gel electrophoresis prior to proceeding to library preparation. Library was prepared as previously described used using total DNA, without any fragment size selection (Foss et al. 2019; Foss et al. 2021).  Chromatin immunoprecipitation followed by sequencing (ChIP-seq)  Cells were grown to log phase in rich medium, YEPD (1% Yeast Extract, 2% Peptone, 2% glucose), synchronized with 3 μM alpha-factor for 1.5 hrs. at 30°C. G1 arrested cells were released into 200 mM hydroxyurea (HU) YEPD and grown for 1hr. Cells were crosslinked with 1% formaldehyde for 30 min at room temperature, quenched with 125 mM glycine for 5 minutes, and centrifuged at 3000 rpm for 5 minutes. Cell pellets were washed three times with TBS (20 mM Tris-HCl, pH 7.6, 150 mM NaCl). Cell pellets were flash frozen.  Chromatin Preparation  The cell pellet was thawed in 300 μL Breaking Buffer (100 mM Tris- HCl, pH 8.0, 20% Glycerol) supplemented with protease inhibitors (Pierce Protease inhibitor EDTA-free tablets [Thermo Scientific A32965] and 1 mM PMSF). 300 μL acid-washed glass beads were added to the mixture. Cells were lysed using a BioSpec Mini-Bead-beater in a cold room: 5x 30-second pulses with 1-minute on ice between rounds. The supernatant was transferred to a new tube and added 600 μL FA buffer (50 mM Hepes-KOH, pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate) with protease inhibitors. The chromatin was sonicated with a F60 Sonic Dismembrator 10 times for 10 seconds at setting 4, with 1-minute rests on ice between rounds. The sonicated chromatin was centrifuged at 14,000 rpm for 15 minutes at 4°C. The supernatant was transferred and spun again at 14,000 rpm for 15 minutes at 4°C.  Immunoprecipitation (IP)  For IP, 20 μL of Dynabeads Protein G beads (Invitrogen 10004D) were washed three times with 500 μL PBS-T (0.8% NaCl, 0.144% Na2HPO4, 0.02% KCl, 0.024% KH2PO, 0.1% Tween 20, pH 7.1), resuspended in PBS-T, and incubated with 5 μL of FLAG M2 (Sigma F1804) antibody for 60 minutes at room temperature. The antibody-conjugated beads were washed and resuspended in 20 μL FA buffer with protease inhibitors. IP chromatin samples (1 μg) were brought up to 400 μL with FA buffer with protease inhibitors and incubated with antibody- conjugated beads for 90 minutes at room temperature. Beads were washed three times with FA buffer with protease inhibitors, twice with FA-HS buffer (50 mM Hepes-KOH, pH 7.6, 500 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate with protease inhibitors), and once with RIPA buffer (10 mM Tris-HCl, pH 8.0, 0.25 M LiCl, 0.5% NP-40, 0.5% Sodium deoxycholate, 5 mM EDTA with protease inhibitors). Input sample was generated by adding 0.1 μg chromatin in 40 μL FA buffer to 40 μL 2X Stop buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 20 mM EDTA, 1% SDS, 2% Tween 20) and reversed crosslinks by overnight incubation at 65°C.  Elution and Crosslink Reversal  Bound chromatin was eluted by incubating the beads in 80 μL 2X Stop buffer at 75°C for 10 minutes. The eluate was collected, and crosslinks were reversed by overnight incubation at 65°C. The IP and Input were treated with 4 μL of 20 mg/mL RNase A and incubated at 55°C for 1 hour, followed by 4 μL of 20 mg/mL Proteinase K and incubation at 55°C for at least 3 hours. DNA was purified using a MinElute PCR Purification Kit  Paired-Ends NGS  Chromatin immunoprecipitation samples were prepared for sequencing following the protocol for the Kapa Hyper Kit. For Input samples, 2 ng were processed for End Repair. For IP samples, the entire volume was used. PCR amplification was performed with 10- 14 cycles based on DNA quantity.  Quantification of rDNA replication using the thymidine analogue EdU  Cells were grown to log phase in rich YEPD medium from a 50 mL culture and synchronized in G1 using 3 μM alpha factor for 2 hours at 30°C. Using 500 μg/mL pronase, arrested cells were released into rich media containing 200 mM hydroxyurea and 130 μM EdU (5-Ethynyl-2’- deoxyuridine). Cells were harvested in G1 and after 60 minutes released into HU. DNA was extracted using YeaStar Genomic DNA Kit (Zymo Research) and sonicated. Separate sequencing primers were ligated to each of the samples. Samples were pooled together, and subjected to click chemistry reaction in 10 mM Tris pH 7.4, 50mM NaCl, 20 μM biotin-TEG azide (Vector Laboratories), 5mM THPTA (tris-hydroxypropyltriazolylmethylamine) (Vector Laboratories), 1mM copper sulfate, and 15 mM sodium ascorbate (total volume 200 μL). Click reaction proceeded for 30 minutes at room temperature prior to DNA ethanol precipitation. Streptavidin magnetic beads (New England Biolabs) were washed 3 times with B&W buffer I (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.05% Tween20, 0.5% SDS) before adding 100 μL of bead suspension to 180 μL DNA plus B&W buffer I. After a 30-minute incubation at room temperature while gently inverting tubes, bead bound DNA was washed with 200 μL B&W buffer I followed by 100 μL Stringency wash buffer (0.1% SDS, 0.1% SSC buffer) and 200 μL Wash Buffer II (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.05% Tween 20). DNA was eluted off the beads by denaturation at 98°C for 5 minutes, followed by cooling at 25°C. Edu-labeled DNA was PCR amplified post-pulldown and subjected to sequencing.  Hydoxyurea ChEC time course experiment  Cells grown in synthetic media to logarithmic phase were arrested in G1 using alpha factor for 90 minutes, centrifuged, washed in water to remove alpha factor, and released into media containing 200 mM Hydroxyurea. Cells were harvested in G1 and at indicated time points following release into HU-containing media, chilled at 4 degrees and analyzed by ChEC as described above.

first: Eric

last: Foss


title: ChIPseq on bulk striatal tissue of 15-20 week-old HD R6/1 mice

summary: To characterize striatal epigenome of HD R6/1 mice

overall_design: ChIPseq data (H2AK119ub) were generated using the striatum of WT or HD R6/1 female and male mice at 15-20 weeks of age.


title: Droplet-based high-throughput 3D genome structure mapping of single cells with simultaneous transcriptomics (b3-mouse-brain)

first: Xiaoliang

last: Xie

summary: Single-cell three-dimensional (3D) genome techniques have advanced our understanding of cell-type-specific chromatin structures in complex tissues, yet current methodologies are limited in cell throughput. Here we introduce a high-throughput single-cell Hi-C approach (dscHi-C) and its transcriptome co-assay (dscHi-C-multiome) using droplet microfluidics. Using dicHi-C, we investigated chromatin structural changes during mouse brain aging by profiling 32,777 single cells across three developmental stages (3 month, 12 month and 23 month), yielding a median of 78,220 unique contacts. Our results show that genes with significant structural changes are enriched in pathways related to metabolic process and morphology change in neurons, and innate immune response in glial cells, highlighting the role of 3D genome organization in physiological brain ageing. Furthermore, our multi-omics joint assay, dscHi-C-multiome, enables precise cell type identification in the adult mouse brain and uncovers the intricate relationship between genome architecture and gene expression. Collectively, we developed the sensitive, high-throughput dscHi-C and its multi-omics derivative, dscHi-C-multiome, demonstrating their potential for large-scale cell atlas studies in development and disease.

overall_design: In this study, we performed droplet-based single-cell Hi-C (dscHi-C) and its transcriptome co-assay (dscHi-C-multiome) on human and mouse cell line, and mouse brain cortex tissues.

first: Honggui

last: Wu

first: Maoxu

last: Wang


title: ChIPseq of 15-20 week-old R6/1 HD mice in neuronal and non-neuronal striatal cell populations

last: DECRAENE

summary: To characterize the epigenome of R6/1 HD mice in specific striatal cellular populations

overall_design: ChIPseq data were generated using sorted nuclei from the striatum of WT or R6/1 HD male and female mice at 15-20  weeks of age.  Two different histone marks were evaluated: H3K27ac and H3K27me3 coming from the same sample.

first: Rafael

last: Alcala Vida

first: J

last: Seguin


title: Cut&Tag of 20 week-old HD R6/1 mice in neuronal and non-neuronal striatal cell populations.

first: Charles

summary: To characterize the epigenome of HD R6/1 mice in striatal cells using CUT&TAG on neuronal and non-neuronal nuclei

overall_design: NeuN+/- nuclei from females, WT and R6/1 mice striatum were used (50000 nuclei). Two different histone marks were evaluated: H3K9ac and H3K18ac coming from the same sample, in order to perform correlation analysis (n>=2 replicates for NeuN+, n=1 for NeuN-). Rabbit IgGs were used as control.

first: Karine

last: Merienne

first: Baptiste

last: Brule

last: Le Gras


title: LEDGF/p75 promotes transcriptional pausing through preventing SPT5 phosphorylation

city: nanjing

first: shuhan

last: si

state: jiangsu

address: sipailou

institute: Southeast University

summary: The transcriptional progression is regulated by SPT5, which is involved in both promoter-proximal RNA polymerase II (Pol II) pausing and elongation, which serve as rate-limiting steps in metazoan. However, the mechanism for SPT5 coordinating these processes remains unclear. Here we report that the disordered sub-regions within SPT5 contain a prion-like domain (PLD) that is required for Pol II pausing, while the phosphorylation site-rich domain (PRD) is essential for transcript elongation. Mechanically, SPT5-PLD helps stabilize Pol II pausing by preventing SPT5-PRD phosphorylation mediated by the super elongation complex (SEC). SPT5-PLD also maintains Pol II pausing by recruiting a newly identified transcriptional pause regulator, PSIP1, which inhibits phosphorylation required for elongation. Additionally, SPT5-PLD prevents premature release of Pol II pausing through PSIP1’s IBD domain by counteracting SEC. Our results demonstrate that SPT5 possesses intrinsic characteristics that balance the transcriptional process by preventing premature pause release and promoting progression to elongation.

overall_design:
Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for AFF4,Pol II, SPT5,SPT5-pCTR1, NELFE in HCT116 cells.
Cleavage Under Targets and Tagmentation (CUT&Tag) for LEDGF in HCT116 cells.

first: Chenghao

last: Guo

first: Shuhan

last: Si

first: Zhuojuan

last: Luo

first: Chengqi

last: Lin


title: Predicting patient outcome based on gene-expression biomarkers learned from colorectal cancer organoids and cell lines

first: Sergey

last: Nikulin

address: 7, Vavilova st.

department: Faculty of Biology and Biotechnology

institute: HSE University

summary: Colorectal cancer is characterized by an extremely high mortality rate, mainly caused by the high metastatic potential of this type of cancer. To date chemotherapy remains the backbone of the treatment of metastatic colorectal cancer. Three main chemotherapeutic drugs used for treatment of metastatic colorectal cancer are 5-FU, oxaliplatin and irinotecan which is metabolized to an active compound SN-38. The main goal of this study was to find the genes connected to the resistance to the aforementioned drugs and to construct predictive gene expression-based classifier to separate responders and non-responders. In this study we used 7 established colorectal cancer organoids to conduct correlational analysis of the expression and drug resistance. We also included in the study publicly available datasets of colorectal cancer cell lines. The obtained results demonstrated that the expression of just a small group of genes consistently correlate with resistance to standard chemotherapeutic drugs in different datasets. Some of these genes have been previously connected to prognosis or response to anticancer drugs, but some of them were linked to the drug resistance for the first time. The classifier constructed on the expression levels of some of these genes allows to discriminate between patients with favorable and unfavorable 5-year OS prognosis and can be further validated in clinic to improve the results of the treatment.

overall_design: Total mRNA profiles of 7 metastatic colorectal cancer organoids from 6 patients were measured to identify correlations of gene expression with sensetivity to standard of care drugs.


title: Dependence of RBPJ genomic binding on the formation of macromolecular complexes

city: Philadelphia

first: Tobias

last: Friedrich

state: Pennsylvania

address: 421 Curie Boulevard

institute: University of Pennsylvania

summary: RBPJ is the central transcription factor involved in the Notch-dependent transcriptional response. Ist repressive function is strongly dependent on its interaction with the corepressor SHARP. Here we analyzed the gomic binding of RBPJ wt, and different RBPJ mutants making use of RBPJ or SHARP depleted HeLa cells. and

overall_design: Overexpression of RBPJ, wild type or different mutants, was achieved in RBPJ or SHARP depleted cells. RBPJ genomic binding was determined via ChIP-Seq.

first: Benedetto

middle: D

last: Giaimo

first: Francesca

last: Ferrante

first: Tilman

last: Borggrefe


title: Heightened expression of type I interferon signaling genes in CD4+ T cells from acutely HIV-1 infected women is associated with lower viral loads

status: Public on Jan 07 2025

first: Gregory

last: Tharp

state: GA

address: 954 Gatewood Dr

department: Developmental and Cognitive Neuroscience

institute: Yerkes National Primate Research Center

summary: In this study, we have identified a number of differentially expressed genes in naïve, central memory, and effector memory CD4 T cells of HIV-infected women compared to men, with consistent elevated expression of genes linked to type 1 interferon signaling. Moreover, after controlling for differences in VL and CD4+ T cell count genes within the type I interferon (IFN) signaling pathway were further shown to be more highly expressed in women, whereas, those genes more highly expressed in men showed no such enrichment. A subset of the genes highly expressed in women were further identified, including several involved in type I IFN signaling in response to viral infections (IRF7, DDX58, SAMHD1, OAS2, and TRIM14), that are both more highly expressed in CD4+ T cells from women and negatively correlated with VL, suggesting that they play a role in the comparative control of VL observed in women.

overall_design: We have performed bulk RNA-seq experiments comparing gene expression between CD4+ T cells from acutely HIV-1 infected men and women in Zambia, since we observe lower viral load (VL) despite higher CD4+ T cell activation in these women during acute/early infection. We performed analysis on both individual CD4+ T cell subsets (Naive, Central Memory and Effector Memory) as well as combined CD4 T cells) controlling for CD4 T cell counts and viral load.

first: Elina

last: El-Badry

first: Luxiao

last: Chen

first: Khader

last: Ghneim

first: Ziyi

last: Li

first: Kelsie

last: Brooks

first: Jake

last: Rhodes

first: Rafick

last: Sekaly

first: William

last: Kilembe

first: Susan

last: Allen

first: Hao

last: Wu

first: Eric

last: Hunter


title: Nuclear Talin-1 Provides a Bridge Between Cell Adhesion and Gene Expression

city: Turku

first: Lea

last: Sistonen

address: Tykistökatu 6 A

department: Cell Biology

country: Finland

institute: Åbo Akademi University

summary: Talin-1 (TLN1) is best known to activate integrin receptors and transmit mechanical stimuli to the actin cytoskeleton at focal adhesions. However, the localization of TLN1 is not restricted to focal adhesions. By utilizing both subcellular fractionations and confocal microscopy analyses, we show that TLN1 localizes to the nucleus in several human cell lines, where it is tightly associated with the chromatin. Importantly, siRNA-mediated depletion of endogenous TLN1 triggers extensive changes in the gene expression profile of human breast epithelial cells. To determine the functional impact of nuclear TLN1, we expressed a TLN1 fusion protein containing a nuclear localization signal. Our findings revealed that accumulation of nuclear TLN1 alters the expression of a subset of genes and impairs the formation of cell-cell clusters. This study introduces an additional perspective on the canonical view of TLN1 subcellular localization and function.

overall_design: HS578T cells co-transfected with GFP and either non-targeting control siRNA (SCR) or siRNA against TLN1 (siTLN1), 2 biological replicas.

first: Alejandro

last: Da Silva

first: Hendrik

last: Hästbacka

first: Mikael

middle: C

last: Puustinen

first: Jenny

last: Pessa

first: Jens

last: Luoto

first: Erika

last: Sundström

first: Benjamin

last: Goult

first: Guillaume

last: Jacquemet

first: Eva

last: Henriksson


title: Bulk RNA-seq of human knee osteoarthritis synovium

city: Bunkyo-ku

first: Junya

last: Miyahara

address: Hongo 7-3-1

department: Sensory & Motor system Medicine, Graduate School of Medicine

institute: The University of Tokyo

summary: To classify knee osteoarthritis (OA) synoviums according to gene expression patterns,  we performed RNA-seq using synovial tissues obtained from OA patients and non-OA patients.

overall_design: We performed RNA-seq using synovial tissues obtained from 36 knee OA patients undergoing primary total knee arthroplasty (TKA) and 5 patients undergoing arthroscopy one year after anterior cruciate ligament reconstruction.

first: Hisatoshi

last: Ishikura

first: Ryota

last: Chijimatsu

first: Taku

last: Saito


title: Gene expression analysis following RRP therapy INO-3107

city: Plymouth Meeting

first: Elisabeth

last: Gillespie

state: PA

address: 660 W Germantown Pike, Suite 110

institute: Inovio Pharmaceuticals

summary: Recurrent respiratory papillomatosis (RRP) is a debilitating chronic disease of the airway caused by Human Papillomavirus (HPV). Here we describe the efficacy, safety, and immunogenicity from a Phase 1/2 trial of INO-3107, a DNA immunotherapy designed to elicit T-cell responses against HPV-6 and HPV-11, in adults with RRP (NCT04398433). Thirty-two eligible patients with HPV-6 and/or HPV-11 RRP, who required ≥2 surgical interventions in the year preceding dosing, were administered 4 doses of INO-3107 by intramuscular (IM) injection followed by electroporation (EP). The primary endpoint was safety and tolerability. Efficacy and immunology endpoints included the frequency of surgical interventions in the year following the first dose of INO-3107 and assessment of immune responses. The majority (26/32; 81%) of patients experienced a reduction in the number of surgeries in the year following initiation of treatment with INO-3107, when compared with the year prior. Peripheral blood assessments revealed HPV-6 and HPV-11 antigen-specific T-cell induction, inclusive of cytolytic CD8+ T-cells. RNA sequencing identified the presence of an inflammatory response in patient papillomas after treatment with INO-3107, inclusive of cytolytic CD8+ T-cell signatures. T-cell receptor sequencing revealed emergent T-cell clones in the blood and assessment of airway tissue confirmed trafficking of these clones to papillomas, which was associated with clinical benefit. Treatment was well tolerated, with 13/32 (41%) patients reporting a treatment-related adverse event (AE), all of which were low-grade. INO-3107 provides clinical benefit to adults with HPV-6 and/or HPV-11 associated RRP, is well tolerated, and generates an antigen-specific immune response against HPV. Importantly, treatment-induced peripheral T-cell responses traffick to airway tissue and are associated with clinical response, consistent with the proposed mechanism of action.

overall_design:
RRP-001 (ClinicalTrials.gov identifier: NCT04398433) was a Phase 1/2, single-arm, open-label study conducted across 8 centers in the United States.  This was a single dose-level trial using a 6-patient safety run-in with a one-week waiting period between each patient.  Eligible patients were men and women 18 years or older with histologically documented HPV-6 or HPV-11 positive RRP, who required frequent RRP surgical interventions.
Patients received one dose of INO-3107 on Day 0 and Weeks 3, 6, and 9 administered intramuscularly followed immediately by electroporation using the CELLECTRA proprietary device.
Peripheral blood mononuclear cells were cryopreserved, and tissue collected during surgical intervention or biopsy was formalin fixed and embedded in paraffin for use in RNA seq.

first: Matthew

last: Morrow

first: Albert

last: Sylvester MS

first: Milan

middle: R

last: Amin

first: Peter

last: Belafsky

first: Simon

last: Best

first: Aaron

last: Friedman

first: Adam

middle: M

last: Klein

first: David

middle: G

last: Lott

first: Ted

last: Mau

first: Randal

last: Paniello

first: Seth

last: Pransky

first: Nabil

middle: F

last: Saba

first: Grace

last: Tan

first: Sadie

last: Wisotsky

first: Sarah

middle: A

last: Marcus

first: Emma

middle: L

last: Reuschel

first: Katherine

last: Reed

middle: B

last: Weiner

first: Michael

last: Dallas

first: Jeffrey

last: Skolnik


title: Single cell RNA-seq of human knee osteoarthritis synovium

first: Junya

last: Miyahara

summary: To classify knee osteoarthritis (OA) synoviums according to gene expression patterns,  we performed single cell RNA-seq using synovial tissues obtained from OA patients.

overall_design: We performed single cell RNA-seq using synovial tissues obtained from 4 knee OA patients undergoing primary total knee arthroplasty (TKA).

first: Hisatoshi

last: Ishikura

first: Ryota

last: Chijimatsu

first: Taku

last: Saito


title: PRMT1-methylated MSX1 phase separates to control palate development

city: Nanjing

first: yucheng

last: jiang

address: No.140 Hanzhong Road, Nanjing

institute: Nanjing medical university

summary: Little is known about the regulation and function of liquid‒liquid phase separation (LLPS) in craniofacial developmental disorders, where Msh homeobox 1 (MSX1) and protein arginine methyltransferase 1 (PRMT1) are two important regulators. MSX1 mutations are associated with human cleft palate, the most common craniofacial birth defect. Here, we show that MSX1 LLPS, regulated by PRMT1-catalyzed methylation, is a vertebrate-conserved mechanism underlying the proliferation of embryonic palatal mesenchymal cells (EPMs) and palatal fusion. MSX1 LLPS is triggered by its N-terminal intrinsically disordered protein region (IDR) and regulated by PRMT1-catalyzed methylation, specifically asymmetric dimethylation of arginine (R). Biochemically, PRMT1 methylates MSX1 by dimethylating the residues R150 and R157 in the MSX1 IDR. Physiochemically, the R157 to serine (R157S) mutant, R150S mutant, and hypomethylated MSX1 under PRMT1 deficiency consistently cause abnormal MSX1 LLPS to form less dynamic gel-like condensates. PRMT1 and its methylation target R residues in MSX1 are conserved in vertebrates, from zebrafish to humans. Phenotypically, less dynamic gel-like condensates formed by abnormal MSX1 LLPS cause EPM proliferation defects and cleft palate.In this study, rna-seq was used to reveal the differences between the effects of Msx1 and its mutants on hepm cells. Our findings provide a new paradigm for further exploration of LLPS underlying genetic disorders for future innovation of genetic disorder treatment by direct modulation of LLPS.

overall_design: Twelve samples from hepm cells transfected with four different plasmids: MSX1, R157S, R157K, and R157F were analyzed and compared with eachother.

first: Junqing

last: Ma

first: Li

last: Meng

first: Yucheng

last: Jiang

first: Liming

last: Chen

first: Jiawen

last: You