title: Antiviral drugs prolong survival in murine recessive dystrophic epidermolysis bullosa

city: Madison

first: Andrew

middle: Perry

last: South

state: Wisconsin

address: 1111 Highland Avenue

institute: University of Wisconsin-Madison

summary: Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin disease characterized by defects in type VII collagen leading to a range of fibrotic pathologies resulting from skin fragility, aberrant wound healing and altered dermal fibroblast physiology. Using a novel in vitro model of fibrosis based on endogenously produced extracellular matrix, we screened an FDA-approved compound library and identified antivirals as a class of drug not previously associated with anti-fibrotic action. Pre-clinical validation of our lead hit, daclatasvir, in a mouse model of RDEB demonstrated significant improvement in fibrosis as well as overall quality of life with increased survival, weight gain and activity, and a decrease in pruritus-induced hair loss. Immunohistochemical assessment of daclatasvir-treated RDEB mouse skin showed a reduction in fibrotic markers, which was supported by in vitro data demonstrating TGFβ pathway targeting and a reduction of total collagen retained in the extracellular matrix. Our data support clinical development of antivirals for treatment of patients with RDEB and potentially other fibrotic diseases.

overall_design: EB samples in triplicate treated with vehicle control (DMSO) or daclatasvir. There are 6 samples total.

first: Grace

last: Tartaglia


status: Public on Mar 01 2024

city: Washington,DC

first: Nino

last: Gustavo

state: Washington,DC,

address: Sheikh Zayed Campus,111 Michigan Avenue,

department: Center for Precision Medicine and Genomics Research

country: USA

institute: Childrens National Hospital

type: Expression profiling by high throughput sequencing

summary: Introduction: Thymic Stromal Lymphopoietin (TSLP) is a primarily epithelial-derived cytokine that drives type 2 allergic immune responses. Early life viral respiratory infections elicit high TSLP production, which leads to the development of type 2 inflammation and airway hyperreactivity. The goal of this study was to examine in vivo and in vitro the human airway epithelial responses leading to high TSLP production during viral respiratory infections in early infancy. Methods: A total of 129 infants (<1m – 24m, median age 10m) with severe viral respiratory infections were enrolled for in vivo (n=113), and in vitro studies (n=16). Infants were classified as “high TSLP” or “low TSLP” for values above or below the 50th percentile. High vs. low TSLP groups were compared in terms of type I-III IFN responses and production of chemokines promoting antiviral (CXCL10), neutrophilic (CXCL1, CXCL5, CXCL8), and type 2 responses (CCL11, CCL17, CCL22). Human infant airway epithelial cell (AEC) cultures were used to define the transcriptomic (RNAseq) profile leading to high vs. low TSLP responses in vitro. Results: Infants in the high TSLP group had greater in vivo type III IFN airway production (median type III IFN in high TSLP 183.2 pg/ml vs. 63.4 pg/ml in low TSLP group, p= 0.007) and increased in vitro type I-III IFN AEC responses after stimulation with a viral mimic (poly I:C). Our RNAseq data showed that infants in the high TSLP group had significant baseline upregulation of IFN signature genes (e.g., IFIT2, IFI6, MX1) and pro-inflammatory chemokine genes prior to viral mimic stimulation. Infants in the high TSLP group also showed a baseline AEC pro-inflammatory state characterized by increased production of all the chemokines assayed (e.g., CXCL10, CXCL8) in the absence of viral stimuli. Conclusion: High TSLP responses in the human infant airways are associated with pre-activated airway epithelial IFN antiviral immunity and increased baseline AEC production of pro-inflammatory chemokines. These findings present a new paradigm underlying the high production of TSLP in the human infant airway epithelium and shed light on the pathogenesis of viral respiratory illnesses during early infancy and beyond childhood.

overall_design: To investigate the immune response initiated by TSLP, in infants, before and after introduction of viral mimic (Poly I:C; PIC), differential expression was performed between high and low TSLP groups at no treatment (NT), 3 hr PIC and 24 hour PIC treatment respectively.

first: Allison

last: Welham

first: Elizabeth

last: Chorvinsky

first: Surajit

last: Bhattacharya

first: Bethelehem

middle: S

last: Bera

first: Kyle

last: Salka

first: Jared

last: Winestock

first: Xilie

middle: X

last: Chen

first: Geovanny

middle: F

last: Perez

first: Dinesh

middle: K

last: Pillai

first: Maria

middle: J

last: Gutierrez

first: Hiroki

last: Morizono

first: Jyoti

last: Jaiswal

first: Gustavo

last: Nino


title: MCL1 inhibition targets Myeloid Derived Suppressors Cells, promotes antitumor immunity and enhances the efficacy of immune checkpoint blockade

city: Aurora

first: Richard

last: Tobin

state: Colorado

institute: University of Colorado Anschutz Medical Campus

summary: Immune checkpoint inhibitors (ICIs) are now the first line treatment for patients with advanced melanoma. Despite promising clinical results, many patients fail to respond to these therapies. BH3 mimetics, a novel class of small molecule inhibitors that bind and inhibit anti-apoptotic members of the BCL2 family proteins such as BCL2 or MCL1, have been very successful in treating hematologic malignancies. However, there are limited studies on the immunomodulatory role of the BH3 mimetics. Several factors contribute to ICI resistance including myeloid-derived suppressor cells (MDSCs) that exert immunosuppressive effects through direct and indirect inhibition of antitumor immunity. Thus, targeting MDSCs to enhance antitumor immunity has the potential to enhance the efficacy of ICIs. In this study, we show that the MCL1 inhibitor S64315 reduces melanoma tumor growth in an immune cell dependent manner in mice. Specifically, S64315 enhances antitumor immunity by reducing MDSC frequency and by promoting the activity of CD8+ T cells. Additionally, human MDSCs are 10 times more sensitive to S64315 than cutaneous melanoma lines. Further, we found that a higher expression of MCL1 is associated with poor survival for patients treated with anti-PD-1. Finally, combining S64315 and anti-PD-1 significantly slowed tumor growth compared to either agent alone. Together, this proof-of-concept study demonstrates the potential of combining MCL1 inhibitor with anti-PD-1 in the treatment of melanoma. It justifies the further development of next generation MCL1 inhibitors to improve efficacy of ICIs in treating malignant melanoma.

overall_design: Myeloid cells were isolated from melanoma tumors by FACS on Live/CD3-/CD19-/CD56-/CD11b+ cells, single cell libraies were prepared using the 10X genomics chromium system using the 10x 3' chemistry (library) and sequenced on novaseq 6000.

first: Yiqun

last: Shellman

first: Tonya

last: Brunetti

first: Martin

last: McCarter


title: Tissue-Resident Memory T Cells in Human Kidney Transplants have alloreactive potential

city: Rotterdam

first: Hector

last: Tejeda Mora

address: Doctor Molewaterplein 40

department: Developmental Biology

country: Netherlands

institute: Erasmus MC

summary: The extent to which tissue-resident memory T (TRM) cells in transplanted organs possess alloreactivity is uncertain. This study investigates the alloreactive potential of TRM cells in kidney explants from four patients who experienced severe acute rejection leading to graft loss. Alloreactive T-cell receptors (TCRs) clones were identified in pre-transplant blood samples through mixed lymphocyte reactions, followed by single-cell RNA and TCR sequencing of the proliferated recipient T cells. Subsequently, these TCR clones were traced in the TRM cells of kidney explants, which were also subjected to single-cell RNA and TCR sequencing. The proportion of TRM cells expressing an alloreactive TCR in the four kidney explants varied from 0% to 9%. Notably, these alloreactive TCRs were predominantly found among CD4+ and CD8+ TRM cells with an effector phenotype. Intriguingly, alloreactive clones were present not only in recipient-derived TRM cells but also in donor-derived TRM cells, constituting up to 4% of the donor population, suggesting the presence of self-reactive TRM cells. Overall, our study demonstrates that  T cells with alloreactive potential present in the peripheral blood prior to transplantation can infiltrate the kidney transplant and adopt a TRM phenotype.

overall_design: Mixed lymphocyte reactions (MLRs) were performed to identify alloreactive T cells. In brief, frozen donor splenocytes and recipient PBMCs were thawed on the day of the experiment. Donor splenocytes were labeled with CellTrace™ Violet Cell Proliferation dye (VPD; Thermo Fisher Scientific, Waltham, MA, USA) and subsequently exposed to gamma irradiation (40 Gy). Recipient PBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE; Thermo Fisher Scientific). Subsequently, irradiated VPD-labeled donor cells and CFSE-labeled recipient cells were cocultured for 6 days. After 6 days of coculture, cells were harvested and stained with 7-AAD (BD Biosciences, Franklin Lakes, NJ, USA) and CD3 (Biolegend, San Diego, CA, USA). Viable CD3+Violet-lowCFSE-low cells were then sorted using a FACS Aria II cell sorter (BD Biosciences). To verify the purity of the gated population, a fraction of the sorted population was subjected to flow cytometry analysis. This assessment demonstrated that all samples had a purity level above 97%.

first: Eric

middle: M

last: Bindels

first: Daphne

last: Hullegie-Peelen


title: Exchange of subtelomeric regions between chromosomes 4q and 10q: a novel way to revert the FSHD genotype and phenotype

city: Villejuif

first: Anna

last: Schwager

state: Please Select

address: 114 Rue Edouard Vaillant

country: France

institute: Institut Gustave Roussy

summary: In this study we used the CRISPR-Cas9 technique to induce the targeted 4q35;10q26 translocation in FSHD myoblasts. We were able to demonstrate that t(4;10)(q35;q26) can generate recombinant genotypes which revert the pathological phenotype of the FSHD cells.

overall_design: RNA-seq was performed for the control (AB1190) and FSHD (AB1080) cell lines, as well as the translocation clones with the improved phenotype (AB1080-T5, AB1080-T6). The cells were collected at the myoblasts stage and at the stage of myotubes after four full days of differentiation. For the non-differentiated samples, 5 × 10^5 cells were collected and subjected to the RNA extraction right away. For the differentiated samples,  5 × 10^5 cells were seeded to 6-well plates to obtain ~90% confluency on the next day, when the differentiation was induced by changing the proliferation medium to the differentiation medium with low serum content (98% DMEM (Sigma-Aldrich #D0822), 2% FBS (Life technology #10270), 1 % penicillin– streptomycin (Gibco #15140-122)). On the 5th day of differentiation, the myotubes were detached with Trypsin-EDTA (Sigma-Aldrich #T2601) and subjected to the RNA-extraction. Total RNA was isolated using the Nucleospin RNA isolation kit (Macherey–Nagel #740955). All samples were prepared in triplicates. mRNA libraries were prepared using Novogene NGS RNA Library Prep Set (PT042) and sequenced on the Illumina NovaSeq 6000 system.


title: CHD7 and SOX2 Act in a Common Gene Regulatory Network During Mammalian Semicircular Canal and Cochlear Development (CUT&Tag)

city: Ann Arbor

first: Donna

last: Martin

address: 1150 Medical Center Dr

institute: University of Michigan

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Inner ear morphogenesis requires tightly regulated epigenetic and transcriptional control of gene expression. CHD7, an ATP-dependent chromodomain helicase DNA-binding protein, and SOX2, an SRY-related HMG box pioneer transcription factor, are known to contribute to vestibular and auditory system development, but their genetic interactions in the ear have not been explored. Here, we analyzed inner ear development and the transcriptional regulatory landscapes in mice with variable dosage of Chd7 and/or Sox2. We show that combined haploinsufficiency for Chd7 and Sox2 results in reduced otic cell proliferation, severe malformations of semicircular canals, and shortened cochlea with ectopic hair cells. Examination of mice with conditional, inducible Chd7 loss by Sox2CreER reveals a critical period (~E9.5) of susceptibility in the inner ear to combined Chd7 and Sox2 loss. Data from genome-wide RNA-sequencing and CUT&Tag studies in the otocyst show that CHD7 regulates Sox2 expression and acts early in a gene regulatory network to control expression of key otic patterning genes, including Pax2 and Otx2. CHD7 and SOX2 directly bind independently and cooperatively at transcription start sites and enhancers to regulate otic progenitor cell proliferation and function. Together, our findings reveal essential roles for Chd7 and Sox2 in the early inner ear and may be applicable for CHD7 and SOX2 related syndromic and other forms of hearing or balance disorders.

overall_design: CUT&Tag for CHD7, SOX2 and no primary antibody control on cells from microdissected E10.5 microdissected otocysts.

first: Jingxia

last: Gao

first: Jennifer

last: Skidmore

first: Jelka

last: Cimerman

first: K

middle: E

last: Ritter

first: Jingyun

last: Qiu

first: Lindsey

middle: Q

last: Wilson

first: Yehoash

last: Raphael

first: Kelvin

middle: Y

last: Kwan


title: CHD7 and SOX2 Act in a Common Gene Regulatory Network During Mammalian Semicircular Canal and Cochlear Development (RNA-Seq)

overall_design:
To investigate how Chd7 and Sox2 cooperatively regulate the downstream gene expression in the mammalian inner ear development, we dissected otocysts from E10.5 embryos from timed pregnancies between Chd7Gt/+;Sox2+/+ males and Chd7+/+;Sox2CreER/+ females.
We then performed bulk RNA sequencing on E10.5 otocysts from all four genotypes (WT, Sox2CreER/+ , Chd7Gt/+, and Chd7Gt/+;Sox2CreER/+).
Differential expressed genes were analyzed from RNA-seq data.


title: Initiation of a ZAKa-dependent Ribotoxic Stress Response by the Innate Immunity Endoribonuclease RNase L

city: Cleveland

first: JIAJIA

last: XI

state: OH

address: 9620 Carnegie Avenue

department: Cancer Biology

institute: Cleveland Clinic

summary: RNase L is a regulated endoribonuclease in higher vertebrates that functions in antiviral innate immunity. Interferons induce OAS enzymes that sense double-stranded RNA of viral origin leading to synthesis of 2’,5’-oligoadenylate (2-5A) activators of RNase L. However, it is unknown precisely how RNase L modulates host transcriptome through its endoribonuclease activity. To isolate effects of RNase L from other effects of double-stranded RNA or virus, 2-5A was directly introduced into cells.  Here we report that RNase L activation by 2-5A causes a ribotoxic stress response that requires the ribosome-associated MAP3K, ZAK. Subsequently, the stress-activated protein kinases (SAPK) JNK and p38 are phosphorylated. RNase L activation profoundly altered the transcriptome by widespread depletion of mRNAs associated with different cellular functions, but also by SAPK-dependent induction of inflammatory genes.  Our findings show that 2-5A is a ribotoxic stressor that causes RNA damage through RNase L triggering a ZAK kinase cascade leading to proinflammatory signaling and apoptosis.

overall_design: To examine the impact of 2-5A induced RNase L activity on transcriptome, the WT or RNaseLKO bone-marrow-derived macrophages will be mock transfected or transfected with 2-5A or its analog A3; 2 hours after transfection, the cells were harvested and the total mRNA were purified for total RNA sequencing; the mRNA from two sets of experiments were sequenced;the RNA-seq data were then analysed for the gene expression profiling; the gene expression profiling between Mock transfected and 2-5A/A3 transfected cells were compared as well as the difference between WT and RNaseLKO cells were analysed.

first: Jiajia

last: Xi

first: Robert

middle: H

last: Silverman

first: Adrian

last: Reich


title: Hypoxia induces transcriptomic changes in bone marrow cells leading to increased erythropoiesis [TFPI]

city: Hangzhou

first: Jun-Kai

last: Ma

address: Yuhangtang road No.866

country: China

institute: Zhejiang University

summary: Erythropoiesis takes place mostly in bone marrow and ends in blood. Previous studies have shown that hypoxia has direct effects on the bone marrow, which could promotes erythropoiesis by modulating erythroid progenitor maturation. However, how bone marrow microenvironment participates in erythropoiesis under hypoxia still need further clarification. In this study, we analyzed transcriptional changes of bone marrow cells of mice exposed to 5000 m altitude hypoxia for 1 week. We profiled the top 20 up-regulated secreted factor genes after treatment with hypoxia, and found TFPI was one of the secreted factor genes with the highest expression.

overall_design: Bone marrow cells were obtained from mice exposed to 5000 m hypoxia for 1 week.

first: Xin

last: Lu

first: Lin

middle: L

last: Feng

first: Jun


title: Early measurable residual disease dynamics and B cell receptor reconstitution following venetoclax and obinutuzumab in chronic lymphocytic leukemia

first: Paul

last: Hengeveld

address: Dr Molewaterplein 40

department: Immunology

summary: In chronic lymphocytic leukemia, measurable residual disease (MRD) depth following fixed-duration treatment is associated with progression-free survival (PFS). Often, MRD is reported as a static parameter, but successive MRD measurements may more accurately capture MRD dynamics and reveal valuable prognostic information. We used our recently developed IGHV leader-based NGS assay to measure MRD at four timepoints in 60 patients treated in the HOVON-139/GIVE trial, in which previously untreated patients received one year induction treatment with obinutuzumab and venetoclax, followed by randomization to consolidation treatment with venetoclax or MRD-conditional consolidation. Induction treatment resulted in MRD undetectability (<10-5) in 93% of assessed patients, with 85% and 74% of patients retaining MRD undetectability at six and twelve months after randomization. Uniquely, we measured MRD at a very early timepoint, during the fourth week of venetoclax ramp-up after obinutuzumab pre-induction. Here, MRD levels showed large interindividual variation, and, importantly, were associated with the probability of achieving MRD undetectability after induction treatment, MRD conversion after randomization and PFS. Additionally, the IGHV leader-based NGS assay allows characterization of the healthy background IGHV repertoire. Of note, venetoclax consolidation treatment, compared to no consolidation treatment, significantly impaired polyclonal IGHV repertoire reconstitution after induction treatment with venetoclax and obinituzumab.

overall_design: B-cell receptor immunoglobulin heavy chain gene repertoire sequencing of post-treatment samples from CLL patients enrolled in the HOVON139/GIVE trial

first: A

middle: W

last: Langerak

first: P


title: Cerebral organoids display dynamic clonal growth with lineage replenishment

city: Vienna

first: Simon

middle: Emanuel

last: Haendeler

address: Dr. Bohr Gasse 9

department: Max Perutz Labs

country: Austria

institute: University of Vienna

summary: During development tissue stem cells expand by symmetrical divisions while asymmetric divisions self-renew a tissue stem cell and produce differentiating cell types that assemble into functional organs. In brain development this process is variable for individual neural stem cells, resulting in differentially sized stem cell clones, but it is unclear how overall brain size is reproducibly generated during neurogenesis. Imaging-based lineage tracing allows for lineage analysis at high cellular resolution but systematic approaches to analyze clonal relationships in an entire tissue are currently lacking. Here we implement whole-tissue lineage tracing by genomic DNA barcoding in 3D human cerebral organoids and show that individual stem cell clones produce progeny on a vastly variable scale. We find that symmetrically dividing cells in a subpopulation of lineages continuously resides within the developing human tissue and drive the variable lineage size distribution. We show that stem cell output is tunable to tissue demands by chemical ablation or genetic fate restriction in chimeric organoids in which perturbed organoid development is completely compensated for by unaffected lineages. This data reveals adaptive plasticity of stem cell populations in developing human brain tissue dependent on tissue needs to ensure normal development.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Barcode-based Lineagetracing to measure the lineage size distribution in brain organoids, chimeric WT/KO brain organoids, chimeric puro-resistent/puro-susceptible brain organoids, 2D cell cultures and mouse. Chimeric knockout conditions are WT/WT, WT/KO-ASPM, WT/KO-PAX6 and WT/KO-TP53 with each 50/50 mixing conditions. Puromycin was performed in 90/10 and 80/20 (susceptible/resistent) mixing conditions, with and without puromycin administration. Two 2D cell cultures were analyzed: 2D stem cell culture and 2D cell culture with neural induction. Mouse samples were seperated by left, right and mid brain and sequenced seperately. All samples are available as timecourse. Organoid samples and 2D cell culture with neural induction were measured after 0, 1, 3, 6, 9, 11, 13, 16, 21, 25, 32, 42 days. 2D stem cell culture was measured after 7, 14, 21, 28, 35, 42 days. All mouse samples were measured on the same day, but the three injection timepoints E9.5, E10.5 and E13.5 were measured. All day 0 samples are generally available in 6 replicates and all other samples in 3 replicates.

first: Dominik

last: Lindenhofer

first: Christopher

last: Esk

first: Florian

last: Pflug

first: Eline

middle: G

last: van de Ven

first: Daniel

last: Reumann

first: Arndt

last: von Haeseler


title: TFPI from Erythroblasts Drives Heme Production in Central Macrophages Promoting Erythropoiesis in Polycythemia Vera

summary: Erythropoietic homeostasis is coordinated by erythroblast development and challenged by a number of genetic diseases including polycythemia vera. Erythroblasts and central macrophages form erythroblastic islands to provide a specific environment for erythropoiesis. However, how central macrophages interplay with erythroblasts during erythropoiesis remains to be further clarified. In this study, we found that erythroid-specific TFPI knockout decreased the number of erythroblasts under both steady-state and stress conditions, and the function of TFPI in erythropoiesis was mediated by macrophages. TFPI affects the downstream heme synthesis pathway of central macrophages, as shown by RNA sequencing analysis, and the deletion of TFPI reduced the heme content of macrophages by inhibiting ferrochelatase (Fech) expression. Our results show that TFPI plays an important role in the regulation of erythropoiesis and reveal a new mechanism of interplay between erythroblasts and macrophages. TFPI will also provide a new potential treatment strategy for polycythemia vera.

overall_design: Central macrophages were sorted from TFPIf/f mice or TFPIf/f;EpoR mice.


title: Effect of depletion of HIF-1α, FACT or RNF20/40 on gene expression in hypoxia of MCF7 breast cancer cells

city: Baltimore

first: YONGKANG

last: YANG

state: Maryland

address: Room 673, MRB, 725 North Broadway

department: ICE

institute: Johns Hopkins University

summary:
To investigate how HIF-1 recruits FACT and RNF20/40 to mediate histone ubiquitination and facilitate transcription of target genes, we established stable cell lines in which HIF-1, FACT or RNF20/40 has been knocked down by shRNA.
We performed gene expression profiling analysis using data obtained from RNA-seq of 4 different cells at 20% and 1% oxygen condition.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for MCF7 subclone of NTC, HIF-1α, FACT and RNF20/40 knockdown that were exposed in 20% or 1% oxygen condition for 24 hours.

first: Yajing

last: Lyu

first: Gregg

last: Semenza


title: Regulation of T helper cell differentiation by the interplay between histone modification and chromatin interaction

city: Bethesda

first: Yaqiang

last: Cao

address: 9000 Rockville Pike

department: System Biology Center

institute: NHLBI

summary: The development and function of the immune system are controlled by temporospatial gene expression programs, which are regulated by cis-regulatory elements, chromatin structure, and trans-acting factors. In this study, we cataloged the dynamic histone modifications and chromatin interactions at regulatory regions during T helper (Th) cell differentiation. Our data revealed that the H3K4me1 landscape established by MLL4 in naive CD4+ T cells is critical for restructuring the regulatory interaction network and orchestrating gene expression during the early phase of Th differentiation. GATA3 plays a crucial role in further configuring H3K4me1 modification and the chromatin interaction network during Th2 differentiation. Furthermore, we demonstrated that HSS3-anchored chromatin loops function to restrict the activity of the Th2 locus control region (LCR), thus coordinating the expression of Th2 cytokines. Our results provide insights into the mechanisms of how the interplay between histone modifications, chromatin looping, and trans-acting factors contributes to the differentiation of Th cells.

overall_design: ChIP-seq, RNA-seq and Hi-TrAC were used to profile the epigenetic, transcriptional and chromatin structure dynamics during CD4+  T cell differentiation.

first: Shuai

last: Liu

first: Kairong

last: Cui

first: Chengyu

first: Keji

last: Zhao


title: RNA-seq of miR-375 overexpression in prostate cancer cells

city: Changsha

first: Dingxiao

last: Zhang

state: Hunan

address: Yuelushan

department: School of BioMedical Sciences

institute: Hunan University

summary: Prostate cancer (PCa) is the most frequently diagnosed malignancy in men worldwide. miRNAs play nonnegligible function in PCa progression. Although several groups studied the effect of miR-375 on PCa phenotypes, some controversial findings emerged. Here, we seek to explore the effect of miR-375 on transcriptome profile in both AR+ LNCaP and AR- PC3 cells via RNA-seq.

overall_design: miR-375 overexpression versus control in two prostate cancer cell lines, two replicates for each group.

first: Cheng

last: Zou

first: Mengjie

last: Chen


title: Transcriptomic and proteomic analyses of distinct Arabidopsis organs reveal high PSI-NDH complex accumulation in stems

city: Turku

first: Laura

last: Laihonen

address: Tykistökatu 6 A 6th floor

country: Finland

institute: University of Turku

summary: In addition to leaves, the main site of photosynthetic reactions, active photosynthesis also takes place in stems, siliques and tree trunks. Although non-foliar photosynthesis has a marked effect on plant growth and yield, only limited information on the expression patterns of photosynthesis-related genes and the structure of photosynthetic machinery in different plant organs has been available. Here, we report the results of transcriptomic analysis of various organs of Arabidopsis thaliana and compare the gene expression profiles of young and mature leaves with a special focus on photosynthetic genes. Further, we analyzed the composition and organization of the photosynthetic electron transfer machinery in leaves, stems and green siliques at the protein level using BN-PAGE. RNA-Seq analysis revealed unique gene expression profiles in different plant organs and showed major differences in the expression of photosynthesis-related genes in young as compared to mature rosettes. Gel-based proteomic analysis of the thylakoid protein complex organization further showed that all studied plant organs contain the necessary components of the photosynthetic electron transfer chain. Intriguingly, stems accumulate high amounts of PSI-NDH complex, which has previously been implicated in cyclic electron transfer.

overall_design: Arabidopsis thaliana Col-0 plants were grown on a peat:vermiculite mixture (2:1) in 8 h light/16 h darkness for short day conditions (mature (5 weeks) and young (3 weeks) rosette) and 12 h light/12 h darkness for long day conditions (stems, flowers, green siliques) at photosynthetic photon flux density (PPFD) of 120 µmol m−2 s−1 (Osram Powerstar HQI®-BT 400W/D PRO Daylight) , 50% humidity, and 23°C. Different biological replicates were grown on separate trays at different times. To extract RNA from roots, surface sterilized seeds were plated on half-strength Murashige and Skoog (1/2 MS, Duchefa) plates with 0.8 % plant agar. The plants were grown under the light rhythm of 8 h light/16 h darkness at PPFD of 50 μmol m−2 s −1, 50 % humidity, and 23 °C in a vertical position in an incubator for six weeks. Four biological replicates were conducted for each material. RNA was extracted from 100 mg of each plant material using innuPREP Plant RNA kit (Analytik Jena, Germany) according to manufacturer’s protocol.

first: Esa

last: Tyystjärvi

first: Umanga

last: Ranasinghe


title: Relapse to cocaine-seeking is regulated by medial habenula Nr4a2

city: Irvine

first: Samuel

last: Morabito

state: CA

address: Biological Sciences III

department: Neurobiology and Behavior

institute: University of California, Irvine

summary: After exposure to drugs of abuse, the reward circuit can experience persistent changes that are thought to underlie relapse behavior. These changes can be epigenetic in nature, and here we identify an epigenetic regulator of memory processes, nuclear orphan receptor subfamily4 groupA member2 (NR4A2 aka NURR1), as necessary for relapse to cocaine-seeking behavior.  Nr4a2 is an epigenetically regulated immediate early gene and transcription factor that is highly expressed in the medial habenula (MHb). Despite the well-studied contributions of the MHb to nicotine-associated behaviors, the MHb is not classically included in reward circuits. Therefore, the role of MHb NR4A2 in cocaine-seeking and relapse behavior remains largely unknown. This is a key open question as NR4A2 is a powerful regulator of memory processes dependent on epigenetic mechanisms. In this study, we examined the role of MHb NR4A2 in cued reinstatement of cocaine self-administration in mice, and found that over-expressing a dominant negative form of Nr4a2 (Nurr2c) in the MHb results in a near complete block of   relapse-like behavior. We used single-nucleus transcriptomics to characterize the molecular cascade following the manipulation of Nr4a2, revealing changes in transcriptional networks related to addiction, neuroplasticity, and glutamatergic signaling. Together, these results place the MHb in the reward circuit as a pivotal regulator of relapse behavior and demonstrate the importance of NR4A2 as a key mechanism driving relapse behavior in the MHb.

overall_design: Adult male heterozygous ChAT-Cre mice were used to contain AAV expression MHb cholinergic neurons, allowing us to increase the expression of the Nurr2c isoform of Nr4a2.

first: Jessica

last: Childs

first: Vivek

last: Swarup

first: Marcelo

last: Wood


title: Translation-dependent and independent mRNA decay occur through mutually exclusive pathways that are defined by ribosome density during T Cell activation [RNA-Seq]

city: Evry-Courcouronnes

first: Emmanuel

last: Labaronne

address: Genopôle Campus  4 rue Pierre Fontaine - Pépinière du Génopôle

institute: ADLIN science

summary: Post-transcriptional control is crucial for regulating protein expression, both basally and in response to extracellular cues. Proper signal transduction requires tight control of both response induction and termination. One way protein expression might be attenuated is by targeting mRNAs to translation-dependent degradation (TDD), thus making any increase in protein expression self-limiting. However, the extent to which TDD is a general mechanism for limiting protein expression is currently unknown. Here we describe a comprehensive analysis of basal and signal-induced TDD in mouse primary CD4 T cells. Our data indicate that most cellular transcripts are decayed to some extent in a translation-dependent manner, both in resting and activated cells. Analysis of transcript features revealed that 3’UTR length and ribosome density are major determinants of the magnitude of TDD as well as GC content and amino acid identity. Consistently, upon T cell activation, all transcripts that undergo changes in ribosome density display a corresponding change in their level of TDD. Surprisingly, the amplitude of translation-independent mRNA decay appears as a mirror image of TDD. Moreover, translation-independent decay also responds to changes in ribosome density upon T cell activation but in the opposite direction to those observed for TDD. Our data demonstrate a strong interconnection between mRNA translation and decay in mammalian cells. Furthermore they indicate that ribosome loading is a major determinant of the pathway by which transcripts are degraded within cells

overall_design: Primary macrophages and T-cell from mice were activated or not and incubated with an transcription (5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole or Triptolide) and translation (Cycloheximide or Harringtonine) during 0, 1 or 3h to monitor translation dependent and indenpendent decay of mRNA

first: Blandine

middle: C

last: Mercier

first: David

last: Cluet

first: Alicia

last: Bicknell

first: Antoine

last: Corbin

last: Guiguettaz

first: Fabien

last: Aube

first: Laurent

last: Modolo

first: Didier

last: Auboeuf

first: Melissa

last: Moore

first: Emiliano

middle: P

last: Ricci


title: Translation-dependent and independent mRNA decay occur through mutually exclusive pathways that are defined by ribosome density during T Cell activation [Ribo-Seq]

first: Laura


title: Functional analysis and transcriptome profile of meninges and skin fibroblasts from human aged donors

status: Public on Feb 29 2024

city: Pavia

first: Matteo

last: Bordoni

address: Via Mondino 2

country: Italy

institute: IRCCS Mondino Foundation

summary: The central nervous system (CNS) is surrounded by three membranes called meninges. Specialized fibroblasts, originating from the mesoderm and neural crest, primarily populate the meninges and serve as a binding agent. Our goal was to compare fibroblasts from meninges and skin obtained from the same human-aged donors, exploring their molecular and cellular characteristics related to CNS functions. We isolated meningeal fibroblasts (MFs) and skin fibroblasts (SFs) from brain donors. A functional analysis was performed measuring cell appearance, metabolic activity, and cellular orientation. We examined fibronectin, serpin H1, β-III-tubulin, and nestin through qPCR and immunofluorescence. A whole transcriptome analysis was also performed to characterize the gene expression of MFs and SFs. MFs appeared more rapidly post-tissue processing, while SFs have an elevated cellular metabolism and a well-defined cellular orientation. The four markers were mostly similar between the MFs and SFs, except for nestin, more expressed in MFs. Transcriptome analysis reveals significant differences, particularly in cyclic adenosine monophosphate (cAMP) metabolism and response to forskolin, both of which are upregulated in MFs. This study highlights MFs' unique characteristics, such as the timing of appearance, metabolic activity, and gene expression patterns, particularly in cAMP metabolism and response to forskolin. These findings contribute to a deeper understanding of non-neuronal cells' involvement in CNS activities and potentially open avenues for therapeutic exploration.

overall_design: We isolated meningeal fibroblasts (MFs) and skin fibroblasts (SFs) from brain donors. We used RNA extracted from fibroblasts.

first: Valentina

last: Fantini

first: Riccardo

middle: R

last: Ferrari

first: Eleonora

last: Spampinato

first: Cecilia

last: Pandini

first: Annalisa

last: Davin

last: Medici

first: Stella

last: Gagliardi

first: Antonio

last: Guaita

first: Orietta

last: Pansarasa

first: Cristina

last: Cereda

first: Tino

last: Poloni


title: Translation-dependent and independent mRNA decay occur through mutually exclusive pathways that are defined by ribosome density during T Cell activation [PAS-Seq]

first: Emmanuel

last: Labaronne

first: Blandine

last: Mercier

first: David

last: Cluet

first: Alicia

last: Bicknell

first: Antoine

last: Corbin

last: Guiguettaz

first: Fabien

last: Aube


title: FOXO1 is a master regulator of CAR T memory programming [RNA_FOXO1_DBO]

city: Philadelphia

first: Katherine

middle: Paige

last: Mueller

state: Pennsylvania

address: 3501 Civic Center Blvd

department: Pediatrics

institute: Children's Hospital of Philadelphia

summary: Poor CAR T persistence limits CAR T cell therapies for B cell malignancies and solid tumors1,2. The expression of memory-associated genes such as TCF7 (protein name TCF1) is linked to response and long-term persistence in patients3–7, thereby implicating memory programs in therapeutic efficacy. Here, we demonstrate that the pioneer transcription factor, FOXO1, is responsible for promoting memory programs and restraining exhaustion in human CAR T cells. Pharmacologic inhibition or gene editing of endogenous FOXO1 in human CAR T cells diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype, and impaired antitumor activity in vitro and in vivo. FOXO1 overexpression induced a gene expression program consistent with T cell memory and increased chromatin accessibility at FOXO1 binding motifs. FOXO1-overexpressing cells retained function, memory potential, and metabolic fitness during settings of chronic stimulation and exhibited enhanced persistence and antitumor activity in vivo. In contrast, TCF1 overexpression failed to enforce canonical memory programs or enhance CAR T cell potency. Importantly, endogenous FOXO1 activity correlated with CAR T and TIL responses in patients, underscoring its clinical relevance in cancer immunotherapy. Our results demonstrate that memory reprogramming through FOXO1 can enhance the persistence and potency of human CAR T cells and highlights the utility of pioneer factors, which bind condensed chromatin and induce local epigenetic remodeling, for optimizing therapeutic T cell states.

overall_design: Transcriptomic study of HA.28z CAR T cells with FOXO1 (wild type), FOXO1-DBDmut (K245>A, K248>A), or NGFR empty vector transcription factors over expressed to evaluate transcriptional activity of a FOXO1 mutant that has lower affinity for DNA binding

first: Alexander

last: Doan

first: Andy

last: Chen

first: Geoffrey

last: Rouin

first: Evan

last: Weber


title: FOXO1 is a master regulator of CAR T memory programming [ATAC_FOXO1_OE]

overall_design: Epigenetic study of CD19.28z or HA.28z CAR T cells overexpressing TCF1, FOXO1, or NGFR (empty vector control) transcription factors


title: Applying a non-GMO breeding approach with an identified natural variation to reduce food allergen Len c3 in Lens culinaris seeds

city: Saskatoon

first: Ioanni

last: Mavraganis

address: 110 Gymnasium Place

country: Canada

institute: National Research Council

summary: Lentils (Lens culinaris) are produced in diverse agroecological regions and are consumed as one of the most important food legumes at world-wide. Lentils possess a nutritional profile from a human health perspective that is not only nutrient-dense but also offers a better balance between protein and carbohydrates. However, lentil causes food allergy which has been a significant concern due to increased consumption in parts of the world. Len c3, a none-specific Lipid Transfer Protein (LTP), was identified as one of the allergens in lentil seeds. In this study, we firstly identified the Len c3 encoding gene LcLTP3b via peptide sequence blasting. We then focused on screening natural variations to search for lentil germplasms that harbors natural mutated allergen-encoding genes. A natural variation 11 was identified with mutations at LcLTP3b and low accumulation of vicilin through genomic-assisted approaches. Furthermore, we generated a pool of lentil germplasms with Len c3-free background through crossing the identified natural variation 11 with two lentil cultivars, CDC Redmoon and CDC Gold. These Len c3-free lentil germplasms can be used as a breeding resource targeting at reducing allergen risk in lentil consumption.

overall_design: Total RNA was extracted from dissected embryos and seed coats containing endosperms of CDC Redmoon, CDC Gold, and M11, using a RNeasy plant mini kit (Qiagen, Germany) according to the manufacturer’s instruction. For transcriptome sequencing, cDNA libraries were constructed from the isolated RNA samples by using a TruSeq RNA Sample Preparation kit v2 (Illumina). The cDNA libraries were used for RNAseq. RNAseq were conducted on an Illumina NOVAseq 6000 pair-end sequencing.


title: FOXO1 is a master regulator of CAR T memory programming [RNA_Dual_OE-KO]

overall_design: Transcriptomic study of CD19.28z CAR T cells with TCF1 (gene name Tcf7) knocked out via CRISPR/Cas9 or AAVS1 sgRNA controls in the setting of FOXO1 or NGFR (empty vector) transcription factor overexpression


title: Skin & Pancreas chronic inflammation versus Normal Skin & Pancreas

city: Charleston

first: Heehwa

last: Son

state: MA

address: 149 13 street

institute: MASSACHUSETTS GENERAL HOSPITAL

summary: To find which signaling is activated in chronic inflammation, we tried to induce chronic skin and pancreas inflammation compared to normal skin and pancreas.

overall_design: To induce skin inflammation, mice were treated with DNFB (contact allergen) and this group was compared to acetone-treated mice (as a control). To induce pancreas inflammation, mice were injected with caerulein and this group was compared to PBS-injected mice (as a control).

first: Jongho

last: Park

first: Shadmehr

last: Demehri


title: FOXO1 is a master regulator of CAR T memory programming [RNA_FOXO1_OE]

overall_design: Transcriptomic study of HA.28z or CD19.28z CAR T cells overexpressing empty vector (NGFR) or FOXO1 or TCF1 transcription factors, 3 independent donors per condition


title: FOXO1 is a master regulator of CAR T memory programming [RNA_FOXO1_KO]

overall_design: Transcriptomic study of CD19.BBz CAR T cells with CRISPR/Cas9-mediated FOXO1 knockout vs. AAVS1 negative sgRNA control.


title: Hepatic follistatin increases basal metabolic rate and attenuates diet-induced obesity during hepatic insulin resistance

city: Boston

first: rongya

last: tao

state: Ma

address: 300 longwood ave

institute: Boston children's hospital

summary: Insulin resistance not compensated by secretion reduces energy storage, but little is known about its effect upon energy expenditure (EE). Insulin receptor substrates Irs1 and Irs2 mediate signaling in all tissues, resulting in the inhibition of FoxO transcription factors. We found that hepatic disruption of Irs1 and Irs2 (LDKO mice) attenuated high-fat diet (HFD)-induced obesity and increased whole-body EE in a FoxO1-dependent manner. Hepatic disruption of Fst (follistatin), a FoxO1-regulated hepatokine, normalized EE in LDKO mice and restored adipose mass during HFD consumption. Moreover, hepatic Fst disruption alone increased fat mass accumulation, whereas hepatic overexpression of Fst attenuated high HFD-induced obesity. Excess circulating Fst in overexpressing mice neutralized Mstn (myostatin), activating mTORC1-promoted pathways of nutrient uptake and EE in skeletal muscle. Similar to Fst overexpression, direct activation of muscle mTORC1 also reduced adipose mass. We conclude that Fst-promoted EE in muscle attenuates obesity during hepatic insulin resistance.

overall_design: We generated the LDKO (Irs1L/L:Irs2L/L:CreAlb)  as previously described. We obtained FstL/L mice from Dr. Martin Matzuk (Baylor College of Medicine, Houston, TX). FstL/L mice were designed with loxP sites inserted before exon 2 and after exon 5 of the Fst gene. We generated LTKOFstKO mice by crossing FstL/L mice with LDKO mice.Expression of selected genes by RNAseq in skeletal muscle from Cntr, LDKO and LTKOFstL/L mice fed chow between 5 and 16 weeks.

first: Rongya

last: Tao

first: Morris

last: White

first: Kyle

last: Copps

first: Oliver

last: Stöhr


title: Small RNA sequencing for leiomyosarcoma cells and their extracellular vesicles

city: Nagoya

first: Kosuke

last: Yoshida

state: Aichi

address: 65 Tsuruma-cho

country: Japan

institute: Nagoya University

type: Non-coding RNA profiling by high throughput sequencing

summary: Uterine leiomyosarcoma is a rare malignant tumor and has a poor prognosis even during its early stages. Extracellular vesicles (EVs) carry cargo, such as microRNAs (miRNAs), which are involved in intercellular communication in the tumor microenvironment and other processes. EVs are classified based on their biogenesis mechanism, and small EVs have a diameter of less than 200 nm, whereas medium/large EVs have a diameter of 200 nm or greater. This study investigated the expression of miRNAs in leiomyosarcoma cells and their EVs.

overall_design: SKN and SK-LMS-1 cells were cultured, and small and medium/large EVs were purified using the ultracentrifuge. Then, total RNA was extracted, and small RNA sequencing was performed.

first: Yukari

last: Nagao

first: Akira

last: Yokoi


title: Bioenergetic mechanisms of alcohol-associated cirrhosis in patient derived hepatocytes

city: Rockville

first: Bani

last: Mukhopadhyay

state: MD

address: 5625 Fishers lane.Rockville.MD, 3S24-

department: LNG

institute: NIH,NIAAA

summary: One in five heavy drinkers develop alcohol-associated cirrhosis. Metabolism, inflammation, signaling, gut microbiome and genetic variations have all been tied to pathogenesis but alcohol-associated cirrhosis (AC) models in rodents are limited by differences between rodent species and humans in genetic background and bioenergetics. Here, we used iPSC derived hepatocytes (iHLCs) as a tool to understand mechanisms of AC injury, hypothesizing that iHLC’s derived from patients would preserve genetic and bioenergetic differences leading to AC, and as compared to iHLC’s from patients free of liver disease.   Bioenergetically, AC iHLCs had lower spare capacity for mitochondrial respiration and produced less ATP. Metabolic capacities of AC iHLC mitochondria for glutamine and fatty acids were reduced. Together with genome wide association (GWAS) studies, these metabolic and histologic profiles of AC derived iHLCs suggest that differences in mitochondrial oxidative phosphorylation and lipid droplet formation predispose to AC.

overall_design: Hepatocyte was derived from patient derived iPSC

first: David

last: Goldman


title: FOXO1 is a master regulator of CAR T memory programming [ATAC_FOXO1]

first: Katherine

last: Mueller

overall_design: Epigenetic study of HA.28z CAR T cells with FOXO1 (wild type), FOXO1-DBDmut (K245>A, K248>A), or NGFR empty vector transcription factors over expressed to evaluate epigenetic remodeling activity of a FOXO1 mutant that has lower affinity for DNA binding

first: Alexander

last: Doan

first: Andy

last: Chen


title: FOXO1 is a master regulator of CAR T memory programming

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

overall_design: Refer to individual Series


title: Identification of a clinically efficacious CAR T cell subset by dynamic multi-dimensional single-cell profiling

city: Houston

first: Navin

last: Varadarajan

state: TX

address: 4726 Calhoun Rd

department: William A Brookshire Chemical and Biomolecular Engineering Department

institute: University of Houston

summary: Adoptive immunotherapy with T cells expressing chimeric antigen receptors (CARs) for B-cell malignancies serves as a model for identifying subsets with superior clinical activity. We profiled the infusion products (IP) of 9 patients with large B-cell lymphoma (LBCL) using scRNA-sequencing to reveal the therapeutic potential of CD19-specific CAR+ T cells. ScRNA-seq demonstrated that T cells from responders were enriched in pathways related to T-cell killing, migration and actin cytoskeleton, and TCR clustering.

overall_design: Using a modified transwell assay, migratory and unmigratory CD19-CD28z-CAR T cells from two healthy donors were analyzed using scRNA sequencing.

first: Melisa

last: Montalvo

first: Irfan

last: Bandey


title: Comparison of iPSC-derived alveolar epithelial cell culture methods  (B2-3 SFTPC-GFP reporter iPSCs) and compound treatment in the alveolar epithelial spheroids (B2-3-dual-42-7 SFTPC-GFP AGER-mCherryHiBiT reporter iPSCs)

city: Kyoto

first: Shimpei

last: Gotoh

address: 53 Kawaharacho, Shogoin, Sakyo-ku

department: Center for iPS cell Research and Application

institute: Kyoto University


title: The iPSC-derived pulmonary epithilial cells in micro-patterned culture plate

summary: We established a micro-patterned respiratory epithelial cell culture system in vitro. In this culture system, various types of lung epithelial cells were identified.

overall_design: Human iPSC-derived lung progenitor cells were seeded onto micro-patterned plates and cultured in "DCIK+3i" (CHIR99021, SB431542 and Y27632) medium or "PAL" medium for 7 or 14 days to induce alveolar or airway epithelial cell colonies, respectively. These cells as well as their parental progenitor cells, were dissociated and subjected to single-cell RNA sequencing (scRNA-Seq) analysis.

first: Atsushi

last: Masui

first: Takuya

last: Yamamoto

first: Satoko

last: Sakurai


title: FOXO1 is a master regulator of CAR T memory programming [RNA_FOXO1_OE_CD4]

overall_design: Transcriptomic study of HA.28z CD4 CAR T cells overexpressing empty vector (NGFR) or FOXO1 or TCF1 transcription factors, 3 independent donors per condition

first: Geoffrey

last: Rouin

first: Evan

last: Weber


title: Serum exosomal miRNA profile in pregnancies with gestational diabetes based on pancreatic β-cell function

city: Mexico City

first: Melissa

last: Razo-Azamar

address: Calz. de los Tenorios 235 Col. Rinconada de las Hadas, Alcaldía Tlalpan, Ciudad de México

department: Centro de Investigación sobre el Envejecimiento

country: Mexico

institute: Instituto Nacional de Medicina Genómica (INMEGEN)

summary:
Pancreatic β-cell function impairment is a key mechanism for developing gestational diabetes mellitus (GDM). Maternal and placental exosomes regulate maternal and placental responses during hyperglycemia. Studies have associated exosomal micro RNAs (miRNAs) with GDM development. To date, no studies have been reported that evaluate the profile of miRNAs present in maternal and placental exosomes in the early stages of gestation from pregnancies that develop GDM.
We used microarrays to assess whether early pregnancy maternal and placental exosomal miRNA profiles vary according to pancreatic β-cell function in women who will develop GDM (preGDM).

overall_design: A prospective nested case-control study was used to identify exosomal miRNAs that vary in early pregnancy stages (<18 weeks of gestation) from women with normoglycemia and those that developed GDM based on their pancreatic β-cell function using HOMA-%β index. Early pregnancy maternal and placental serum exosomes were isolated to obtain miRNA profiles.

first: M

first: B

last: Palacios-González


title: Micro-patterned culture of iPSC-derived airway epithelial cells [airway]

summary: We established a micro-patterned respiratory epithelial cell culture system in vitro. In this culture system, the iPSC-derived lung progenitor cells were differentiated into airway epithelial cells in a position-specific manner.

overall_design: The human iPSC-derived lung progenitor cells were seeded onto micro-patterned plates and cultured for 7 days. Nuclei were stained with Hoechst-33342, and the Edge and Center cell populations of the colony were separated and collected according to the Hoechst intensity. The Hoechst-high intensity population corresponded to the peripheral cells of the colony (Edge cells), and the Hoechst-low intensity population corresponded to the central cells of the colony (Center cells).


title: Comparison of iPSC-derived alveolar epithelial cell culture methods

summary: We established a novel alveolar epithelial culture method, called "On-Gel" culture. To characterize the "On-Gel" culture, we compared each transcriptome of the cultured cells in "On-Gel", fibroblast dependent-alveolar organoids (FD-AO) and fibroblast-free alveolar organoids (FF-AO) and their progenitor cells (CPMhigh Lung Progenitors).

overall_design: Human iPSCs were differentiated into lung progenitor cells. After isolated with anti-Carboxypepdidase M (CPM) antibody, CPMhigh Lung Progenitors were co-cultured with human fetal lung fibroblasts (HFLFs) to generate FD-AOs in which surfactant protein C (SPC)-positive cells appeared (Passage 0: P0). SPC-positive cells were isolated by FACS and cultured in each condition for generating FD-AOs, FF-AOs, or "On-Gel" spheroids (P1). EpCAM-positive cells of FD-AOs derived from B2-3 SFTPC-GFP reporter iPSCs were isolated by MACS and the transcriptomes of P1 epithelial cells in each culture method were analyzed.

first: Yuko

last: Ohnishi


title: Compound treatment in the alveolar epithelial spheroids derived from the dual reporter iPSC in "On-Gel" culture.

summary: We established a dual reporter (SFTPC:GFP AGER:mCherry-HiBiT) human iPSC line and the "On-Gel" spheroid culture system suitable for finding the essential signals for alveolar type 2 (AT2) to type 1 (AT1) cell conversion comprehensively. After compound screening, we found that LATS-IN-1 and BAY1125976 synergistically promoted AT1 marker gene expression.

overall_design: Alveolar epithelial spheroids in "On-Gel" culture were treated with DMSO, LATS-IN-1, BAY1125976, and  both of them to investigate the transcriptomes of each condition to find the mechanism of the differentiation from AT2 to AT1 cells.


title: Human iPSC-derived pulmonary epithelial cells infected with SARS-CoV-2 variants in the micro-patterned culture plates [SARS-CoV-2]

summary: We infected iPSC-derived pulmonary epithelial cells in the micro-patterned culture plate with the SARS-CoV-2 variants. Analyses of infected alveolar and airway epithelial cells, respectively, revealed the tropism of the variants.

overall_design: The iPSC-derived pulmonary epithelial cells infected with SARS-CoV-2 variants in the micro-patten culture plate were obtained from the culture plates and each total RNA was collected and subjected to RNA-seq.

first: Kazuo

last: Takayama


title: Transcriptomes of human iPSC-derived alveolar and airway cells in the micro-patterned culture plates and their responses to SARS-CoV-2 variant infection


title: A-MYB substitutes for B-MYB in activating cell cycle genes and in stimulating proliferation

city: Leipzig

first: Robin

last: Kohler

address: Semmelweisstr. 14

department: Medical School

country: Germany

institute: University Leipzig

summary: A-MYB (MYBL1) is a transcription factor with a role in meiosis in spermatocytes. The related B-MYB protein is a key proto-oncogene and a master regulator activating late cell cycle genes. To activate genes, B-MYB forms a complex with MuvB and is recruited indirectly to cell cycle genes homology region (CHR) promoter sites of target genes. Activation through the B-MYB-MuvB (MMB) complex is essential for successful mitosis. Here, we discover that A-MYB has a function in transcriptional regulation of the mitotic cell cycle and can substitute for B-MYB. Knockdown experiments in cells not related to spermatogenesis show that B-MYB loss alone only delays cell cycle progression. Only dual knockdown of B-MYB and A-MYB causes cell cycle arrest. A-MYB can substitute for B-MYB in binding to MuvB. The resulting A-MYB-MuvB complex activates genes through CHR sites. We find that A-MYB activates the same target genes as B-MYB. Many of the corresponding proteins are central regulators of the cell division cycle. In summary, we demonstrate that A-MYB is an activator of the mitotic cell cycle by activating late cell cycle genes.

overall_design: To investigate the function of A-MYB and B-MYB in the mitotic cell cycle, siRNA knockdowns were performed on HCT116 cells as well as U2OS cells. Cells were treated with siRNA targeting MYBL1 (A-MYB), or MYBL2 (B-MYB) as well as a combination of MYBL1 and MYBL2 targeting siRNAs for 48 h. Non-targeting siRNA was used as a negative control. Total RNA expression was analzed by paired-end RNA-seq. Per cell line and sample two biological replicates were generated.

first: Kurt

last: Engeland


title: PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects

city: Newcastle Upon Tyne

first: Majlinda

last: Lako

address: International Centre for Life, Central Parkway

department: Institute of Genetic Medicine and Institute for Ageing

country: United Kingdom

institute: Newcastle University

summary: Mutations in the carboxy terminal of the core spliceosome factor PRPF8 cause autosomal dominant retinitis pigmentosa (RP) 13. Comprehensive cellular, biochemical, and molecular investigations of iPSC-derived retinal organoids, retinal pigment epithelium (RPE) and kidney organoids from four patients carrying the pathogenic PRPF8 RP type 13 c.6926A>C (p.H2309P) heterozygous missense mutation, revealed retinal tissue-specific effects including lower splicing specificity, ciliary abnormalities, altered apical-basal polarity, rod degeneration and loss of photoreceptors. The p.H2309P mutation affected the 5’ splice site recognition by PRPF8 of transcripts encoding ciliary proteins, altered spliceosome kinetics and organisation of nuclear speckles as well as PRPF8 binding to spliceosomal U6 snRNAs and snoRNAs, leading to accumulation of unspliced poly A+ mRNAs specifically in RPE cells and retinal organoids. Together these data provide the most comprehensive characterisation of splicing factor causing RP disease, providing molecular insights into the tissue specificity of pathomechanisms and informing future therapeutic approaches.

overall_design: Control and patient-specific ROs were subjected to single-cell RNA-Seq at day 210 of differentiation.

first: Robert

last: Atkinson

first: Maria

last: Georgiou

first: Chunbo

last: Yang

first: Katarzyne

last: Szymanska

first: Marina

last: MoyaMolina

first: Birthe

last: Hilgen

first: Avril

last: Watson

first: Joseph

last: Collin

first: Elton

last: Rosas de Vasconcelos

first: Albert

last: Lahat

first: Ross

last: Laws

first: Chia

last: Beh

first: Rachel

last: Queen

first: David

last: Elliott

first: Chile

last: Siachisumo

first: Martin

last: McKibbin

first: Chris

last: Inglehearn

first: Henning

last: Urlaub

first: Sushma

last: Nagaraja Grellscheid

first: Colin

last: Johnson

first: Sina

last: Mozaffari-Jovin


title: Micro-patterned culture of iPSC-derived alveolar epithelial cells [alveolar]

first: Shimpei

last: Gotoh

summary: We established a micro-patterned respiratory epithelial cell culture system in vitro. In this culture system, the iPSC-derived lung progenitor cells were differentiated into alveolar epithelial cells in a position-specific manner.

overall_design: The human iPSC-derived lung progenitor cells were seeded onto micro-patterned plates and cultured for 8 or 14 days. Nuclei were stained with Hoechst-33342, and the Edge and Center cell populations of the colony were separated and collected according to the Hoechst intensity. The Hoechst-high intensity population corresponded to the peripheral cells of the colony (Edge cells), and the Hoechst-low intensity population corresponded to the central cells of the colony (Center cells).

first: Atsushi

last: Masui


title: Proteome-scale movements and compartment connectivity during the eukaryotic cell cycle

first: Mary

middle: T

last: Couvillion

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

summary: Cell cycle progression relies on coordinated changes in the composition and subcellular localization of the proteome. By applying two distinct convolutional neural networks on images of millions of live yeast cells, we resolved proteome-level dynamics in both concentration and localization during the cell cycle, with resolution of ~20 subcellular localization classes. We show that a quarter of the proteome displays cell cycle periodicity, with proteins tending to be controlled either at the level of localization or concentration, but not both. Distinct levels of protein regulation are preferentially utilized for different aspects of the cell cycle, with changes in protein concentration being mostly involved in cell cycle control, while changes in protein localization in the biophysical implementation of the cell cycle program. We present a resource for exploring global proteome dynamics during the cell cycle, which will aid in understanding a fundamental biological process at a systems level.

overall_design: To understand how periodicity in protein concentration emerges during the cell cycle, we generated cell cycle-specific ribosome profiling and RNA-seq datasets in duplicate. Briefly, we synchronized cells in G1 phase with alpha factor, and isolated cells at five time-points after release into fresh medium, corresponding to the phases used for in silico synchrony of our proteomics data (G1 Post-Start; S/G2; Metaphase; Anaphase and Telophase; G1 Pre-Start was excluded due to the method of synchrony).

first: Athanasios

last: Litsios

first: Benjamin

last: Grys

first: Oren

middle: Z

last: Kraus

first: Helena

last: Friesen

first: Catherine

last: Ross

first: Myra

last: Masinas

first: Duncan

last: Forster

first: Stefanie

last: Timmermann

first: Maximilian

last: Billmann

first: Chad

last: Myers

first: Nils

last: Johnsson

first: Stirling

last: Churchman

first: Charles

last: Boone

first: Brenda

last: Andrews


title: Transcriptomic profilings of bovine mammary epithelial cells stimulated by ten different essential amino acids

city: Qingdao

first: Lianbin

last: Xu

address: No.700, Changcheng Road

department: College of Animal Science and Technology

institute: Qingdao Agricultural University

summary: In order to investigate the transcriptomic responses of bovine mammary epithelial cells to various essential amino acid administrations

overall_design: Bovine mammary epithelial cells cultured in 6-well plates were first serum-starved overnight and subsequently assigned to 1 of 12 treatment media (n = 6). DMEM-F12 medium is the positive control treatment (POS), while DMEM-F12 medium devoid of all essential amino acids served as the negative control treatment (NEG). 10 treatments were NEG individually supplemented with arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan or valine. Individual essential amino acid were supplemented to achieve concentrations equal to those of POS. After 6-h treatment, all cell samples were taken at the same time, frozen in liquid nitrogen, and stored at -80°C until required.

first: Dengpan

last: Bu

first: Xiuli

last: Li


title: Transcriptome analysis in PHf2 KO C2C12 cells

city: Paris

first: iori

last: sakakibara

address: 22 rue Méchain

institute: Cochin institut

summary: To evaluate the role of Phf2 in C2C12 myotube, we generated Phf2 KO C2C12 cells and analyzed the transcriptome by RNA-sequencing.

overall_design: Examination of the role of Phf2 in C2C12 myotubes.

first: Iori

last: Sakakibara

first: Taku

last: Fukushima


title: The protective role of the HSP90 inhibitor, STA-9090, in lungs of SARS-CoV-2-infected Syrian golden hamsters

city: Berlin

first: Luiz Gustavo

last: Teixeira Alves

address: Hannoversche Strasse 28

institute: MDC Berlin

summary: The emergence of new SARS-CoV-2 variants, capable of escaping the humoral immunity acquired by the available vaccines, together with waning immunity and vaccine hesitancy, challenges the efficacy of the vaccination strategy in fighting COVID-19. Improved therapeutic strategies are urgently needed to better intervene particularly in severe cases of the disease. They should aim at controlling the hyper-inflammatory state generated upon infection, reducing lung tissue pathology, and inhibiting viral replication. Previous research has pointed to a possible role for the chaperone HSP90 in SARS-CoV-2 replication and COVID-19 pathogenesis. Pharmacological intervention through HSP90 inhibitors was shown to be beneficial in the treatment of inflammatory diseases, infections and reducing replication of diverse viruses. In this study, we investigated the effects of the potent HSP90 inhibitor Ganetespib in vitro on alveolar epithelial cells and alveolar macrophages to characterize its effects on cell activation and viral replication. Additionally, the Syrian hamster animal model was used to evaluate its efficacy in controlling systemic inflammation and viral burden after infection in vivo. In vitro, Ganetespib reduced viral replication on alveolar epithelial cells in a dose-dependent manner and lowered significantly the expression of pro-inflammatory genes, in both alveolar epithelial cells and alveolar macrophages. In vivo, although no reduction in viral load was observed, administration of Ganetespib led to an overall improvement of the clinical condition of infected animals, with decreased systemic inflammation, reduced edema formation and lung tissue pathology. Altogether, we show that Ganetespib could be a potential treatment option for moderate and severe cases of COVID-19.

sample_organism:
Homo sapiens
Mesocricetus auratus

platform_organism:
Homo sapiens
Mesocricetus auratus

overall_design: Total RNA sequencing was performed on AECs, alveolar macrophages and blood from hamsters, infected with SARS-CoV-2, to evaluate the effects of the HSP90 inhibitor Ganetespib on cell activation and viral replication. Differentially expressed genes analysis was performed, aiming at genes related to cell activation and inflammation.

first: Luiz

first: Morris

last: Baumgardt

first: Christine

last: Langner

first: Mara

last: Fischer

first: Julia

last: Adler

first: Judith

last: Hoppe

first: Theresa

last: Firsching

first: Guido

last: Mastrobuoni

first: Jenny

last: Grobe

first: Katja

last: Hoenzke

first: Stefan

last: Kempa

first: Achim

middle: D

last: Gruber

first: Andreas

last: Hocke

first: Jakob

last: Trimpert

first: Emanuel

last: Wyler

first: Markus

last: Landthaler


title: Dual antagonism of the endothelin A and angiotensin II type 1 receptors protects from glomerular hypercellularity in a model of IgA nephropathy

city: Birmingham

first: Colin

middle: Robert

last: Reily

state: Alabama

department: Medicine

institute: University of Alabama at Birmingham

summary: IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, is characterized by glomerular deposition of immune complexes (IC) consisting of IgA1 with some O-glycans deficient in galactose (Gd-IgA1) and Gd-IgA1-specific IgG autoantibodies. These IC induce kidney injury, and in the absence of disease-specific therapy, up to 40% of IgAN patients progress to kidney failure. IgA1 with its clustered O-glycans is unique to humans, which hampered development of small-animal models of IgAN. To address this issue, we developed a model wherein engineered IC (EIC) formed from human Gd-IgA1 and recombinant IgG autoantibody are injected into nude mice to induce glomerular injury mimicking human IgAN. Here, we used this animal model to assess the protective effects of sparsentan, a single-molecule dual antagonist of endothelin A receptor (ETAR) and angiotensin II type 1 receptor (AT1R) (DEARA). Oral administration of sparsentan (60 or 120 mg/kg) to mice intravenously injected with EIC attenuated the EIC-induced glomerular hypercellularity.

overall_design: In vitro formed immune complexes were intravenously (i.v.) injected into the lateral tail vein of NCr nude mice, every other day for a total of six injections. Kidneys were harvested the day after last injection for immunohistochemistry and NGS.

first: Zina

last: Moldoveanu

first: Tiziano

last: Pramparo

first: Stacy

last: Hall

first: Zhi-Qiang

last: Huang

first: Terri

last: Rice

first: Lea

last: Novak

first: Radko

last: Komers

first: Celia

last: Jenkinson

first: Jan


title: Prolonged exposure to lung-derived cytokines is associated with activation of microglia in patients with COVID-19

status: Public on Feb 28 2024

city: Chicago

first: Rogan

middle: A

last: Grant

state: IL

address: 303 E Superior St, Simpson Querrey Biomedical Research Center 5-522

department: Pulmonary and Critical Care Medicine

institute: Northwestern University

summary: Neurological impairment is the most common finding in patients with post-acute sequelae of COVID-19. Furthermore, survivors of pneumonia from any cause have an elevated risk of dementia. In rodent models, cognitive dysfunction following pneumonia has been linked to the release of pro-inflammatory cytokines from the injured lung. As the primary immune cell in the brain, microglia are poised to respond to inflammatory signals from the circulation, and dysfunction in microglia has been linked to cognitive impairment in murine models of dementia and in humans. Here, we report a transcriptional response in human microglia collected from patients who died following COVID-19 suggestive of their activation by TNF-ɑ and other circulating pro-inflammatory cytokines. Consistent with these findings, the levels of 55 alveolar and plasma cytokines were elevated in a cohort of 341 patients with respiratory failure, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. While peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, cumulative cytokine exposure was higher in patients with COVID-19. Corticosteroid treatment, which has been shown to be beneficial in patients with COVID-19, was associated with lower levels of CXCL10, CCL8, and CCL2—molecules that sustain inflammatory circuits between alveolar macrophages harboring SARS-CoV-2 and activated T cells. These findings suggest that corticosteroids may break this cycle and decrease systemic exposure to lung-derived cytokines and inflammatory activation of microglia in patients with COVID-19.

overall_design: We flow-cytometry sorted neuroimmune cells from brain samples obtained at autopsy from 5 patients who died from COVID-19 and 3 patients who died from other causes for single-cell RNA-sequencing.

first: Grant

last: Rogan


title: Xenografted human microglia display diverse transcriptomic states in response to Alzheimer’s disease-related Aβ pathology

city: Leuven

first: Bart

last: De Strooper

address: Herestraat 49 box 602

department: Center for Brain & Disease Research

country: Belgium

institute: VIB-KU Leuven

summary: Microglia are central players in Alzheimer’s Disease (AD) pathology, but analyzing microglia states in human brain samples is challenging due to genetic diversity, postmortem delay and admixture of pathologies. To circumvent these issues, here we generated 138,577 single cell expression profiles of human stem cell-derived microglia xenotransplanted in the brain of the AppNL-G-F model of amyloid pathology and wild type controls. Xenografted human microglia adopt a disease-associated (DAM) profile similar to that seen in mouse microglia, but display a more pronounced HLA state, likely related to antigen presentation in response to amyloid plaques. The human microglial response also involves a pro-inflammatory cytokine/chemokine CRM response to oligomeric Aβo. Genetic deletion of TREM2 or APOE, as well as APOE polymorphisms and TREM2R47H expression in the transplanted microglia modulate these responses differentially. The expression of other AD risk genes is differentially regulated across the distinct cell states elicited in response to amyloid pathology. Thus, we have identified multiple transcriptomic cell states adopted by human microglia in response to AD-related pathology, which should be taken into account in translational studies.

overall_design: The main goal of this project is to decipher the complex human microglial response towards amyloid aggregates. Here, we transplanted human microglia progenitors from several stem cell lines (UKBIO11-A, BIONi010-C and H9) into the mouse brain of AppNL-G-F, Apphu/hu or AppNL-G-F ApoE-/-  mice to mimic several amyloid pathology conditions, including injection of oligomeric Aβo. After exposure to diseased conditions, we isolated human microglia and sequenced more than 138,000 microglia across 106 mice.

first: Renzo

last: Mancuso

first: Nicola

last: Fattorelli

first: Anna

last: Martinez-Muriana

first: Emma

last: Davis

first: Leen

last: Wolfs

first: Johanna

last: Van Den Daele

first: Ivana

last: Geric

first: Jessie

last: Premereur

first: Paula

last: Polanco

first: Baukje

last: Bijnens

first: Pranav

last: Preman

first: Lutgarde

last: Serneels

first: Suresh

last: Poovanthigal

first: Sriram

last: Balusu

first: Catherine

last: Verfaille

first: Mark

last: Fiers


title: Non-canonical WNT5A controls the activation of latent TGFβ to drive fibroblast activation and tissue fibrosis

city: Düsseldorf

first: Jörg

last: Distler

address: Merowingerplatz 1A

department: Department of Rheumatology

institute: University Hospital Düsseldorf, Medical Faculty of Heinrich Heine University

summary: Transforming growth factor β (TGFβ) signaling is a core pathway of fibrosis, but the molecular regulation of the activation of latent TGFβ remains incompletely understood. Here, we demonstrate a crucial role of WNT5A/JNK/ROCK signaling that rapidly coordinates the activation of latent TGFβ in fibrotic diseases. WNT5A was identified as predominant non-canonical WNT ligand in fibrotic diseases such as systemic sclerosis, sclerodermatous chronic graft-versus-host disease and idiopathic pulmonary fibrosis, stimulating fibroblast-to-myofibroblast transition and tissue fibrosis by activation of latent TGFβ. The activation of latent TGFβ requires rapid JNK- and ROCK-dependent cytoskeletal rearrangements and integrin αV (ITGAV). Conditional Knockout of WNT5A or its downstream targets prevented activation of latent TGFβ, rebalanced TGFβ signaling and ameliorated experimental fibrosis. We thus uncovered a novel mechanism for the aberrant activation of latent TGFβ in fibrotic diseases and provided evidence for targeting WNT5A/JNK/ROCK signaling in fibrotic diseases as a new therapeutic approach.

overall_design: Gene expression profile of healthy dermal fibroblasts stimulated with or without recombinant WNT5A protein or recombinant WNT3A protein or recombinant TGFβ and pretreated with the JNK inhibitor SP600125, the ROCK inhibitor Y27632, the TGFβRI kinase inhibitor SD-208, ITGAV inhibitor CWHM12, or Cytochalasin D.

first: Thuong

last: Trinh-Minh


title: ATAC-Seq timelessnon-coding DNA contains cell-type preferential enhancers important for proper Drosophila circadian regulation

city: Waltham

first: Albert

middle: Dayuan

last: Yu

address: 415 South St.

institute: Brandeis University

summary: To address the contribution of transcriptional regulation to Drosophila clock gene expression and to behavior, we generated a series of CRISPR-mediated deletions within two regions of the circadian gene timeless (tim), an intronic E box region and an upstream E box region that are both recognized by the key transcription factor Clock (Clk) and its heterodimeric partner Cycle. The upstream deletions but not an intronic deletion dramatically impact tim expression in fly heads; the biggest upstream deletion reduces peak RNA levels and tim RNA cycling amplitude to about 15% of normal, and there are similar effects on tim protein (TIM). The cycling amplitude of other clock genes is also strongly reduced, in these cases due to increases in trough levels. These data underscore the important contribution of the upstream E box enhancer region to tim expression and of TIM to clock gene transcriptional repression in fly heads. Surprisingly, tim expression in clock neurons is only modestly affected by the biggest upstream deletion and is similarly affected by a deletion of the intronic E box region. This distinction between clock neurons and glia is paralleled by a dramatically enhanced accessibility of the intronic enhancer region within clock neurons. This distinctive feature of tim chromatin was revealed by ATAC-seq (Assay for Transposase-Accessible Chromatin with sequencing) assays of purified neurons and glia as well as of fly heads. The enhanced cell type-specific accessibility of the intronic enhancer region explains the resilience of clock neuron tim expression and circadian behavior to deletion of the otherwise more prominent upstream tim E box region.

overall_design: ATAC-Seq was performed on frozen fly heads, FACS sorted clock neurons and glial cells. Flies were entrained in 12:12LD and were collected at ZT02 and ZT14 timepoints. ATAC-Seq was performed as described in Methods.

first: Dingbang

last: Ma

last: Ojha

first: Weifei

last: Luo

first: Michael

last: Rosbash


title: RNA-Seq of Drosophila tim promoter/enhancer mutants around the clock

summary: The contribution of cis-regulatory elements to Drosophila circadian gene expression is poorly understood. We generated a series of CRISPR-mediated deletions within the regulatory regions of the circadian gene timeless (tim) and characterized them through multiple high-throughput sequencing experiments. We isolated heads from wild-type and mutant flies around the clock and performed RNA-Seq on them to determine the effects of regulatory element deletions on circadian gene expression.

overall_design: Male and female flies were entrained in 12:12 LD conditions for 4 days at 25°C, then collected at ZT2, ZT6, ZT10, ZT14, ZT18 and ZT22. Total RNA from fly heads was extracted using TRIzol reagent (Invitrogen) following the supplier’s protocol. The resulting RNA was used to make libraries for transcriptome sequencing using the TrueSeq RNA Sample Prep Kit (v2; Illumina). The quality of the libraries was assessed using a Bioanalyzer 2100 (Agilent) and then sequenced on a NextSeq 550 (Illumina). RNA libraries were mapped to Drosophila genome dm6 using STAR with default setting and expression levels were quantified using FeatureCounts. Two independent biological replicates were done with the exception of yw at ZT18.


title: Aspergillus nidulans gfdB, encoding the hyperosmotic stress protein glycerol-3-phosphate dehydrogenase, disturbs osmoadaptation in Aspergillus wentii

city: Debrecen

first: Tamas

last: Emri

address: Egyetem ter 1

country: Hungary

institute: University of Debrecen

summary: The genome of the osmophilic Aspergillus wentii, unlike that of the osmotolerant Aspergillus nidulans, contains only the gfdA but not the gfdB glycerol 3-phosphate dehydrogenase gene. Here, we studied transcriptomic changes of A. nidulans (reference strain and DgfdB gene deletion mutant) and A. wentii (reference strain and An-gfdB expressing mutant) elicited by high osmolarity. A. nidulans showed canonic hyperosmotic stress response characterized by upregulation of trehalose and glycerol metabolism genes (including gfdB) as well as genes of the high-osmolarity glycerol (HOG) map kinase pathway. Deletion of gfdB caused only negligible alterations in the transcriptome suggesting that the glycerol metabolism was flexible enough to compensate for the missing GfdB activity in this species. A. wentii responded differently to increased osmolarity than A. nidulans: E.g.; bulk upregulation of glycerol and trehalose metabolism genes as well as HOG pathway genes were not detected. Expression of An-gfdB in A. wentii did not abolish osmophilia, but it reduced growth and caused much bigger alterations in the transcriptome than the missing gfdB gene did in A. nidulans. Flexible glycerol metabolism and hence two differently regulated gfd genes may be more beneficial for osmotolerant (living under changing osmolarity) than for osmophilic (living under constantly high osmolarity) species.

sample_organism:
Aspergillus wentii
Aspergillus nidulans

platform_organism:
Aspergillus wentii
Aspergillus nidulans

overall_design: Comparative gene expression profiling analysis of RNA-seq data for mycelia of A. nidulans (reference strain and DgfdB gene deletion mutant) and Aspergillus wentii (reference strain and A. nidulans-gfdB expressing mutant) at normal osmolarity, in the presence of 1 M sorbitol, 2 M NaCl, or 1 M sorbitol + 2 M NaCl with three biological replicates.

first: Veronika

last: Bodnár

first: Károly

last: Antal

first: István

last: Pócsi

first: Tamás


title: ChIP-Seq of V5-Clk in wildtype and tim promoter mutant Drosophila heads around the clock

summary: To address the contribution of transcriptional regulation to Drosophila clock gene expression and to behavior, we generated a series of CRISPR-mediated deletions within two regions of the circadian gene timeless (tim), an intronic E box region and an upstream E box region that are both recognized by the key transcription factor Clock (Clk) and its heterodimeric partner Cycle. The upstream deletions but not an intronic deletion dramatically impact tim expression in fly heads; the biggest upstream deletion reduces peak RNA levels and tim RNA cycling amplitude to about 15% of normal, and there are similar effects on tim protein (TIM). The cycling amplitude of other clock genes is also strongly reduced, in these cases due to increases in trough levels. To examine the effect of promoter E-box deletions on Clk binding, we performed Clk-ChIP around the clock in wild-type and 126 mutant flies.

overall_design: ChIP-seq was performed on yw;; WT dCLK-V5(wt) andyw; tim_up126; dCLK-V5. 3-5 day old flies were entrained in LD condition for at least 3 days, then collected every 4 hours throughout the day.

first: Pranav


title: Retinal gene expression changes from post-natal 21 (p21) to p60 during retinal degeneration in RCS rat.

city: Los Angeles

first: Kabir

last: Ahluwalia

address: University of Southern California Accounts Payable

institute: University of Southern California

summary: Molecular mechanisms underlying retinal degeneration are not well characterized including in the widely utilized Royal College of Surgeons (RCS) rat model of retinal degeneration. To better understand molecular pathways driving retinal degeneration we performed mRNA transcriptomics on RCS retinas following the natural progression of the disease. This data can identify novel therapeutic targets for future development.

overall_design: Retinas from RCS rats at p21 (n=6) and p60 (n=6) were collected and processed for RNA-Seq. Each timepoint consisted of n=3 immunocompetent and n=3 immunodeficient RCS rats to identify the effects of the immunodeficient RCS variant.

first: Ahluwalia

last: Kabir


title: Allele-specific ATAC-seq of SmcHD1 KO cell lines upon Xist induction with NPC differentiation

city: Oxford

first: Joseph

middle: S

last: Bowness

address: South Parks Road

department: Department of Biochemistry

institute: University of Oxford

summary:
X chromosome inactivation (XCI) in mammals is mediated by Xist RNA which functions in cis to silence genes on a single X chromosome in XX female cells, thereby equalising levels of X-linked gene expression relative to XY males. XCI progresses over a period of several days, with some X-linked genes silencing faster than others. Chromosomal location of a gene is an important determinant of silencing rate, but uncharacterised gene-intrinsic features also mediate resistance or susceptibility to silencing.

In this study, we examine mouse embryonic stem cell lines with an inducible Xist allele (iXist-ChrX mESCs) and integrate allele-specific data of gene silencing and decreasing inactive X (Xi) chromatin accessibility over time-courses of Xist induction with cellular differentiation. Our analysis reveals that motifs bound by the transcription factor YY1 are associated with persistently accessible regulatory elements, including many promoters and enhancers of slow-silencing genes. We further show that YY1 is evicted relatively slowly from target sites on Xi, and that silencing of X-linked genes is increased upon YY1 degradation. Together our results suggest that YY1 acts as barrier to Xist-mediated silencing until late stages of the XCI process.

overall_design: This series contains data of ATAC-seq from time-courses of inducible XCI in iXist-ChrX mESCs comparing WT with SmcHD1 KO cell lines. The genotypes of these model cell lines are female interspecific hybrids between M.m.Domesticus and M.m.Castaneous mouse strains, with dox-inducible Xist engineered onto either the Domesticus (iXist-ChrX-Dom) or Castaneous (iXist-ChrX-Cast) allele (Nesterova et al, 2019). SmcHD1 KO clones were derived from each of these parental lines as documented in our previous publication, SmA3 from iXist-ChrX-Dom and SmD5 from iXist-ChrX-Cast (Bowness et al, 2022). Time-courses of Xist induction with differentiation from mESCs to NPCs were conducted in parallel for WT and SmcHD1 KO, and timepoints of 0, 3, 5, 7 and 15 days were taken for comparison. At later timepoints of the time-courses, more accessibility is retained at regulatory elements on the inactive X chromosome (Xi) in SmcHD1 KO compared to WT.

first: Tatyana

middle: B

last: Nesterova

first: Neil

last: Brockdorff


title: Hypoxia stimulates CTC-platelet cluster formation to promote breast cancer metastasis

city: Nanjing

first: Xiaoxiang

last: Guan

state: None Selected

address: Guangzhoulu

department: Department of Oncology

institute: The First Affiliated Hospital of Nanjing Medical University

summary: Circulating tumor cell clusters/micro-emboli (CTM) possess greater metastatic capacity and survival advantage compared to individual circulating tumor cell (CTCs). However, the formation of CTM subtypes and their role in tumor metastasis remain unclear. In this study, we used a microfluidic Cluster-chip with easy operation and high efficiency to isolate CTM from peripheral blood, which confirmed their correlation with clinicopathological features and identified the critical role of CTC-platelet clusters in BC metastasis. The correlation between platelets and CTM function was further confirmed in a mouse model and RNA-seq of CTM identified high-expressed genes related to hypoxia stimulation and platelet activation which possibly suggested the correlation of hypoxia and CTC-platelet cluster formation.  In conclusion, we successfully developed the Cluster-chip platform to realize the clinical capture of CTMs and analyze the biological properties of CTC-platelet clusters, which could benefit the design of potential treatment regimens to prevent CTM-mediated metastasis and tumor malignant progression.

overall_design: In order to  further understand the mechanism of CTM hematogenous metastasis, examination of the molecular characteristics of CTM can aid in elucidating the mechanism of CTM-mediated hematogenous metastasis. For this purpose, we used the Cluster-chip to recover CTM in mice with lung metastasis secondary to breast cancer for RNA sequencing to investigate the biological characteristics of hematogenous metastasis with a control group comprised of the 4T1 cell line.

first: Xin

last: Han

first: Yanni

last: Song

first: Fang

last: Yang

first: Jacqueline

middle: F

last: Wang

first: Weijia

last: Zhou

first: Chengjun

last: Zhu

first: Peiliang

last: Shen


title: Cell-free DNA 5-hydroxymethylcytosine for immunotherapy response prediction in lung cancer

city: Houston

first: Zejuan

last: Li

state: Texas

address: 6565 Fannin St, MC227

department: Department of Pathology and Genomic Medicine

institute: Houston Methodist Hospital

type: Methylation profiling by high throughput sequencing

summary: Immune checkpoint inhibitors (ICIs) drastically improve therapeutic outcomes for lung cancer, but accurate prediction of individual patient responses to ICIs remains a challenge. We performed a genome-wide analysis of 5-hydroxymethylcytosine (5hmC) in plasma cell-free DNA (cfDNA) samples from 83 lung cancer patients. Using machine learning approaches, we developed a 5hmC signature to predict ICI treatment response and calculated a weighted-predictive score (wp-score) based on the 5hmC levels of signature genes in each sample. A low wp-score was significantly correlated with longer progression-free survival across three independent patient sample sets, and demonstrated superior predictive capability to tumor programmed death-ligand 1. Moreover, we identified novel 5hmC-associated genes and signaling pathways integral to ICI treatment response in lung cancer. Our study suggests that cfDNA 5hmC analysis is a minimally invasive, innovative strategy for guiding treatment selection in lung cancer patients.

overall_design:
Analyze 5-hydroxymethyl cytosine (5hmC) genome-wide distribution in plasma cell-free DNA from lung cancer patients treated with immune checkpoint inhibitors.

Please note that 17 samples from GSE202988 were re-used in the current study, resulting in total 85 data columns in the processed '5hmC_ReadCount_Genebody_LungCancer.txt' data file.
Re-analysed Samples in GSE202988/PRJNA837976:
GSM6146017 Z117 SRX15269868 SAMN28239535
GSM6146018 Z36 SRX15269869 SAMN28239534
GSM6146019 Z41 SRX15269870 SAMN28239533
GSM6146020 Z22 SRX15269871 SAMN28239532
GSM6146021 Z29 SRX15269872 SAMN28239531
GSM6146024 Z30 SRX15269875 SAMN28239528
GSM6146025 Z32 SRX15269876 SAMN28239527
GSM6146027 Z12 SRX15269880 SAMN28239525
GSM6146030 Z42 SRX15269726 SAMN28239522
GSM6146031 Z5 SRX15269727 SAMN28239521
GSM6146032 Z25 SRX15269728 SAMN28239520
GSM6146033 Z54 SRX15269729 SAMN28239519
GSM6146034 Z26 SRX15269730 SAMN28239518
GSM6146035 Z17 SRX15269731 SAMN28239517
GSM6146036 Z46 SRX15269732 SAMN28239516
GSM6146037 Z38 SRX15269733 SAMN28239515
GSM6146068 Z106 SRX15269456 SAMN28239484

first: Jianming

last: Shao


title: A Novel Approach for Pancreas Transcriptomics Reveals the Cellular Landscape in Homeostasis and Acute Pancreatitis

city: Boston

first: Sahar

last: Nissim

institute: Brigham and Women's Hospital

summary: BACKGROUND & AIMS: Acinar cells produce digestive enzymes that impede transcriptomic characterization of the exocrine pancreas. Thus, single-cell RNA-sequencing (scRNA-seq) studies of the pancreas underrepresent acinar cells relative to histological expectations, and a robust approach to capture pancreatic cell responses in disease states is needed. We sought to innovate a method that overcomes these challenges to accelerate study of the pancreas in health and disease. METHODS: We introduce FixNCut, a scRNA-seq approach where tissue is reversibly fixed with dithiobis(succinimidyl propionate) prior to dissociation and single-cell preparation. We apply FixNCut to an established mouse model of acute pancreatitis, validate findings using GeoMx whole transcriptome atlas (WTA) profiling, and integrate our data with prior studies to benchmark our method in both mouse and human pancreas datasets. RESULTS: FixNCut achieves unprecedented definition of challenging pancreatic cells including acinar and immune populations in homeostasis and acute pancreatitis, and identifies changes in all major cell types during injury and recovery. We define the acinar transcriptome during homeostasis and acinar-to-ductal metaplasia and establish a unique gene set to measure deviation from normal acinar identity. We characterize pancreatic immune cells, and analysis of T-cell subsets reveals a polarization of the homeostatic pancreas towards type-2 immunity. We report immune responses during acute pancreatitis and recovery, including early neutrophil infiltration, expansion of dendritic cell subsets, and a substantial shift in the transcriptome of macrophages due to both resident macrophage activation and monocyte infiltration. CONCLUSIONS: FixNCut preserves pancreatic transcriptomes to uncover novel cell states during homeostasis and following pancreatitis, establishing a broadly applicable approach and reference atlas for study of pancreas biology and disease.

overall_design: Pancreatitis was induced in C57BL/6 mice at 8-9 weeks of age using intraperitoneal caerulein injections on two consecutive days once every hour for 8 hours. Formalin-fixed paraffin-embedded microarray tissue sections (5 µm) were used for Nanostring's GeoMX Whole Transcriptome Atlas Digital Spatial Profiling.

first: Katherine

middle: J

last: Aney

first: Woo-Jeong

last: Jeong

first: Ethan

middle: E

last: Chen

first: Kirk

last: Jensen

first: Kellie

last: Wise

first: Luciano

last: Martelotto


title: Transcriptional response to Notch activation in SVG-A cells

first: Julia

middle: M

last: Rogers

state: Massachusetts

address: 45 Shattuck Street

department: BCMP

summary: SVG-A cells are immortalized fetal astrocytes that respond to Notch signaling. We characterized the transcriptional response to Notch activation in this cell line by performing RNA-seq on cells grown on tissue culture dishes coated with JAGGED1, a Notch ligand. Differential gene expression analysis shows induction of both canonical Notch responsive genes, as well as cell-type specific Notch responsive genes.

overall_design: Cells were plated overnight on the JAGGED-coated tissue culture plates in the presence of 100 nM gamma secretase inhibitor (GSI), a small molecule that prevents the final proteolytic cleavage of the Notch receptor. The following morning, GSI was washed out by 3 media changes, and cells were collected at 2, 4, and 24 hours after GSI washout. Additionally, a mock washout control was performed in which GSI was maintained in the media and cells were harvested after the mock washout. Three replicates were collected of these 4 conditions. RNA-seq was performed on these 12 samples. Genes that were differentially expressed at 2, 4, and 24 hours were identified.

first: Emily

last: Egan

first: Stephen

last: Blacklow


title: Circadian rhythm-dependent programs control inflammatory responses and drug metabolism in primary human hepatocytes

city: Cambridge

first: Charles

middle: Arthur

last: Whittaker

institute: Koch Institute

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

overall_design: Refer to individual Series


title: Molecular regulation of PPARγ/RXRα signaling by the novel cofactor ZFP407

status: Public on Feb 27 2024

city: Cleveland

first: David

last: Buchner

address: 10900 Euclid Ave

department: Genetics and Genome Sciences

institute: Case Western Reserve University

summary: Cofactors interacting with PPARγ can regulate adipogenesis and adipocyte metabolism by modulating the transcriptional activity and selectivity of PPARγ signaling. ZFP407 was previously demonstrated to regulate PPARγ target genes such as GLUT4, and its overexpression improved glucose homeostasis in mice. Here, using a series of molecular assays, including protein-interaction studies, mutagenesis, and ChIP-seq, ZFP407 was found to interact with the PPARγ/RXRα protein complex in the nucleus of adipocytes. Consistent with this observation, ZFP407 ChIP-seq peaks significantly overlapped with PPARγ ChIP-seq peaks, with more than half of ZFP407 peaks overlapping with PPARγ peaks. Transcription factor binding motifs enriched in these overlapping sites included CTCF, RARα/RXRγ, TP73, and ELK1, which regulate cellular development and function within adipocytes. Site-directed mutagenesis of frequent PPARγ phosphorylation or SUMOylation sites did not prevent its regulation by ZFP407, while mutagenesis of ZFP407 domains potentially necessary for RXR and PPARγ binding abrogated any impact of ZFP407 on PPARγ activity. These data suggest that ZFP407 controls the activity of PPARγ, but does so independently of post-translational modifications, likely by direct binding, establishing ZFP407 as a newly identified PPARγ cofactor. In addition, ZFP407 ChIP-seq analyses identified regions that did not overlap with PPARγ peaks. These non-overlapping peaks were significantly enriched for the transcription factor binding motifs of TBX19, PAX8, HSF4, and ZKSCAN3, which may contribute to the PPARγ-independent functions of ZFP407 in adipocytes and other cell types.

overall_design: Chromatin Immunoprecipitation DNA Sequencing (ChIP-Seq) of Zfp407 in differentiated 3T3-L1 adipocytes

first: Alyssa

last: Charrier

first: Jeremiah

last: Ockunzzi

first: Leighanne

last: Main

first: Siddharth

middle: V

last: Ghanta