title: Axonal ER tubules control local translation via P180/RRBP1-mediated ribosome interactions

city: Amsterdam

first: Max

last: Koppers

address: de Boelelaan 1085

department: CNCR

institute: Vrije Universiteit Amsterdam

summary: Local mRNA translation in axons is critical for the spatial and temporal regulation of the axonal proteome. A wide variety of mRNAs are localized and translated in axons, however how protein synthesis is regulated at specific subcellular sites in axons remains unclear. Here, we establish that the axonal endoplasmic reticulum (ER) supports axonal translation in developing neurons. Axonal ER tubule disruption impairs local translation and ribosome distribution. Using nanoscale resolution imaging, we find that ribosomes make frequent contacts with axonal ER tubules in a translation-dependent manner and are influenced by specific extrinsic cues. We identify P180/RRBP1 as an axonally distributed ribosome receptor that regulates local translation and binds to mRNAs enriched for axonal membrane proteins. Importantly, the impairment of axonal ER – ribosome interactions causes defects in axon morphology. Our results establish an important role for the axonal ER in dynamically localizing mRNA translation which is important for proper neuron development.

overall_design: To identify mRNAs interacting with P180/RRBP1 in neurons we performed RNA-sequencing after pulldown of P180-GFPbio using GFPbio as a control condition in primary cortical neurons/. Two biological replicates were obtained and sequenced.

first: Ginny

last: Farias

first: Mai

last: Nguyen

first: Chun

last: Li


title: Direct neuronal reprogramming of mouse astrocytes is associated with multiscale epigenome remodeling and requires Yy1

status: Public on Apr 30 2024

city: Neuherberg

first: Boyan

last: Bonev

state: Deutschland

address: Ingolstädter Landstr. 1

country: Germany

institute: Helmholtz Zentrum München

type: Expression profiling by high throughput sequencing

type: Genome binding/occupancy profiling by high throughput sequencing

type: Methylation profiling by high throughput sequencing

summary: Direct neuronal reprogramming is a promising approach to regenerate neurons from local glial cells. However, mechanisms of epigenome remodeling and co-factors facilitating this process are unclear. Here, we combine single-cell multiomics with genome-wide profiling of 3D nuclear architecture and DNA methylation in mouse astrocyte-to-neuron reprogramming mediated by Neurogenin2 (Ngn2) and its phosphorylation-resistant form (PmutNgn2), respectively. We show that Ngn2 drives multilayered chromatin remodelling at dynamic enhancer-gene interaction sites. PmutNgn2 leads to higher reprogramming efficiency and enhances epigenetic remodeling associated with neuronal maturation. However, the differences in binding sites or downstream gene activation cannot fully explain this effect. Instead, we identify Yy1, a transcriptional co-factor, recruited by direct interaction with Ngn2 to its target sites. Upon deletion of Yy1, activation of neuronal enhancers, genes, and ultimately reprogramming are impaired without affecting Ngn2 binding. Thus, our work highlights the key role of interactors of proneural factors in direct neuronal reprogramming.

overall_design: Single cell multiome (scRNA-seeq + scATAC-seq), bulk ATAC-seq, bulk RNA-seq, ChIP-seq, Cut&Run and Methyl-HiC  was performed on primary mouse astrocytes (2 days post induction) transduced with doxycycline-inducible lentiviral constructs expressing either GFP, Ngn2 and PmutNgn2 respectively. Since the constructs also expressed GFP, the astrocytes were FACS sorted based on GFP expression. To profile the effect of Yy1 on direct neuronal reprogramming. scRNA-seq and bulk ATAC-seq was performed on Yy1 KO primary mouse astrocytes (2 days post induction) transduced with  a doxycycline-inducible lentiviral construct expressing Ngn2.

first: Allwyn

last: Pereira

first: Jeisimhan

last: Diwakar

first: Giacomo

last: Masserdotti

first: Sude

last: Beşkardeş

first: Tatiana

last: Simon

first: Younju

last: So

first: Lucía

last: Martín-Loarte

first: Franziska

last: Bergemann

first: Lakshmy

last: Vasan

first: Tamas

last: Schauer

first: Anna

last: Danese

first: Riccardo

last: Bocchi

first: Maria

last: Colomé-Tatché

first: Carol

last: Schuurmans

last: Philpott

first: Tobias

last: Straub

first: Magdalena

last: Götz


title: Fetal Hypoglycemia Induced by Placental SLC2A3 RNA Inter-ference Alters Fetal Pancreas Development and Transcriptome at Mid-Gestation

status: Public on Apr 26 2024

city: Fort Collins

first: Victoria

last: Kennedy

state: Colorado

address: 1683 Campus Delivery

country: USA

institute: Colorado State University

summary: Glucose, the primary energy substrate for fetal oxidative processes and growth, is transferred from maternal to fetal circulation down a concentration gradient by placental facili-tative glucose transporters. In sheep, SLC2A1 and SLC2A3 are the primary transporters available in the placental epithelium, with SLC2A3 located on the maternal-facing apical trophoblast membrane and SLC2A1 located on the fetal-facing basolateral trophoblast membrane. We have previously reported that impaired placental SLC2A3 glucose transport resulted in smaller, hypo-glycemic fetuses with reduced umbilical artery insulin and glucagon concentrations, in addition to diminished pancreas weights. These findings led us to subject RNA derived from SLC2A3-RNAi (RNA interference) and NTS-RNAi (non-targeting sequence) fetal pancreases to qPCR followed by transcriptomic analysis. We identified a total of 771 differentially expressed genes (DEGs). Upregulated pathways were associated with fat digestion and absorption, partic-ularly fatty acid transport, lipid metabolism, and cholesterol biosynthesis, suggesting a potential switch in energetic substrates due to hypoglycemia. Pathways related to molecular transport and cell signaling, including PPAR and PI3K-Akt signaling, as well as negative regulation of cellular proliferation were also impacted. A few genes directly related to gluconeogenesis were also dif-ferentially expressed. Our results suggest that fetal hypoglycemia during the first half of gestation impacts fetal pancreas development and function that is not limited to beta cell function.

overall_design: To investigate the impact of placental SLC2A3 deficiency, we performed a transcriptomic analysis on fetal pancreases at mid-gestation.

middle: C

first: Cameron

middle: S

last: Lynch

first: Amelia

middle: R

last: Tanner

first: Quinton

middle: A

last: Winger

first: Ahmed

last: Gad

first: Paul

middle: J

last: Rozance

first: Russell

middle: V

last: Anthony


title: Genome-wide methylation profile of mitochondrial DNA across bovine preimplantation development

status: Public on Apr 24 2024

city: Québec City

first: Marc-André

last: Sirard

state: Quebec

address: Offfice 2732, 2440 Hochelaga Blvd.

department: Sciences Animales

country: Canada

institute: Université Laval

summary: This study characterized variations in the methylation profile of mitochondrial DNA (mtDNA) during initial bovine embryo development and correlated the presence of methylation with mtDNA transcription. Bovine oocytes were obtained from abattoir ovaries and submitted to in vitro culture procedures. Oocytes and embryos were collected at various stages (immature oocyte, IM; mature oocyte, MII; zygote, ZY; 4-cells, 4C; 16-cells, 16C and blastocysts, BL). Total DNA (including mtDNA) was used for Whole Genome Enzymatic Methyl Sequencing and for quantification of mtDNA copy number. Extracted RNA was used for quantification of mitochondrial transcripts (ND6, CYTB, tRNA-Phe and tRNA-Gln) using Droplet Digital PCR. The number of mtDNA copies per oocyte/embryo was found to be similar, while methylation levels in mtDNA varied among stages. Higher total methylation levels were found mainly at 4C and 16C. In specific gene regions, higher methylation levels were also observed at 4C and 16C (ND6, CYTB and tRNA-Phe), as well as an inverse correlation with the quantity of transcripts for these regions. This is a first description of epigenetic changes occurring in mtDNA during early embryonic development. Our results indicate that methylation might regulate the mtDNA transcription at a local level, particularly around the time of embryonic genome activation.

overall_design: Bovine oocytes and embryos were collected at different developmental stages accross inital development (from immature oocyte to day 7 blastocyst)

first: Camila

middle: B

last: de Lima

first: Marcella

middle: P

last: Milazzotto

first: Clement

last: Plessis

first: Hélène

last: Martin


title: Pervasive nuclear envelope ruptures precede ECM signaling and disease onset without activating cGAS-STING in Lamin-cardiomyopathy mice

status: Public on Apr 25 2024

city: Cincinnati

first: Kohta

last: Ikegami

state: OH

address: 240 Albert Sabin Way, Cincinnati, OH 45229

department: Division of Molecular Cardiovascular Biology

institute: Cincinnati Children's Hospital Medical Center

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

overall_design: Refer to individual Series


title: Pervasive nuclear envelope ruptures precede ECM signaling and disease onset without activating cGAS-STING in Lamin-cardiomyopathy mice [mRNA-seq]

summary: Nuclear envelope (NE) ruptures are emerging observations in Lamin-related dilated cardiomyopathy, an adult-onset disease caused by loss-of-function mutations in Lamin A/C, a nuclear lamina component. Here, we test a prevailing hypothesis that NE ruptures trigger the pathological cGAS-STING cytosolic DNA-sensing pathway using a mouse model of Lamin cardiomyopathy. The reduction of Lamin A/C in cardiomyocytes of adult mice causes pervasive NE ruptures in cardiomyocytes, preceding inflammatory transcription, fibrosis, and fatal dilated cardiomyopathy. NE ruptures are followed by DNA damage accumulation without causing immediate cardiomyocyte death. However, cGAS-STING-dependent inflammatory signaling remains inactive. Deleting cGas or Sting does not rescue cardiomyopathy. The lack of cGAS-STING activation is likely due to the near absence of cGAS expression in adult cardiomyocytes at baseline. Instead, extracellular matrix (ECM) signaling is activated and predicted to initiate pro-inflammatory communication from Lamin-reduced cardiomyocytes to fibroblasts. Our work nominates ECM signaling, not cGAS-STING, as a potential inflammatory contributor in Lamin cardiomyopathy.

overall_design: mRNA-seq

first: Atsuki

last: En


title: Expression profile of bone marrow HSCs from healthy donors

city: Rio de Janeiro

first: Renata

last: Binato

address: Praça da Cruz Vermelha 23

country: Brazil

institute: INCA

summary: mRNA samples from bone marrow CD34+ cells (HSC) from healthy donors after co-culture assays (Control condition, with hMSCs and CD34+ cells from healthy donors; and AML condition, with hMSCs-AML and CD34+ cells from donors) were amplified, labeled and hybridized to the ClariomTM S Array human (Thermo Scientific, USA). Normalization and analysis of microarray data was performed using the Transcriptome Analysis Console (TAC) software (Affymetrix, USA).

overall_design: We analyzed gene expression from 1 CD34+ cells (HSC) sample  from control condition and 3 CD34+ cells (HSC) sample  from AML condition after co-culture assays using the Clariom S Array human (Thermo Scientific, USA). Array data was processed and analyzed using Transcriptome Analysis Console (TAC) software (Affymetrix, USA).

first: Pedro

middle: L

last: Azevedo

first: Eliana

last: Abdelhay


title: Secukinumab for the treatment of adult-onset pityriasis rubra pilaris: a single-arm clinical trial with transcriptomic analysis

status: Public on Apr 23 2024

city: Rochester

first: Tao

last: Ma

state: MN

address: 200 1st ST SW

institute: Mayo Clinic

summary: Background: The pathogenesis of pityriasis rubra pilaris (PRP) is not completely understood, but interleukin (IL)-17 has been shown to play a critical role. There are no reliable immunomodulatory agents to treat PRP. We conducted an open-label, single-arm clinical trial of secukinumab, a monoclonal antibody that inhibits IL-17A, for the treatment of PRP. Objectives: To evaluate the clinical efficacy of secukinumab and define the transcriptomic landscape of PRP and its response to IL-17A blockade. Methods: Twelve patients with PRP were recruited for an open-label trial of secukinumab. Patients received a 24-week course of secukinumab. The primary endpoint was a ≥ 75% reduction in Psoriasis Area and Severity Index (PASI 75) from baseline to week 28. Secondary endpoints included PASI 90, change in Physician's Global Assessment (PGA), and change in Dermatology Life Quality Index (DLQI). RNA sequencing was performed on lesional and nonlesional skin biopsies obtained at baseline and week 2. Sample groups were compared to identify differential gene expression and pathway enrichment. This trial was registered with ClinicalTrials.gov: 'Cosentyx (secukinumab) for the treatment of adult onset pityriasis rubra pilaris' - NCT03342573. Results: At week 28, six of 11 patients (55%) achieved PASI 75, and three patients (27%) achieved PASI 90. PGA (P = 0.008) and DLQI scores (P = 0.010) showed significant improvement with treatment. No serious treatment-related adverse events were encountered. Treatment with secukinumab normalized transcriptional differences between lesional and nonlesional skin. Transcriptomic data from nonresponsive patients suggest that overactivity of innate immune pathways may be driving resistance to secukinumab. Conclusions: Secukinumab appears to be an effective treatment for PRP and warrants further investigation. PRP is a transcriptionally heterogeneous disease, reflecting its variable response to therapy. Agents targeting other IL-17 isoforms and innate immune mediators should be considered for future clinical trials. What is already known about this topic? The pathogenesis of pityriasis rubra pilaris is incompletely understood. Successful treatment has been reported with a variety of immunomodulatory agents, but disease is often refractory to therapy. Interleukin (IL)-17 is thought to drive keratinocyte proliferation and vascular dysfunction in this disease. A previous trial demonstrated efficacy of the anti-IL-17A drug ixekizumab for pityriasis rubra pilaris. What does this study add? Herein we describe the findings of a clinical trial of secukinumab, an anti-IL-17A monoclonal antibody, for the treatment of pityriasis rubra pilaris. Secukinumab was effective in treating pityriasis rubra pilaris. Our transcriptomic data give new insight into the expressional changes that occur in response to secukinumab and suggest mechanisms of treatment resistance.

overall_design: Whole transcriptomic analysis was performed on PRP (n=12) and control (normal skin) tissue (n=10)  pre-therapy (week 0) and ctrl (n=11) and PRP (n=12) skin tissue post-therapy (week 2) on 12 patients.

first: Blake

middle: W

last: Boudreaux

first: Thais

last: Pincelli

first: Puneet

last: Bhullar

first: Meera

middle: H

last: Patel

first: Caitlin

middle: M

last: Brumfiel

first: Xing

last: Li

first: Michael

middle: G

last: Heckman

first: Mark

last: Pittelkow

first: Aaron

last: Mangold

first: Jason

last: Sluzevich


title: Genetic dependencies associated with transcription factor activities in human cancer cell lines

status: Public on Apr 29 2024

city: Vienna

first: Malgorzata

middle: Emilia

last: Kamuda

address: Dr.Boehringer-Gasse 5-11

department: Global Computational Biology and Digital Sciences

country: Austria

institute: Boehringer-Ingelheim


title: Non-coding RNA and mRNA expression profile in multi-drug resistance of colorectal cancer

status: Public on Apr 28 2024

city: wuhan

first: Qingming

last: Wu

state: Hubei

address: huangjiahu east

country: China

institute: wuhan university of science and technology

type: Non-coding RNA profiling by high throughput sequencing

summary: Background: Non-coding RNAs have been proved to play an essential role in the development and progression of various cancers. However, the functions and mechanisms of non-coding RNA and mRNA  in multi-drug resistance of colorectal cancer have not been fully elucidated.Methods: We performed RNA-sequencing to screen differentially expressed non-coding RNA and mRNA in CRC chemo-resistant and chemo-sensitive cell lines.  Results: The results showed that 1779 mRNAs, 64 miRNAs, 11 circRNAs and 295 lncRNAs were common differentially expressed in two chemo-resistant cell lines.Conclusions:The results of the study may provide new theories and therapeutic targets for multi-drug resistance in colorectal cancer.

overall_design: To detect the non-coding RNA and mRNA  expression profiles in  colorectal cancer  chemo-resistant and chemo-sensitive cell lines.

first: Fei

last: Yao

first: Qiang

last: Wang

first: Xiaochen

last: Xiang


title: Pervasive nuclear envelope ruptures precede ECM signaling and disease onset without activating cGAS-STING in Lamin cardiomyopathy mice [SLAM-IT-seq]

first: Kohta

last: Ikegami

overall_design: SLAM-IT-seq

first: Atsuki

last: En


title: Multiomic profiling of medulloblastoma reveals subtype-specific targetable alterations at the proteome and N-glycan level [methylation 1]

city: Hamburg

first: Julia

middle: Elisabeth

last: Neumann

address: Martinistrasse 52

department: ZMNH

institute: University Hospital Hamburg Eppendorf

type: Methylation profiling by genome tiling array

summary: Medulloblastomas (MBs) are malignant pediatric brain tumors that are molecularly and clinically very heterogenous. To unravel phenotypically relevant MB subtypes, we compiled a harmonized proteome dataset of 167 MBs and integrated findings with DNA methylation and N-glycome data. Six proteome MB subtypes emerged, that could be assigned to two main molecular programs: transcription/translation (pSHHt, pWNT and pGroup3-Myc), and synapses/immunological processes (pSHHs, pGroup3 and pGroup4). Multiomic analysis revealed different conservation levels of proteome features across MB subtypes at the DNA-methylation level. Aggressive pGroup3-Myc MBs and favorable pWNT MBs were most similar in cluster hierarchies concerning overall proteome patterns but showed different protein abundances of the vincristine resistance associated multiprotein complex TriC/CCT and of N-glycan turnover associated factors.  The N-glycome reflected proteome subtypes and complex-bisecting N-glycans characterized pGroup3-Myc tumors. Our results shed light on new targetable alterations in MB and set a foundation for potential immunotherapies targeting glycan structures.

overall_design: Bisulphite converted DNA from the 34 samples were hybridised to the Illumina Infinium 850k Human Methylation Beadchip and 6 older cases  were hybridised to the Illumina Infinium 450k Human Methylation Beadchip

first: Shweta

last: Godbole

first: Hannah

last: Voss


title: Multiomic profiling of medulloblastoma reveals subtype-specific targetable alterations at the proteome and N-glycan level [methylation 2]

summary: Medulloblastomas (MBs) are malignant pediatric brain tumors that are molecularly and clinically very heterogenous. To unravel phenotypically relevant MB subtypes, we compiled a harmonized proteome dataset of 167 MBs and integrated findings with DNA methylation and N-glycome data. Six proteome MB subtypes emerged, that could be assigned to two main molecular programs: transcription/translation (pSHHt, pWNT and pGroup3-Myc), and synapses/immunological processes (pSHHs, pGroup3 and pGroup4). Multiomic analysis revealed different conservation levels of proteome features across MB subtypes at the DNA-methylation level. Aggressive pGroup3-Myc MBs and favorable pWNT MBs were most similar in cluster hierarchies concerning overall proteome patterns but showed different protein abundances of the vincristine resistance associated multiprotein complex TriC/CCT and of N-glycan turnover associated factors.  The N-glycome reflected proteome subtypes and complex-bisecting N-glycans characterized pGroup3-Myc tumors. Our results shed light on new targetable alterations in MB and set a foundation for potential immunotherapies targeting glycan structures.To biologicallyy validate these results we further generated methylome and proteome data for 29 samples.

overall_design: Bisulphite converted DNA from the 29 samples were hybridised to the Illumina Infinium 850k Human Methylation Beadchip

first: Godbole

last: Shweta

first: Neumann

last: Julia


title: Effect of bioengineered niches on perivascular stem cell phenotype.

city: Glasgow

last: Donnelly

address: Glasgow University

department: Centre for the Cellular Microenvironment

country: United Kingdom

institute: University of Glasgow

summary: Long-term reconstituting haematopoietic stem cells (LT-HSCs) are used to treat blood disorders via allogeneic stem cell transplantation (alloSCT), to engraft and repopulate the blood system. The very low abundance of LT-HSCs and their rapid differentiation during in vitro culture hinders their clinical utility. Previous developments using stromal feeder layers, defined media cocktails, and bioengineering have enabled HSC expansion in culture, but of mostly short-term HSCs (ST-HSC) and progenitor populations at the expense of naïve LT-HSCs. Here, we report the creation of a bioengineered LT-HSC maintenance niche that recreates physiological extracellular matrix organisation, using soft collagen type-I hydrogels to drive nestin expression in perivascular stromal cells (PerSCs or pericytes). We demonstrate that nestin, which is expressed by HSC-supportive bone marrow stromal cells, is cytoprotective and, via regulation of metabolism, is important for HIF-1 expression in PerSCs. When CD34+ve HSCs were added to the bioengineered niches comprising nestin/HIF-1 expressing PerSCs, LT-HSC numbers were maintained with normal clonal and in vivo reconstitution potential, without media supplementation. We provide proof-of-concept that our bioengineered niches can support the survival of CRISPR edited HSCs. Successful editing of LT-HSCs ex vivo can have potential impact on the treatment of blood disorders.

overall_design: PerSCs were cultured in 2D and 3D niches for 7 days to establish the effect of different extracellular niche components on phenotype.

first: Matthew

last: Dalby


title: Hematopoietic stem cell niche generation and maintenance are distinguishable by an epitranscriptomic program

city: New York

first: Longfei

last: Gao

state: NY

address: 650 West 168th Street

institute: Columbia University

summary: The niche is typically considered as a pre-established structure sustaining stem cells. Therefore, the regulation of its formation remains largely unexplored. It is unknown whether distinct molecular mechanisms control the establishment versus maintenance of a stem cell niche. To address this, we compared perinatal with adult bone marrow mesenchymal stromal cells (MSCs), a key component of the hematopoietic stem cell (HSC) niche. MSCs exhibited an enrichment in genes mediating m6A mRNA methylation regulation at the perinatal stage and downregulated the expression of Mettl3, the m6A methyltransferase, shortly after birth. Deletion of Mettl3 from developing MSCs led to excessive osteogenic differentiation and a severe HSC niche formation defect, which was rescued by deletion of Klf2, a target of m6A. In contrast, deletion of Mettl3 from MSCs postnatally did not affect HSC niche or hematopoiesis. Stem cell niche generation and maintenance thus depend on divergent molecular mechanisms, which may be exploited for regenerative medicine.

overall_design: We performed scRNA-seq on non-hematopoietic cells in the limb bone marrow of two-week-old control and Prx1-cre;Mettl3-fl/fl mice

first: Heather

last: Lee

first: Joshua

last: Goodman

first: Lei

last: Ding


title: Comparing Gut Microbial Composition and Functional Adaptations between SPF and Non-SPF pigs

status: Public on Apr 22 2024

city: Wanju-gun, Jeollabuk-do

first: Haesun

state: Jeollabuk-Do

country: South Korea

institute: National institute of animal science

summary: The gut microbiota is closely associated with digestion, metabolism, immunity, and host health. The imbalance of the microbial community in livestock directly affects their well-being and, consequently, productivity. The composition and diversity of the gut microbiota are influenced not only by host genetics but also by environmental factors such as the microbial complexity of the rearing environment, feeds, and antibiotics. Here, we focus on the comparison of gut microbial communities in miniature pigs developed for xenotransplantation in specific pathogen-free (SPF) and conventional (non-SPF) facilities. To identify the disparities in gut microbial composition and functionality between these two environments, 16S RNA metagenome sequencing was conducted using fecal samples. The results revealed that the non-SPF pigs had higher gut microbiota diversity than the SPF pigs. The genera Streptococcus and Ruminococcus were more abundant in SPF pigs than in non-SPF pigs. Blautia, Bacteroides, and Roseburia were exclusively observed in SPF pigs, whereas Prevotella was exclusively found in non-SPF pigs. Carbohydrate and nucleotide metabolism, as well as environmental information processing, were predicted to be enriched in SPF pigs. In addition, energy and lipid metabolism, along with processes related to genetic information, cellular communication, and diseases, were predicted to be enriched in non-SPF pigs. This study makes an important contribution to elucidating the impact of environments harboring a variety of microorganisms, including pathogens, on the gut microbiota of miniature pigs. Furthermore, we sought to provide foundational data on the characteristics of the gut microbiota in genetically modified pigs, which serve as source animals for xenotransplantation.

overall_design: A total of 8 Massachusetts General Hospital (MGH) miniature pigs (4 SPF and 4 non-SPF pigs) from National Institute of Animal Science were enrolled into this study. The SPF facility employs high efficiency particulate air (HEPA) filters to introduce filtered air, preventing exposure to animals in the general environment and thereby mitigating the risk of pathogens that could infect pigs. In order to support SPF pig production and management, the facility conducts regular testing for 42 types of pathogens. SPF pigs were bred in sterilized isolator (the positive pressure of 5 mmH2O, humidity of 50%) and fed artificially sterilized milk. The temperature was maintained at 38ºC for the first week and then decreased by 3℃ every week until 5th week after birth. After weaning, they were raised in the SPF level breeding cage with sterilized pellet-type commercial feed and sterilized water. Non-SPF pigs were naturally fed by the sow until 5 weeks of age and then gradually weaned in conventional facility. The fecal samples were obtained from SPF and non-SPF pigs at the age of 18 months.


title: The temporal progression of lung immune remodeling during breast cancer metastasis

city: Palo Alto

first: Christopher

last: McGinnis

state: CA

address: Biomedical Innovations Building 4600, 240 Pasteur Drive

department: Pathology

institute: Stanford University

summary: The dynamics of immune reprogramming in the breast cancer lung metastastic niche remains incomplete. We used MULTI-seq (McGinnis, Patterson, et al., Nature Methods, 2019) to perform multiplexed single-cell RNA-sequencing analysis of cryopreserved lung immune cells isolated from PyMT mice, freshly-isolated lung immune cells isolated from PyMT mice, and cryopreserved lung immune cells isolated from 4T1 tumor-bearing mice from the onset of primary tumorigenesis through the formation of macrometastatic lesions. Cryopreserved PyMT samples were collected from 6-14 weeks of age, fresh PyMT samples were collected at 6, 10 and 13 weeks of age (with age-matched wild-type controls)

overall_design: MULTI-seq barcoded CD45+ CD31- Ter119- mouse immune cells were isolated using FACS and analyzed using 3' scRNA-seq (10x Genomics)

first: Juliane

last: Winkler

first: Ansuman

last: Satpathy


overall_design: We performed me-RIP on P2 CD45/Ter119/CD31-Cxc12-DsRed+ bone marrow stromal cells


title: MUC1-C PROMOTES CHRONIC INFLAMMATION IN THE PROGRESSION OF HEAD AND NECK SQUAMOUS CELL CARCINOMAS

city: Buffalo

first: Mark

middle: D

last: Long

address: Elm & Carlton Streets

department: Bioinformatics & Biostatistics

institute: Roswell Park Comprehensive Cancer Center

summary: Mucin 1 (MUC1) is aberrantly expressed in adenocarcinomas of epithelial barrier tissues and contributes to their progression. Less is known about involvement of MUC1 in the pathogenesis of squamous cell carcinomas (SCCs). The present work demonstrates that the MUC1 gene is upregulated in advanced head and neck SCCs (HNSCCs) and, as determined by scRNA-seq analysis, is expressed in the malignant cell populations of HNSCC tumors. Studies of HNSCC cell lines demonstrate that the MUC1-C subunit regulates expression of (i) RIG-I and MDA5 pattern recognition receptors, (ii) STAT1 and interferon (IFN) regulatory factors, and (iii) downstream IFN-stimulated genes (ISGs). MUC1-C integrates STAT1 with induction of the ∆Np63 and SOX2 genes that are aberrantly expressed in HNSCCs. We also show that MUC1-C/STAT1 signaling activates the NFE2L2 gene and, in turn, MUC1-C binds directly to NRF2 in regulating redox balance. In extending these HNSCC cell dependencies, we demonstrate that MUC1-C is necessary for NOTCH3 expression, self-renewal capacity and tumorigenicity. These results indicate that MUC1-C is a common effector that integrates STAT1-mediated chronic inflammation with regulation of the ∆Np63, SOX2, NRF2 and NOTCH3 TFs. Our findings indicate that MUC1-C drives progression of the HNSCC CSC state and is a target for advanced HNSCC treatment.

overall_design: RNA sequencing analysis was used to identify changes in HNSCC cells (CAL27 & HSC3) after doxycycline inducible knockdown of MUC1

first: Nakashoji

last: Ayako

first: Donald

last: Kufe


title: RAS Pathway–Mutated Chronic Myelomonocytic Leukemia at the Single Cell Level

status: Public on Apr 21 2024

city: Los Angeles

first: Feiyang

last: Ma

address: 610, Charles Young Dr East, TLSB 3000C

department: Molecular Biology Institute

institute: UCLA

summary: RAS pathway mutations, which are present in 30% of patients with chronic myelomonocytic leukemia (CMML) at diagnosis, confer a high risk of resistance to and progression after hypomethylating agent (HMA) therapy, the current standard of care for the disease. Using single-cell, multi-omics technologies, we sought to dissect the biological mechanisms underlying the initiation and progression of RAS pathway–mutated CMML. We found that RAS pathway mutations induced the transcriptional reprogramming of hematopoietic stem and progenitor cells (HSPCs), which underwent proliferation and monocytic differentiation in response to cell-intrinsic and -extrinsic inflammatory signaling that also impaired immune cells’ functions. HSPCs expanded at disease progression and relied on the NF-KB pathway effector MCL1 to maintain their survival, which explains why patients with RAS pathway–mutated CMML do not benefit from BCL2 inhibitors such as venetoclax. Our study has implications for developing therapies to improve the survival of patients with RAS pathway–mutated CMML.

overall_design: Single cell RNA and ATAC sequencing in Lin-CD34+ stem and progenitor cells and mononuclear cells isolated from patients with CMML and RAS pathway mutations at the time of diagnosis or after failure of hypomethylation agent therapy or venetoclax-based therapy

first: Guillermo

last: Montalban Bravo

first: Simona

last: Colla


title: Multiomic profiling of medulloblastoma reveals subtype-specific targetable alterations at the proteome and N-glycan level

first: Julia

last: Neumann

summary:
Medulloblastomas (MBs) are malignant pediatric brain tumors that are molecularly and clinically heterogenous. The application of omics technologies – mainly studying nucleic acids – has significantly improved MB classification and stratification, but treatment options are still unsatisfactory. The proteome and their N-glycans hold the potential to discover clinically relevant phenotypes and targetable pathways. We compile a harmonized proteome dataset of 167 MBs and integrate findings with DNA methylome, transcriptome and N-glycome data. We show six proteome MB subtypes, that can be assigned to two main molecular programs: transcription/translation (pSHHt, pWNT and pGroup3myc), and synapses/immunological processes (pSHHs, pGroup3 and pGroup4). Multiomic analysis reveals different conservation levels of proteome features across MB subtypes at the DNA methylome level. Aggressive pGroup3myc MBs and favourable pWNT MBs are most similar in cluster hierarchies concerning overall proteome patterns but show different protein abundances of the vincristine resistance-associated multiprotein complex TriC/CCT and of N-glycan turnover-associated factors. The N-glycome reflects proteome subtypes and complex-bisecting N-glycans characterize pGroup3myc tumors. Our results shed light on targetable alterations in MB and set a foundation for potential immunotherapies targeting glycan structures.
This SuperSeries is composed of the SubSeries listed below.


title: An Improved SNAP-ADAR Tool Enables Efficient RNA Base Editing to Interfere with Post-translational Protein Modification

status: Public on Apr 19 2024

city: Tübingen

first: Thorsten

last: Stafforst

state: Baden-Württemberg

address: Auf der Morgenstelle 15

department: IFIB

institute: Eberhard Karls University of Tübingen

summary: RNA base editing applies endogenous or engineered adenosine deaminases to introduce adenosine-to-inosine changes into a target RNA in a highly programmable manner. Recently, notable success was achieved for the repair of disease-causing guanosine-to-adenosine mutations by means of RNA base editing. Here, we propose that RNA base editing could be broadly applied to perturb protein function by removal of regulatory sites of post-translational modification (PTM), like phosphorylation and/or acetylation sites. We demonstrate the feasibility of PTM interference (PTMi) on more than 70 PTM sites in various signaling proteins and identify key determinants for high editing efficiency and potent down-stream effects. Specifically, we demonstrate both negative and positive regulation of the JAK/STAT pathway by PTMi. To identify potent regulatory sites for PTMi, we applied an improved version of the SNAP-ADAR tool, which achieved high editing efficiency over a broad codon scope with tight control of bystander editing. The transient nature of RNA base editing enables the fast, dose-dependent (thus partial) and reversible manipulation of PTM sites, which is a key advantage over DNA editing approaches, where genetic compensation or lethality can conceal a phenotype. In summary, PTM interference might become a valuable field of application of RNA base editing in basic biology and medicine.

overall_design: To study the effect of PTMi on the interferon-induced JAK/STAT pathway,we compared the editing of six different PTM sites at the RNAs of five different players of the pathway (STAT1 Y701C, STAT1 T288A, STAT2 Y690C, JAK1 Y1034C, IFNGR1 Y457C and IFNAR1 Y466C) by transfection of the respective guide RNA (BB180, BB478, BB 349, BB355, BB304 and BB305 respectively) in HeLa-PB-SA1Q cells and global gene expression changes 24 h after IFN-α or -γ addition via next generation sequencing (DESeq analysis) was studied. THE RNA-seq data from all samples with IFN-α or -γ were compared for differential gene expression with the control sample with no IFN induction and an empty transfection.

first: Karthika

last: Kiran Kumar

first: Shubhangi

last: Singh

first: Stella

last: Schmelzle

first: Paul

last: Vogel

first: Carolin

last: Fruhner

first: Alfred

last: Hanswillemenke

first: Adrian

last: Brun

first: Jacqueline

last: Wettengel

first: Yvonne

last: Füll

first: Lukas

last: Funk

first: Valentin

last: Mast

first: Josephine

last: Botsch

first: Philipp

last: Reautschnig

first: Jin

last: Li


title: Contribution of VEGF-B-induced endocardial endothelial cell lineage in physiological versus pathological cardiac hypertrophy.

city: Helsinki

first: Ibrahim

last: Sultan

address: Haartmaninkatu 8

country: Finland

institute: Helsinki University

summary: ScRNA-seq was used to investigate the effects of autocrine versus paracrine VEGF-B signaling in the heart using transgenic mouse models. The paracrine model was further investigated in pregnancy-induced cardiac hypertrophy as well as in mice with ligation of the left anterior descending (LAD) coronary artery.

overall_design: Murine hearts were isolated and digested followed by analysis of the stromovascular fraction (SVF) by scRNA-seq, or FAC sorting of the cardiac enodthelial cells (ECs) by using FITC-tagged Pecam1 antibody and analysis by scRNA-seq.

first: Kari

last: Alitalo


title: Integrative transcriptomic and proteomic profiling of subconfluent and confluent cancer cells

city: Gothenburg

first: Aishe

last: Sarshad

address: Medicinaregatan 9C

department: Dept.of Biochemistry & Cell Biology

country: Sweden

institute: University of Gothenburg

summary: In this study we examine the impact of cell confluency on gene expression. We focused on Argonaute (AGO) protein dynamics and associated gene and protein expression in HEK293, A375, and SHSY5Y cell lines. As a measure of cell confluency, we observed a profound translocation of AGO2 into the nucleus. Therefore, we generated transcriptomic data using RNA sequencing to compare gene expression in subconfluent versus confluent cells, which highlighted significant alterations in gene regulation patterns directly corresponding to changes in cell density. Our study also encompasses miRNA profiling data obtained through small RNA sequencing, revealing miRNA expressional changes dependent on cellular confluency, as well as cellular localization. Finally, we derived proteomic data from mass spectrometry analyses following AGO1-4 immunoprecipitation, providing a comprehensive view of AGO interactome in both nuclear and cytoplasmic compartments under varying confluency. These datasets offer a detailed exploration of the cellular and molecular dynamics, influenced by cell confluency, presenting a valuable resource for further research in cellular biology, particularly in understanding the basic mechanisms of cell density in cancer cells.

overall_design: We performed miRNAseq in subconfluent and confluent HEK293, A375, SHSY5Y lysates from nuclear and cytoplasmic fraction.  In HEK293 cells AGO1-4 was also immunoprecipitated from subconfluent and confluent total lysates

first: V

last: Lobo

first: E

last: Shcherbinina

first: J

middle: O

last: Westholm

first: I

last: Nowak

first: H

last: Huang

first: D

last: Angeletti

last: Anastasakis

first: A


title: Investigating the circular RNA gene expression characteristics in salivary adenoid cystic carcinoma (SACC) cells

status: Public on Apr 18 2024

city: beijing

first: min

last: Fu

address: Peking University School and Hospital of Stomatology, No.22, Zhongguancun South Avenue, Haidian District

summary: To further investigate the circular RNA gene expression characteristics in SACC cell lines, we utilized the SBC Human (4*180K) ceRNA microarray to identify differential circular RNAs between SACC-83 and  SACC-LM cells.

overall_design: We extracted RNA from SACC-83 and SACC-LM cells for SBC Human (4*180K) ceRNA microarray analysis.

first: Xi-Yuan

last: Ge


title: Investigating the circular RNA gene expression characteristics in salivary adenoid cystic carcinoma (SACC) patients.

institute: Peking University School and Hospital of Stomatology

summary: To further investigate the circular RNA gene expression characteristics in SACC patients, we utilized the SBC Human (4*180K) ceRNA microarray to identify differential circular RNAs between SACC tissues and normal SMG tissues from the same patient.

overall_design: We extracted RNA from 10 pairs of SACC tissues and corresponding normal SMG tissues from the same patient for SBC Human (4*180K) ceRNA microarray analysis.

first: Min


title: Body mass index alone may not serve as a reliable indicator of the risk of developing rheumatoid arthritis

city: Shenzhen

first: jianhui

last: liang

state: Guangdong

address: 1068 Xueyuan Avenue, Nanshan District

institute: Shenzhen Institute of Science and Technology

summary: Obesity is recognized as a risk factor for triggering rheumatoid arthritis (RA), and it can worsen joint deformities and diminish the quality of life in patients with RA. The reduction of body weight in obese individuals is believed to alleviate RA symptoms。

overall_design: To further investigate whether obesity influences RA, we established collagen-induced-arthritis (CIA) models in mice using both regular and high-fat diets (HFD), and collected synovial tissue and performed RNA sequencing.

first: Yan

last: Wang

first: Qian

last: Xie

first: Jianhui

last: Liang

first: Kuangyang

last: Yang

first: Yanni

last: Shen

first: Xiao

last: Peng

first: Hao

last: Tan

first: Lichu

last: Liu


title: Integrative transcriptomic and proteomic profiling of the effects of cell confluency on gene expression

first: Aishe

last: Sarshad

summary: In this study we examine the impact of cell confluency on gene expression. We focused on Argonaute (AGO) protein dynamics and associated gene and protein expression in HEK293, A375, and SHSY5Y cell lines. As a consequence of cell confluency, AGO2 protein translocates into the nucleus. Therefore, we generated transcriptomic data using RNA sequencing to compare gene expression in subconfluent versus confluent cells, which highlighted significant alterations in gene regulation patterns directly corresponding to changes in cell density. Our study also encompasses miRNA profiling data obtained through small RNA sequencing, revealing miRNA expressional changes dependent on cellular confluency, as well as cellular localization. Finally, we derived proteomic data from mass spectrometry analyses following AGO1-4 immunoprecipitation, providing a comprehensive view of AGO interactome in both nuclear and cytoplasmic compartments under varying confluency. These datasets offer a detailed exploration of the cellular and molecular dynamics, influenced by cell confluency, presenting a valuable resource for further research in cellular biology, particularly in understanding the basic mechanisms of cell density in cancer cells.

overall_design: We performed gene expression profiling of subconfluent and confluent A375, SHSY5Y and HEK293 cells via RNA sequencing in 3 biological replicates, and carried out differential expression analysis between subconfluent and confluent cells of respective cell line.

first: V

last: Lobo

first: E

last: Shcherbinina

first: J

middle: C


title: Ribosomal protein RPL20B modulates cytokine production by macrophage in reponse to yeast

first: Kai

last: Li

address: 8700 Beverly Boulevard

institute: Cedar-Sinai Medical Center

summary: Phagocytosis is the process by which myeloid phagocytes bind and internalize potentially dangerous microbes. During phagocytosis, innate immune receptors and the associated signaling adapters are localized to the maturing phagosome compartment. We used proximity labeling of phagosomal contents (PhagoPL) to identify proteins localizing to phagosomes containing model yeast and bacteria and identified programmed death-ligand 1 (PD-L1) as a protein that specifically enriches in phagosomes containing yeast. We found that PD-L1 directly binds to yeast upon processing in phagosomes. By surface display library screening, we identified the ribosomal protein RPL20B as a fungal protein ligand for PD-L1. Using an auxin-inducible depletion system, we found that detection of RPL20B by macrophages influences production of a subset of inflammatory cytokines and chemokines produced when macrophages encounter yeast.

overall_design: To investigate whether host detection of RPL20B during phagocytosis has any influence on inflammatory responses of macrophages to yeast, GM-CSF primed bone marrow-derived macrophages (BMDMs) were stimulated with either RPL20B sufficient-yeast (w/oIAA yeast) or the PRL20B depleted-yeast (induced yeast) for 4h. Comparative gene expression profiling analysis were performed using data obtained from RNA-seq of w/oIAA or induced yeast-stimulated BMDMs. 3 biological replicates per condition were independently prepared for RNA-sequencing (different batches of BMDMs stimulated with different batches of yeast preparations).

first: David

last: Underhill


title: Extracellular Vesicles Containing circMYBL1 Induce CD44 in Adenoid Cystic Carcinoma Cells and Pulmonary Endothelial Cells to Promote Lung Metastasis.

first: min

summary: Adenoid cystic carcinoma (ACC) is a rare malignant epithelial neoplasm that arises in secretory glands and commonly metastasizes to the lungs. MYBL1 is frequently overexpressed in ACC and has been suggested to be a driver of the disease. Here, we identified a circRNA derived from MYBL1 pre-mRNA that accompanied overexpression of MYBL1 in ACC. Overexpression of circMYBL1 was correlated with increased lung metastasis and poor overall survival in ACC patients. Ectopic circMYBL1 overexpression promoted malignant phenotypes and lung metastasis of ACC cells. Mechanistically, circMYBL1 formed a circRNA-protein complex with CCAAT enhancer binding protein beta (CEBPB), which inhibited ubiquitin-mediated degradation and promoted nuclear translocation of CEBPB. In the nucleus, circMYBL1 increased the binding of CEBPB to the CD44 promoter region and enhanced its transcription. In addition, circMYBL1 was enriched in small extracellular vesicles (sEVs) isolated from the plasma of ACC patients. Treatment with sEVs containing circMYBL1 in sEVs enhanced pro-metastatic phenotypes of ACC cells, elevated the expression of CD44 in human pulmonary microvascular endothelial cells (HPMECs), and enhanced the adhesion between HPMECs and ACC cells. Moreover, circMYBL1 encapsulated in sEVs increased the arrest of circulating ACC cells in the lung and enhanced the lung metastatic burden. This data suggests that circMYBL1 is a tumor-promoting circRNA that could serve as a potential biomarker and therapeutic target in ACC.

overall_design: Total RNA was isolated from SACC-83 cells with circMYBL1 overexpression(OE) and vector control(Vec), followed by high-throughput sequencing to identify target genes.


title: Caspase-2 is essential for proliferation and self-renewal of nucleophosmin-mutated acute myeloid leukemia

status: Public on Apr 17 2024

city: Houston

first: Lisa

last: Bouchier-Hayes

state: Texas

address: 1102 Bates Ave

institute: Baylor College of Medicine

summary: Mutation in nucleophosmin (NPM1) causes relocalization of this normally nucleolar protein to the cytoplasm (NPM1c+). Despite NPM1 mutation being the most common driver mutation in cytogenetically normal adult acute myeloid leukemia (AML), the mechanisms of NPM1c+-induced leukemogenesis remain unclear. Caspase-2 is a pro-apoptotic protein activated by NPM1 in the nucleolus. Here, we show that caspase-2 is also activated by NPM1c+ in the cytoplasm and DNA damage-induced apoptosis is caspase-2-dependent in NPM1c+ but not in NPM1wt AML cells. Strikingly, in NPM1c+ cells, caspase-2 loss results in profound cell cycle arrest, differentiation, and down-regulation of stem cell pathways that regulate pluripotency including impairment in the AKT/mTORC1 and Wnt signaling pathways, and inhibition of Rictor cleavage. In contrast, there were minimal differences in proliferation, differentiation, or the transcriptional profile of NPM1wt cells lacking caspase-2. Our results show that caspase-2 is essential for proliferation and self-renewal of AML cells expressing mutated NPM1. This study demonstrates that caspase-2 is a major effector of NPM1c+ function.

overall_design:
To investigate the function of caspase-2 in NPM1wt and NPM1c+ AML we established OCI-AML2 (NPM1wt) and OCI-AML3(NPM1c+) cell lines with caspase-2 knocked out by CRISPR/Cas9
We performed RNAseq on the parental cells (OCI-AML2 and OCI-AML3) and two caspase-2 knockout clones of each)

first: Dharaniya

last: Sakthivel

first: Jonathan

last: Flanagan


title: DNA damage hotspots for the Top2i "ICRF193"

city: Illkirch

first: Tao

last: YE

address: 1 rue Laurent Fries

country: France

institute: IGBMC (CNRS/INSERM/UDS)

summary: In our project we are using the Top2 inhibitor, ICRF-193, in order to induce damage in NIH3T3 MEFs. We report the high-throughput identification of genomic hotspots for damage induction by ICRF-193, using γH2AX as a marker. We find that ICRF-193 primarily induces damage in repetitive sequences, which are known to reside in regions of heterochromatin, with a very small portion of damaged sites residing in regions of active genes in euchromatin. Overall, this study provides evidence that catalytic inhibition of Top2 enzymes by ICRF-193, leads to induction of damage in the nuclear sub-compartments of heterochromatin.

overall_design: Examination of genomic hotspots for damage induction by ICRF-193, using γH2AX as a marker

first: Michalis

last: Amoiridis

last: Ye

first: Evi

last: Soutoglou


title: ATAC-seq of E10.5 WT and Chd4-CMko mouse hearts

city: Chapel Hill

first: Wei

last: Shi

state: NC

address: 221 Fordham Hall

department: Biology

institute: UNC-Chapel Hill

summary: Purpose: The goal of this study is to identify the differential  cardiac chromatin accessibility between WT and cardiomyocyte conditional knockout (Chd4-CMko) hearts at E10.5 using ATAC-seq. Methods: Three hearts at E10.5 were pooled per genotype per replicate, and were then dissociated into single cells. 40,000 viable cells were taken for were lysed to isolate nuclei, which were treated with Tn5 transposase (Nextera DNA Sample Prep Kit, Illumina) to isolate DNA. Fragmented DNA was then amplified using bar-coded PCR primers and libraries were seuqenced. Results: 15736 differential peaks (2-fold change) were identified between E10.5 WT and Chd4-CMko hearts.

overall_design: Each embryonic mouse heart was dissected in cold PBS and individually cryopreserved in 200 mL of DMEM medium with 10% FBS and 10% DMSO (vol/vol) using a Mr. Frosty isopropyl alcohol chamber. Hearts from each genotype were thawed in a 37℃-water bath and pooled (three pooled  E10.5 hearts per genotype per biological replicate, four biological replicates for each genotype). Samples were pelleted at 1000g for 3 min at 4℃. The medium was aspirated, and the hearts were washed with cold PBS. Hearts were then dissociated in 200 mL of 0.05% Trypsin/EDTA at 37℃ for 10 min, with gentle agitation every 3 min. Dissociation was stopped by adding 400 mL of DMEM/10% FBS (vol/vol), and the samples were passed through a 100-micron cell strainer to remove connective tissue. Cells were quantified using Trypan blue, and 40,000 viable cells were taken for downstream procedures. Dissociated embryonic cardiac cells were lysed to isolate nuclei, which were treated with Tn5 transposase (Nextera DNA Sample Prep Kit, Illumina) to isolate DNA Fragmented DNA was then amplified using bar-coded PCR primers and libraries were pooled. Pooled libraries were then sequenced with the NovaSeq 6000 platform. Reads were aligned to the mm10 reference genomes using BWA-MEM and peaks were called using HOMERv4.10.3. Annotation and analysis were performed using ChIP-Seeker v1.20.0.

first: Lauren

middle: K

last: Wasson

first: Frank

last: Conlon


title: Pancreatic epithelial IL-17/IL-17RA signaling drives B7-H4 to promote tumorigenesis

status: Public on Apr 15 2024

first: Lourdes

middle: Susana

last: Castro Pando

address: 7777 Greenbriar Drive, Apt 2050

institute: UT MD ANDERSON CANCER CTR.

summary: IL-17 is required for the initiation and progression of pancreatic cancer, particularly in the context of inflammation, as previously shown by genetic and pharmacological approaches. The cellular compartment and downstream molecular mediators of IL-17-mediated pancreatic tumorigenesis have not been fully identified. We interrogated the cellular compartment required by generating transgenic animals with Interleukin 17 receptor A (IL-17RA) genetically deleted from the pancreatic epithelial compartment vs. the hematopoietic compartment via generation of IL-17RA-deficient (IL17-RA-/-) bone marrow chimeras, in the context of embryonically activated or inducible Kras. Deletion of IL-17RA from the pancreatic epithelial compartment, but not from hematopoietic, resulted in delayed premalignant lesions initiation and progression and increased CD8+ cytotoxic T cells infiltration to the tumor microenvironment. Absence of IL-17RA in the pancreatic compartment affected transcriptional profiles of epithelial cells, modulating stemness and immunological pathways. Interestingly, B7-H4, a known inhibitor of T cell activation encoded by the gene Vtcn1, was the most upregulated checkpoint molecule via IL17 early during pancreatic tumorigenesis, and its genetic deletion delayed pancreatic premalignant lesions development and reduced immunosuppression. We reveal pancreatic epithelial IL-17RA requirement for pancreatic tumorigenesis by reprogramming the immune pancreatic landscape which is partially orchestrated by regulation of B7-H4.

overall_design: Gene expression profile at single cell level of cells from the pancreas of 4 IL-17RA genetically deleted from the pancreatic epithelial compartment (KC; IL-17RAfl/fl )and 4 control (KC) mice. Single-cell suspensions were obtained by mincing murine pancreas followed by enzymatic digestion  and analyzed using 10z Genomics scRNAseq plataform.

first: Susana

last: Castro-Pando


title: EVOLUTIONARY CO-OPTION OF AN ANCESTRAL CLOACAL REGULATORY LANDSCAPE WITH THE EMERGENCE OF DIGITS AND GENITALS

city: Geneva

first: Aurelie

last: Hintermann

address: 30 quai Ernest-Ansermet

department: Genetics and Evolution

country: Switzerland

institute: University of Geneva

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

sample_organism:
Danio rerio
Mus musculus

platform_organism:
Danio rerio
Mus musculus

overall_design: Refer to individual Series


title: Dual role of signaling pathways in myeloma requires cell-type specific targeting of ligand-receptor interactions.

city: Boston

first: Julia

last: Frede

address: 450 Brookline Ave.

department: Medical Oncology

institute: Dana-Farber Cancer Institute

summary: Although most patients with multiple myeloma respond to treatment initially, therapy resistance develops almost invariably and only a subset of patients show durable responses to immunomodulatory (IMiD) therapies. While the immune microenvironment has been extensively studied in myeloma patients, its composition is currently not used as prognostic markers in clinical routine. We hypothesized that the outcome of immune signaling pathway engagement can be highly variable, depending on which two cellular populations participate in this interaction. This would have important prognostic and therapeutic implications, suggesting that it is crucial for immune pathways to be targeted in a specific cellular context.  To test this hypothesis, we investigated a cohort of 25 patients with newly diagnosed multiple myeloma. We examined the complex regulatory networks within the immune compartment and their impact on disease progression. Analysis of immune cell composition and expression profiles revealed significant differences in the B cell compartment associated with treatment response. Transcriptional states in patients with short time to progression demonstrated an enrichment of pathways promoting B cell differentiation and inflammatory responses, which may indicate immune dysfunction.  Importantly, the analysis of molecular interactions within the immune microenvironment highlights the dual role of signaling pathways, which can either be associated with good or poor prognosis depending on the cell types involved. Our findings therefore argue that therapeutic strategies targeting ligand-receptor interactions should take into consideration the composition of the microenvironment and the specific cell types involved in molecular interactions.

overall_design:
Cryopreserved CD138- bone marrow samples from 25 treatment-naïve newly-diagnosed myeloma (NDMM) patients were analyzed in this study. Low-input RNA-Sequencing libraries were prepared using the SmartSeq2 protocol from 100 cell pools sorted for different immune  cell populations. Pooled libraries were paired-end sequenced using a 75 cycle kit on a NextSeq 500 (Illumina) with an average sequencing depth of 1 million reads per sample.
-------------------------------
Authors state "As these are primary human samples, I am only submitting the processed data (counts matrix) rather than the raw fast files."

first: Pablo

last: Hernandez Lopez

first: Tushara

last: Vijaykumar

first: Praveen

last: Anand

first: Daniel

last: Auclair

first: Birgit

last: Knoechel

first: Jens

last: Lohr


title: Effect of MDK expressing Cervical Cancer cells(MS751) conditioned media in Human LEC

status: Public on Apr 16 2024

city: Shanghai

first: He

last: Fei

address: 801 Heqing Road

department: Department of Obstetrics and Gynecology

institute: Shanghai Fifth People’s Hospital

summary: Gene expression analysis in HLECs treated with gain of function of MDK in human Cervical Cancer cells(MS751) .

overall_design: Biological triplicates of hLEC treated for 24h with conditioned media of Cervical Cancer cells. Cell line MS751 cell line overexpressing MDK (MDK) or an empty vector (NC) were used to produce the conditioned media.

first: Fei

last: He


title: Comparison of tRNA abundance profiling in primary M395 melanoma cultures SENS or RES to 1µM Vemurafenib

city: Liège

first: Najla

last: El-Hachem

address: 1 avenue de l'hopital

department: Stem Cells

country: Belgium

institute: GIGA Institute

summary: In this project, we aim at studying differential tRNA expression or dynamics in sensitive and resistance melanoma primary cultures.

overall_design: To investigate changes in tRNA profiles in SENS and RES melanoma cells.

first: Pierre

last: Close


title: Ribosome sequencing experiment in control and VARS-depleted RES M395 melanoma cultures

summary: In this project, we identify novel translational targets of VARS in primary melanoma cultures by the use of ribosome sequencing.

overall_design: To investigate changes in translation from M395 RES cells depleted or not of VARS.

first: Coralie

last: Capron


title: Polysome profiling experiment in control and VARS-depleted RES M395 melanoma cultures

first: Arnaud

last: Lavergne

state: Liège

address: Avenue de l'hôpital 11

department: GIGA

institute: University of Liège

summary: In this project, we identify novel translational targets of VARS in primary melanoma cultures by the use of polysome profiling.

overall_design: To investigate changes in polysome bound RNA from M395 RES cells depleted or not of VARS.


title: Generation of human cerebral organoids with a structured outer subventricular zone

city: New York

first: Ryan

last: Walsh

address: 430 E 67th St

department: Developmental Biology

institute: MSKCC

summary:
Outer radial glia (oRG) emerged during mammalian evolution as cortical progenitor cells that directly support the development of an enlarged outer subventricular zone (oSVZ) and, in turn, the expansion of the neocortex. The in vitro generation of oRG is essential to model and investigate the underlying mechanisms of human neocortex development and expansion. By activating the STAT3 pathway using LIF, which is not produced in guided cortical organoids, we developed a cerebral organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The structured oSVZ is composed of progenitor cells expressing specific oRG markers such as GFAP, LIFR, HOPX and closely matching human oRG in vivo.
In this microenvironment, cortical neurons showed faster maturation with enhanced metabolic and functional activity. Incorporation of hPSC-derived brain vascular LIF-producing pericytes in cerebral organoids mimicked the effects of LIF treatment. These data indicate that the cellular complexity of the cortical microenvironment, including cell types of the brain vasculature, favors the appearance of oRG and provides a platform to routinely study oRG in hPSC-derived brain organoids.

overall_design: Cerebral organoids were treated with LIF or used to generate assembloids by fusion with pericyte spheroids. Single cell suspensions were obtained through papain dissociation at days 40, 60, 65 or 100 and used for single cell RNA-sequencing.

first: Elisa

last: Giacomelli

first: Gabriele

last: Ciceri

first: Chelsea

last: Rittenhouse

first: Maura

last: Galimbert

first: Youjun

last: Wu

first: James

last: Muller

first: Elena

last: Vezzoli

first: Johannes

last: Jungverdorben

first: Ting

last: Zhou

first: Roger

middle: A

last: Barker

last: Cattaneo

first: Lorenz

last: Studer

first: Arianna

last: Baggiolini


title: Single cell analysis of mutant Surfactant Protein-C injury and remodeling

city: Salt Lake City

first: Alessandro

last: Venosa

state: UT

department: Pharmacology and Toxicology

institute: University of Utah

summary: Mutation in the alveolar epithelial type 2 cell Surfactant Protein-C gene [SP-CI73T] is associated with pulmonary fibrosis (PF). We have previously demonstrated that inducible expression of the most common PF-linked mutation in the SP-C gene triggers inflammatory exacerbations of lung injury, progressing to fibrosis. We used single-cell sequencing to define the phenotype of epithelial, endothelial, immune and stromal cells accumulating in the lung at a time coordinated with peak injury (14 d post induction) and fibrotic remodeling (42 d post induction). The analysis showed at least 7 phenotypically distinct clusters of monocytes/macrophages during injury. Of these clusters, three exhibited distinctive pro-fibrotic signatures, based on expression of the prototypical markers TGFb1, osteopontin/SPP1 and fibronectin1. Notably SPP1+ cells expressed the highest levels of complement (C1qa, b, and c) and lipid associated genes (apoliporproteinE/ApoE, Cd36, Marco, Ldlr, Lrp1). We then set aim to examine the role of ApoE  in inflammatory cell activation by crossing the SP-CI73T line with ApoE knock out mice. Mice heterozygous and homozygous for mutant ApoE were included to this analysis (both 14 and 42 da post injury). Survival analysis demonstrate unchanged inflammatory cell composition, but flow cytometric analysis noted a shift in myeloid (neutrophil and eosinophil) and lymphoid (B cells) cell recruitment in the ApoE mutant mice. Together, these results demonstrate time related changes in monocyte/macrophage dynamics and supports the notion that lipid homeostasis may be involved in their pro-fibrotic activation.

overall_design: Single cell sequentinc of collagenase digested lung tissue from SP-C mutant mice at steady state and 14 d post injury

first: Philip

last: Moos


title: RNA sequencing for the gene expression profile changes of shPdgf-d and shControl mESCs

city: Guangzhou

first: Weisi

last: Lu

address: No.7 Jinsui Road

department: State Key Laboratory of Ophthalmology

institute: Zhongshan Ophthalmic Center, Sun Yat-sen University

summary: In this study, by RNA sequencing, we found that Pdgf-d knockdown significantly downregulated differentiation associated genes, particularly those of vascular development and angiogenesis. MAPK/ERK signaling related genes level were repressed as well. Collectively, our work reveals that PDGF-D is a novel player in ESCs regulation and suggests potential application for PDGF-D in ESCs culture and vascular lineage derivation.

overall_design: Examination of  differentially expressed genes in shPdgf-d and shControl mESCs, which were performed sequencing in triplicate.

first: Xuri

last: Li

first: Peipei

last: Xu


title: Impact of sunitinib resistance on clear cell renal cell carcinoma therapeutic sensitivity in vitro

city: Rockville

first: Alexis

last: Norris

state: MD

address: 7500 Standish Place

institute: US FDA

summary: Sunitinib resistance creates a major clinical challenge for the treatment of advanced clear cell renal cell carcinoma (ccRCC) and functional and metabolic changes linked to sunitinib resistance are not fully understood. We sought to characterize the molecular and metabolic changes induced by the development of sunitinib resistance in ccRCC by developing and characterizing the transcriptome of two human ccRCC cell lines resistant to sunitinib.

overall_design: Two human advanced renal cell carcinoma cell lines A498 and 786-O were purchased from American Type Culture Collection. Sunitinib (SU11248) malate was purchased from Selleckchem. For the development of sunitinib-resistant cells, parental cells were exposed to increasing concentrations of sunitinib. In brief, the RCC cell lines were treated with varying concentrations of sunitinib (0, 1, 2, 3, 4, 5, 7, 10, 20 and 50 μM). With the passage of time of every 4 days, it has been observed that A498 cells showed stable growth and eventually became confluent at a concentration of 2 µM, while 798-O cells showed stable growth to confluence at a concentration of 4 µM. It can be assumed that cells that grew to confluence had developed stable sunitinib-resistance after a period of 4 months or >20 passages. The cells were constantly in sunitinib containing medium throughout the selection process.

first: Ghosh

last: Susmita

first: Carole

last: Sourbier


title: Condensate-promoting ENL mutation induces tumorigenesis via chromatin remodeling [ChIP-seq and CUT&Tag]

status: Public on Apr 14 2024

city: Philadelphia

first: Liling

last: Wan

state: PA

address: 421 Curie Blvd

department: Cancer Biology

institute: University of Pennsylvania

summary: Gain-of-function mutations in the chromatin ‘reader’ ENL, identified in AML and Wilms tumor, have been shown to induce aberrant formation of transcriptional condensates in cellular systems. However, the precise role of these mutations and their condensate forming property in tumorigenesis remains unclear. By creating a conditional knock-in mouse model for the most prevalent ENL mutation, we establish ENL mutant as a bona fide oncogenic driver of acute myeloid leukemia in vivo. Heterozygous expression of ENL mutant perturbs the normal hematopoietic hierarchy and results in the aberrant expansion of myeloid progenitors with increased self-renewal property. Furthermore, the ENL mutant remodels histone modifications to alter differentiation processes and drive oncogenic gene expression during hematopoietic development. Importantly, targeted point mutagenesis to disrupt the condensate formation property completely abolishes ENL mutant’s oncogenic function in hematopoietic stem and progenitor cells (HSPCs). Lastly, short-term treatment with a small molecule inhibitor that blocks the acetyl-binding activity of ENL mutant reverts its impact on chromatin and significantly delays leukemia development in mice. Our studies reveal the crucial biological function of mutation-induced transcriptional condensates in chromatin regulation and cancer in vivo and provide proof-of-concept for targeting of pathogenic condensates as a promising therapy for certain cancers.

overall_design: To determine chromatin alterations induced by Enl-T1 in hematopoietic populations

first: Yiman

last: Liu

first: Qinglan


title: EVOLUTIONARY CO-OPTION OF AN ANCESTRAL CLOACAL REGULATORY LANDSCAPE WITH THE EMERGENCE OF DIGITS AND GENITALS [ATAC-seq]

first: Aurelie

last: Hintermann

summary: The existence of homologies between fins and tetrapod limb skeletal parts as well as of the nature of those mechanisms underlying the transition of the former towards the latter, have been a rich source of discussion for more than a century. While the recent use of gene expression patterns to try and infer evolutionary scenarios has been a popular and successful approach, in particular the distribution of Hox transcript domains, it has failed to provide clearcut evidence as to whether fishes do have bony elements related by ancestry to tetrapod hands and feet. In tetrapods, posterior Hoxd genes transcription in digits is controlled by a well-characterized series of enhancers forming a large regulatory landscape, which has its syntenic counterpart in zebrafish. We show here that the deletion of the orthologous landscape in fishes does not affect the transcription of these genes in fin buds. Instead, it abrogates hoxd expression in the cloaca, an essential structure related to the mammalian uro-genital sinus. We also report that Hoxd gene function in the mammalian uro-genital sinus depends on enhancers located in the same regulatory landscape and thus conclude that an ancestral Hox ‘cloacal’ regulation was co-opted, in tetrapod, as a playground to subsequently accompany the evolution of both external genitals and digits regulatory landscapes, along with the emergence of these developmentally and phylogenetically related structures.

overall_design:
To assay chromatin accessibility, we conducted ATAC-seq experiments. We collected different mouse and zebrafish tissues. Samples were processed as described in 10.1038/nmeth.2688.

UPDATE: [08-12-2024] For Samples GSM7976428, GSM7976429, and GSM7976430, the stage was corrected from 16hpf to 30hpf in the titles, stage characteristics, and processed data files. The raw data and processed data associated with these Sample accession numbers did not change.

first: Aurélie

first: Chase

last: Bolt

first: Lucille

last: Lopez-Delisle

first: Sandra

last: Gitto

first: Bénédicte

last: Mascrez

first: Denis

last: Duboule


title: Transcriptome analysis of cytotrophoblasts and syncytiotrophoblasts derived from trophoblast stem cells, mock-infected or infected with Toxoplasma gondii

status: Public on Apr 12 2024

city: Pittsburgh

first: Rafaela

middle: Jose

last: da Silva

state: Pennsylvania

address: 4249 Fifth Avenue

department: Biological Sciences

institute: University of Pittburgh

summary: We tested the relevance of an established stem-cell-derived model of trophoblast development to Toxoplasma gondii infection. Human trophoblast stem cells were cultured under conditions suitable for cytotrophoblast cells or for the differentiation into syncytiotrophoblasts, and subsequently infected with T. gondii. RNA-seq data from both mock-treated and infected cells revealed differences between cell types and their respective responses to T. gondii infection.

sample_organism:
Toxoplasma gondii
Homo sapiens

platform_organism:
Toxoplasma gondii
Homo sapiens

overall_design: Human trophoblast stem cells were cultured as cytotrophoblast cells and also differentiated into syncytiotrophoblast using a differentiation medium. Cells were mock-infected or infected with T. gondii (RH-YFP strain) with a multiplicity of infection (MOI) of 5. RNA-seq was performed in order to evaluate the differences in transcriptome between both cells and the changes after infection. The experiment was designed with 3 biological replicates for each condition.


title: Gene expression profiles at single cell level of  lung epithelial cells from the Cracd WT and Cracd KO mice

status: Public on Apr 13 2024

first: Bongjun

last: Kim

address: 6565 MD Anderson BLVD

department: Experimental Radiation Oncology

institute: MD Anderson Cancer Center

summary: Tumor cell plasticity contributes to intratumoral heterogeneity and therapy resistance. Through cell plasticity, lung adenocarcinoma (LUAD) cells transform into neuroendocrinal (NE) cell-like tumor cells. However, the mechanisms of NE cell plasticity remain unclear. Depletion of CRACD tumor suppressor results in the de-repression of NE-related gene expression in the pulmonary epithelium. Similarly, Cracd KO augments intratumoral heterogeneity with upregulation of NE markers in LUAD animal models. Single-cell transcriptomic analysis showed that Cracd KO upregulates NE genes in AT2 pulmonary epithelial cells, accompanied by a cell de-differentiation state and activation of Sox2, Oct4, and Nanog pathways. The single-cell transcriptomes of LUAD patient tumors recapitulate that the distinct LUAD NE cell cluster is co-enriched with NE genes and SOX2, OCT4, and NANOG pathways. This study reveals an unexpected role of CRACD tumor suppressor in restricting cell plasticity inducing cell de-differentiation, which provides new insights into NE cell plasticity of LUAD.

overall_design: Lungs were harvested from euthanized mice (Cracd WT or Cracd KO) after perfusing 10 ml of cold phosphate-buffered saline (PBS) into the right ventricle. The lung was digested in Leibovitz’s medium (Invitrogen) with 2 mg/mL Collagenase Type I (Worthington), 2 mg/mL Elastase (Worthington), and 2 mg/mL DNase I (Worthington) at 37 °C for 45 min. The tissue was triturated with a pipet every 15 min of digestion until homogenous. The digestion was stopped with FBS (Invitrogen) to a final concentration of 20%. The cells were filtered with a 70 μm cell strainer (Falcon) and spun down at 5,000 r/min for 1 min. The cell pellet was resuspended in red blood cell lysing buffer (Sigma) for 3 min, spun down at 5,000 r/min for 1 min, and washed with 1 mL ice-cold Leibovitz’s medium with 10% FBS. In single-cell RNA sequencing (scRNA-seq), digested lung cells were resuspended in 400 μl of buffer with 5 μl of anti-CD31-FITC (BD Biosciences, CA, USA), 5 μl of anti-CD45-APC (BD Biosciences), and 5 μl of anti-CD326 (EpCAM)-PE-Cy7 (BD Biosciences) and incubated for 30 min at 4 C. Cells were then washed twice, followed by sorting of the epithelial cells (EpCAM+ / CD31- / CD45-) by fluorescence-activated cell sorting at the Cytometry and Cell Sorting Core at BCM.

first: Shengzhe

last: Zhang

first: Yuanjian

last: Huang

first: Jae-Il

last: Park


title: Condensate-promoting ENL mutation induces tumorigenesis via chromatin remodeling [RNA-seq]

overall_design: To determine transcriptional changes induced by Enl-T1 in hematopoietic populations

last: Li


title: Lig3 is the primary ligase in AEJ -mediated V(D)J recombination

city: Stanford

first: Jinglong

last: Wang

state: California

address: 269 Campus Drive

department: Radiation Oncology

institute: Stanford Medicine

summary: The critical role of the alternative end joining (AEJ) repair mechanism in V(D)J recombination for B and T lymphocyte development, particularly in the absence of the key non-homologous end joining (NHEJ) component Ku70, is not well understood. AEJ involves functionally redundant factors such as Parp1 vs Polθ and Lig3 vs Lig1, making it unclear which factors are responsible for repairing V(D)J recombination. Additionally, the regulatory mechanism of AEJ remains enigmatic. In this study, we utilized the murine Igκ antigen locus as a model and employed gene editing and HTGTS-Seq pipelines to systematically evaluate potential factors involved in V(D)J recombination. We assessed various aspects of recombination, including efficiency, V gene usage, end resection, joining fidelity, repair patterns, and inter-chromosome translocation. Our findings highlight the Parp1-XRCC1-Lig3 axis as a key mediator of V(D)J recombination, regulated by 53BP1 and ATM. This study advances our understanding of AEJ in DNA damage repair and contributes to our knowledge of lymphocyte development in immunology.

overall_design: Various murine proB cells including WT and Ku70-/-, with/without indicated inhibitors were arrested by STI-571 to induce V(D)J recombination, the V-J recombination in IgK locus were systematically captured using JK1CE and JK1SE by HTGTS.

first: Cheyenne

last: Sadeghi

first: Richard

last: Frock


title: Condensate-promoting ENL mutation induces tumorigenesis via chromatin remodeling

first: Liling

last: Wan


title: EphB2 Receptor Promotes Dermal Fibrosis in Systemic Sclerosis

status: Public on Apr 10 2024

first: Patrice

last: Mimche

state: Utah

address: 26 North Medical Dr

summary:
Objectives: Eph/Ephrin cell-cell signaling is emerging as a key player in tissue fibrogenesis. The aim of this study was to test the hypothesis that the receptor tyrosine kinase EphB2 mediates dermal fibrosis in systemic sclerosis (SSc).
Methods: We assessed normal and SSc human skin biopsies for EphB2 expression. The in vivo role of EphB2 in skin fibrosis was investigated by subjecting EphB2-knockout mice to both bleomycin-induced and tight skin (Tsk1/+) genetic mouse models. EphB2 kinase-dead and overactive point mutant mice were used to evaluate the role of EphB2 forward signaling in bleomycin-induced dermal fibrosis. In vitro studies were performed on dermal fibroblasts from SSc patients and healthy controls, which was followed by in vivo analysis of fibroblast-specific EphB2 deficient mice.
Results: Expression of EphB2 is upregulated in SSc skin tissue and explanted SSc dermal fibroblasts compared to healthy controls. EphB2 expression is elevated in two animal models of dermal fibrosis. In mice, EphB2 drives dermal fibrosis in both the bleomycin and the Tsk1/+ models of skin fibrosis. EphB2 forward signaling is a critical mediator of dermal fibrosis. Transforming growth factor-β (TGFβ) cytokines upregulate EphB2 in dermal fibroblasts via non-canonical TGFβ/SMAD signaling and silencing EphB2 in dermal fibroblasts is sufficient to dampen TGFβ-induced fibroblast-to-myofibroblast differentiation. Moreover, mice with fibroblast-specific deletion of EphB2 showed impaired fibroblast-to-myofibroblast differentiation and reduced skin fibrosis upon bleomycin challenge.
Conclusion: Our data implicate EphB2 overexpression and kinase-mediated forward signaling in the development of dermal fibrosis in SSc. EphB2 thus represents a potential new therapeutic target for SSc.

overall_design: Human dermal fibroblasts (70% confluency) were transfected with EPHB2 Mission®-esiRNA (Sigma Aldrich, USA) delivered into the cells using the lipofectamine® RNAiMAX (Invitrogen, USA) according to the manufacturer’s instruction. Mission®-esiRNA complexes targeting eGFP were used as nontargeting control. After 48H incubation, transfected NHDF were stimulated with recombinant human TGF-β1 at 10 ng/ml for another 24H. Cells were harvested, total RNA was extracted using RNA Stat60® (Tel-Test Inc, USA) following standard phenol-chloroform procedure, and further purified using the NucleoSpin RNA-II purification kit (Machery-Nagel) following the manufacturer’s instructions.

middle: N

first: Severin

last: Kamdem

first: Erika

middle: S

last: Egal

first: Tracy

last: Frech


title: A syngeneic spontaneous zebrafish model oftp53-deficient, EGFRviii, and PI3KCAH1047R-driven glioblastoma reveals inhibitory roles for inflammation during tumor initiation and relapsein vivo

city: Toronto

first: Madeline

middle: Nicole

last: Hayes

state: Ontario

address: 72 Elm St

department: DSCB

institute: Sickkids Research

summary: To build a patient-relevantin vivomodel of human glioblastoma, we expressed common oncogenic variants including activated human EGFRviii or KRAS,and PI3KCAH1047Runder the control of the radial glial-specific promoterher4.1in syngeneictp53loss-of-function mutant zebrafish.Robust tumor formation was observed prior to 45 days of life, with a gene expression signature similar to human glioblastoma of the mesenchymal subtype, along with a strong inflammatory component. Within early stage tumor lesions, and in an intact and endogenous tumor microenvironment, we visualized infiltration of phagocytic cells, as well as internalization of tumor cells bympeg1.1:GFP+ microglia/macrophages, suggesting negative regulatory pressure by pro-inflammatory cell types on tumor growth at early stages of glioblastoma initiationin vivo. Furthermore, CRISPR/Cas9-mediated gene targeting of master inflammatory transcription factorsirf7andirf8led to increased tumor formation in the primary context, while suppression of microglial/macrophage activity led to enhanced tumor cell engraftment following transplantation into otherwise immune competent zebrafish hosts. Altogether, we developed a genetically-relevant model of aggressive human glioblastoma and harnessed the unique advantages of zebrafish including live imaging, high-throughput genetic and chemical manipulations to highlight important tumor suppressive roles for the innate immune system on glioblastoma initiation, with important future significance for therapeutic discovery and optimizations.

overall_design: To capture broad transcriptomic differences in tumor-burdened brains versus normal brains, we performed bulk RNA sequencing and gene expression analysis of whole brains and sorted cells.  EGFRviii derived tumors were induced by microinjection of EGFRviii, PI3KCAH1047R, and mScarlet constructs (defined as EPS). KRAS derived tumors were induced by microinjection of KRAS, PI3KCAH1047R, GFP constructs (defined as KPG). Three whole brains positive for fluorescent tumors (EPS_1-3, KPG_1-3), alongside three control brains from injected siblings who did not develop tumors (CTRL_1-3), were dissected for each condition for bulk RNA sequencing. We also performed fluorescent activated cell sorting on pooled dissociated brains from the EPS condition, resulting in enrichment for mScarlet postive tumor cells (CS_Pos) and mScarlet negative cells (CS_Neg) for comparative analysis.

first: Bret

last: Pearson

first: Alex

last: Weiss

first: Cassandra

last: D'Amata


title: Dynamics and consequences of widespread transcript isoform changes during neuronal differentiation [ONT-Seq]

city: Berlin

first: Dr. Andreas

last: Mayer

address: Ihnestraße 63-73

institute: Max-Plack-Institute for Molecular Genetics

summary: Analysis of isoform expression dynamics during in vitro neuronal differentiation

overall_design: Time-course transcript-level expression profiling with high throughput sequencing


title: Dynamics and consequences of widespread transcript isoform changes during neuronal differentiation

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

overall_design: Refer to individual Series


title: On-chip neural induction boosts neural stem cell commitment: toward a pipeline for iPSC-based therapies

status: Public on Apr 08 2024

city: Solna

first: Anna

last: Herland

address: Tomtebodagatan 23

institute: SciLifelab KTH

summary: The clinical translation of induced pluripotent stem cells (iPSCs) holds great potential for personalized therapeutics. However, the current workflow to generate iPSCs is expensive, time-consuming, and requires standardization. We present a simplified microfluidic approach for reprogramming fibroblasts into iPSCs and their subsequent differentiation into neural stem cells (NSCs). Our method exploits microphysiological technology, providing a 100-fold reduction in reagents for reprogramming and a 9-fold reduction in number of input cells. The iPSCs generated from microfluidic reprogramming of fibroblasts show upregulation of pluripotency markers and downregulation of fibroblast markers, on par with those reprogrammed in the standardized well-conditions. The NSCs differentiated in microfluidic chips show upregulation of neuroectodermal markers (ZIC1, PAX6, SOX1), highlighting their propensity for nervous system development. We then compare the cells obtained on conventional well plates and microfluidic chips for reprogramming and neural induction by bulk RNA sequencing. Pathway enrichment analysis of NSCs generated in the chip showed neural stem cell development enrichment and boosted commitment to the neural stem cell lineage in the initial phases of neural induction, attributed to the confined environment in a microfluidic chip. This method provides a cost-effective pipeline to reprogram and differentiate iPSCs which can be made compliant with the current good manufacturing practices for therapeutics.

overall_design:
To perform the reprogramming of somatic cells intro induced pluripotent stem cells on a microfluidic chip and compare it to a conventional well plate format.
Further, we performed the differentiation of iPSCs into ectodermal lineage using dual-SMAD inhibition protocol on the microfluidic chip and conventional well plates to study the differences between the choice of culture format.

first: Saumey

last: Jain

first: Dimitrios

last: Voulgaris

first: Surangrat

last: Thongkorn

first: Rick

last: Hesen

first: Alice

last: Hägg

first: Mohsen

last: Moslem

last: Falk


title: Dynamics and consequences of widespread transcript isoform changes during neuronal differentiation [RNA-Seq]


title: MUC1-C REGULATES NEAT1 lncRNA EXPRESSION AND PARASPECKLE FORMATION IN CANCER STEM CELLS

first: Atrayee

last: Bhattacharya

state: Massachusetts

address: 450 Brookline Avenue, Boston 02215

institute: Dana Farber Cancer Institute, Harvard Medical School

summary: MUC1-C subunit plays an essential role in regulating NEAT1 expression. MUC1-C activates the NEAT1 gene with induction of the NEAT1_1 and NEAT1_2 isoforms by NF-kB- and MYC-mediated mechanisms. MUC1-C/MYC signaling also induces expression of the SFPQ, NONO and FUS RNA binding proteins (RBPs) that associate with NEAT1_2 and are necessary for paraspeckle formation.

overall_design: RNA Seq in Triple Negative Breast Cancer Cell line with NEAT1 siRNA silencing


title: scRNAseq analysis of cytokine-treated human islet cells

city: Charleston

first: Jennifer

last: Stancill

state: South Carolina

address: 173 Ashley Avenue, CR513

department: Biochemistry and Molecular Biology

institute: Medical University of South Carolina

summary: Pancreatic beta-cells are essential for survival, being the only cell type capable of insulin secretion. While they are believed to be vulnerable to damage by inflammatory cytokines such as interleukin-1 beta (IL-1beta) and IFN-gamma, we have recently identified physiological roles for cytokine signaling in rodent beta-cells that include that stimulation of antiviral and antimicrobial gene expression and the inhibition of viral replication. In this study, we examine cytokine-stimulated changes in gene expression in human islets using single-cell RNA sequencing. Surprisingly, the global responses of human islets to cytokine exposure were remarkably blunted compared to our previous observations in the mouse. The small population of human islet cells that were cytokine responsive exhibited increased expression of IL-1beta-stimulated antiviral guanylate binding proteins, just like in the mouse. Most human islet cells were not responsive to cytokines, and this lack of responsiveness was associated with high expression of genes encoding ribosomal proteins. We further correlated the expression levels of RPL5 with stress response genes, and when expressed at high levels, RPL5 is predictive of failure to respond to cytokines in all endocrine cells. Further, we postulate that donor cause of death and isolation methodologies may contribute to stress of the islet preparation. Our findings indicate that induction of stress responses in human islets (associated with isolation and/or cause of death) limit cytokine stimulated gene expression, and we urge caution in the evaluation of studies that have examined cytokine-stimulated gene expression in human islets without evaluation of stress-related gene expression.

overall_design: Eight samples each (no cytokine treatment, 6 hr IL-1b, 6 hr IFNg, 6 hr IL-1b + IFNg, 18 hr IL-1b, 18 hr IL-1b + IFN-g, 18 hr IL-1b + IFN-g + NMMA, and 18 hr NMMA) from three experimental replicates of human islets were prepared using 10x Genomics Chromium platform

first: John

last: Corbett


title: Effect of mechanical stimulation on immune function of dendritic cells (DCs)

city: Paris

first: Wail

last: Zeitouni

address: 29, rue du Faubourg Saint-Jacques

department: Paris Biotech Santé

institute: GenoSplice technology

summary:
Address the effect of mechanical stimulation by confinement on the status of activation of dendritic cells and the role of cPLA2 in orchestrating this response.
We performed gene expression analysis using data obtained from RNA-seq of 3 independent experiment of DCs from WT and KO of cPLA2.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for bone marrow derived DCs from 2 mouse models.

first: Zahraa

last: Alraies

first: Benoit

last: Albaud

first: Patricia

last: Legoix


title: Oxylipins and metabolites from pyroptotic cells act as promoters of tissue repair

status: Public on Apr 07 2024

city: Ghent

first: Parul

last: Mehrotra

address: Zwijnaarde Technology Lab

department: Center for Inflammation Research

institute: VIB - UGhent

summary: Pyroptosis is a lytic cell death mode that helps limit the spread of infections and is also linked to pathology in sterile inflammatory diseases and autoimmune diseases. During pyroptosis, inflammasome activation and the engagement of caspase-1 lead to cell death, along with the maturation and secretion of the inflammatory cytokine interleukin 1β (IL-1β). The dominant effect of IL-1β in promoting tissue inflammation has clouded the potential influence of other factors released from pyroptotic cells. Here, using a system where macrophages are induced to undergo pyroptosis without IL-1β/⍺  release (denoted Pyro-1), we uncover unexpected beneficial effects of the Pyro-1 secretome. First, we noted that the Pyro-1 supernatants upregulated gene signatures linked to migration, cellular proliferation, and wound healing. Consistent with this gene signature, Pyro-1 supernatants boosted migration of primary fibroblasts and macrophages, as well as faster wound closure in vitro, and improved tissue repair in vivo. In mechanistic studies, lipidomics and metabolomics of the Pyro-1 supernatants identified the presence of both oxylipins and metabolites, linking them to pro-wound healing effects. Focusing specifically on the oxylipin prostaglandin E2 (PGE2), we find that PGE2 synthesis is induced  de novo  during pyroptosis, downstream of caspase-1 activation, and COX2 activity; further, PGE2 synthesis occurs late in pyroptosis, with its release dependent on Gasdermin D pores opened during pyroptosis. As for the pyroptotic metabolites, they link  to immune cell infiltration into the wounds, and polarization to CD301+ macrophages. Collectively, these data advance the concept that the pyroptotic secretome 1 Mehrotra et al., Page No.  possesses oxylipins and metabolites with tissue repair properties that may be harnessed therapeutically.

overall_design: Supernatants from Live, apoptotic or pyroptotic cells were generated by triggering them with LeTox (and their live untreated coounterparts). Mo BMDMs were treated for 4h with the supernatants thus generated followed by RNAseq.

first: Sophia

last: Maschalidi

first: Laura

last: Boeckaerts

first: Christian

last: Maueröder

first: Rochelle

last: Tixeira

first: Jonathan

last: Pinney

first: Javier

last: Burgoa Cardás

first: Vladimir

last: Sukhov

first: Yunus

last: Incik

first: Christopher

middle: J

last: Anderson

first: Bing

last: Hu

first: Burcu

last: Keçeli

first: Amanda

last: Goncalves

first: Lieselotte

last: Vande Walle

first: Nina

last: Van Opdenbosch

first: Alexey

last: Sergushichev

first: Esther

last: Hoste

first: Umang

first: Mohamed

last: Lamkanfi

first: Kodi

last: Ravichandran


title: Yishen Jiangzhuo decoction attenuates cisplatin-induced acute kidney injury by inhibiting inflammation, oxidative stress, and apoptosis through the TNF signal pathway

status: Public on Apr 06 2024

city: Fuzhou

first: Dengyong

last: Zheng

address: 282 Wusi Road Gulou District

department: Department of Nephrology

institute: The Second Affiliated People's Hospital of Fujian University of Traditional Chinese Medicine

summary: We aimed to investigate the therapeutic effects and mechanisms of Yishen Jiangzhuo decoction (YSJZD) in a mouse model of cisplatin-induced acute kidney injury (AKI). The mice were divided into the NC, cisplatin, and cisplatin + YSJZD groups. A concentration-dependent effect of YSJZD on cisplatin-induced AKI was observed, and the optimal concentration for intervention was calculated. Changes in blood urea nitrogen and serum creatinine levels combined with hematoxylin & eosin and periodic acid-Schiff staining, and transmission electron microscopy observations indicated that YSJZD enhanced renal function, reduced pathological injury, and protected renal tubular epithelial cells in cisplatin-induced AKI mice. The results of the transcriptomic and enrichment analyses showed that the mechanisms of YSJZD may be associated with inflammation, oxidation, apoptosis, and the TNF signal pathway. Immunofluorescence, oxidative stress index, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and western blotting (WB) revealed that YSJZD downregulated apoptosis in the renal tissues of AKI mice, and further decreased the expression levels of p-p65, p-p38 MAPK, tumor necrosis factor -α, cleaved-caspase-3, and malondialdehyde, while increasing the levels of SIRT3, GSH, and SOD. Overall, the results showed that YSJZD could effectively abrogate cisplatin-induced AKI in mice through mechanisms primarily related to its anti-inflammatory, antioxidative, and antiapoptotic effects by inhibited the TNF signal pathway. YSJZD warrants further investigation as a clinical empirical prescription.

overall_design: The mice were divided into the NC, cisplatin, and cisplatin + YSJZD groups.Three cortical kidney tissue samples were randomly selected from each group. Total ribonucleic acid (RNA) was isolated from 50 mg of kidney tissue.Only high-quality whole RNA samples were used to generate complementary deoxyribonucleic acid (cDNA) libraries.BGI  Co., Ltd. prepared a cDNA library and performed RNA-Seq on a DNBSEQ platform in accordance with the manufacturer's instructions.DESeq2 software was used to identify differentially expressed genes (DEGs) between groups, with the threshold set at a fold-change threshold ≥ 2 and a Q-value < 0.05. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) enrichment analyses of DEGs were performed using the phyper function in R code.

first: Xinglin

last: Ruan

first: Qiang

last: Wu

first: Yuliang

last: Qiu

first: Shiwei


title: Protein kinase CK2 modulates NRL-mediated gene expression in rod photoreceptors of mammalian retina

city: Bethesda

first: Zachary

last: Batz

address: 6 Center Dr Bldg 6, Rm 303

department: NEI

institute: NIH

summary: Maf-family basic motif leucine zipper protein NRL specifies rod photoreceptor cell fate during retinal development and, in concert with homeodomain protein CRX and other regulatory factors, controls the expression of most rod-expressed genes including the visual pigment gene RHO (rhodopsin). Transcriptional regulatory activity of NRL is modulated by post-translational modifications (PTMs), specially phosphorylation, and mutations in phosphosites can lead to retinal degeneration. During our studies to elucidate NRL-mediated transcriptional regulation, we identified Protein Kinase CK2 in NRL-enriched complexes bound to RHO promoter-enhancer regions and in NRL-enriched high molecular mass fractions from the bovine retina. The presence of CK2 in NRL complexes was confirmed by co-immunoprecipitation from developing and adult mouse retinal extracts. Bioinformatic and mass spectrometry analysis indicated potential phosphorylation of NRL at Ser117 residue by CK2. Co-transfection of Csnk2a1 cDNA encoding CK2 with NRL and CRX reduced the bovine Rho promoter-driven luciferase expression in HEK293 cells, and mutagenesis of NRL-Ser117 residue to Ala restored the reporter gene expression. In concordance, overexpression of CK2 in the mouse retina in vivo by electroporation resulted in reduction of Rho promoter-driven DsRed reporter gene expression. RNA-seq analysis of CK2-electroporated retina revealed altered expression of many transcriptional targets of NRL. Thus, our studies demonstrate an important role of CK2 in context-dependent fine-tuning of NRL function, consequently modulating the expression of rod photoreceptor genes.

overall_design: Electroporated areas of the retinas were collected at postnatal day 8 and disassociated with dissociation solution (30 U/mL papain, HBSS pH 7.4, 1 mg/mL glucose, 10 mM Hepes, 100 U/mL DNase I,  5 µg/mL superoxide dismutase, 5 µg/mL catalase, 10 µg/mL D-alpha-tocopherol acetate, 1 mg/mL glucoscysteine-HCl, 5 µg/mL superoxide dismutase, 50 µg/mL gentamycin). Intact GFP positive rod photoreceptors were manually collected with a micromanipulator. Libraries were generated from 50-100 isolated rod cells using SMARTer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian (Takara Bio USA, Inc). We prepared three independent RNA samples for each treatment and performed paired-end sequencing using HiSeq2500 (Illumina, San Diego, USA).

first: Sharda

middle: P

last: Yadav

first: Xulong

last: Liang

first: Angel

last: Aponte

first: Jacob

last: Nellissery

first: Anand

last: Swaroop


title: Maize root bacteria degrade host-specialized metabolites through the lactonase BxdA

status: Public on Apr 05 2024

city: Bern

first: Marco

last: Kreuzer

state: Bern

address: Baltzerstrasse 6

institute: University of Bern

summary: Root exudates contain specialised metabolites that affect the plant’s root microbiome. How host-specific microbes cope with these bioactive compounds, and how this ability shapes root microbiomes, remains largely unknown. We investigated how maize root bacteria metabolise benzoxazinoids, the main specialised metabolites of maize. Diverse and abundant bacteria metabolised the major compound in the maize rhizosphere MBOA and formed AMPO. AMPO forming bacteria are enriched in the rhizosphere of benzoxazinoid-producing maize and can use MBOA as carbon source. We identified a novel gene cluster associated with AMPO formation in microbacteria. The first gene in this cluster, bxdA encodes a lactonase that converts MBOA to AMPO in vitro. A deletion mutant of the homologous bxdA genes in the genus Sphingobium, does not form AMPO nor is it able to use MBOA as a carbon source. BxdA was identified in different genera of maize root bacteria. Here we show that plant-specialised metabolites select for metabolisation-competent root bacteria. BxdA represents a novel benzoxazinoid metabolisation gene whose carriers successfully colonize the maize rhizosphere and thereby shape the plant’s chemical environmental footprint

overall_design: To find genes that are involved in the transformation of MBOA to AMPO we performed a RNA Seq experiment where five replicates of bacterial culture where either exposed to MBOA or DMSO (control condition) in the growth medium. The RNA was harvested after 16 hours of growth.