title: Immunomodulatory effects of microbiota-derived propionate in systemic lupus erythematosus

status: Public on Sep 30 2024

city: League city

first: Hui

last: Wang

state: Texas

address: 301 University Blvd.

country: USA

institute: UTMB

type: Expression profiling by high throughput sequencing

summary: We performed RNAseq on total RNA extracted from kidney tissues from MRL-lpr micetreated with propionate and controls.We then performed differential expression analysis, meta-analysis, and gene set enrichment anaylsis using data obtained by RNA-seq of control and PA-treated mice.

overall_design: Mice were treated with PA for 6 weeks and kidney tissues were used for RNAseq.

first: Wang

last: Hui

first: M

last: Firoze Khan


title: 3D-Printed PCL Scaffolds Loaded with bFGF and BMSCs(PCLMF) Enhance Tendon-Bone Healing in Rat Rotator Cuff Tears by Immunomodulation and Osteogenesis Promotion

city: Suzhou

first: Yichao

last: Ni

state: --- Select One ---

address: No.1055 Sanxiang Road, Gusu District, Soochow , Jiangsu, China.

country: China

institute: Second affiliated hospital of Soochow university

summary: Rotator cuff tears are the most common conditions in sports medicine and attract increasing attention. Scar tissue healing at the tendon-bone interface results in a high rate of retears, making it a major challenge to enhance the healing of the rotator cuff tendon-bone interface. Biomaterials currently employed for tendon-bone healing in rotator cuff tears still exhibit limited efficacy. 3D printing, a promising technology, enables the customization of scaffold shapes and properties. Bone marrow mesenchymal stem cells (BMSCs) have multi-differentiation potential and valuable immunomodulatory effects. Basic fibroblast growth factor (bFGF), known for its role in proliferation, has been reported to promote osteogenesis. These properties make them applicable in tissue engineering. In this study, we developed a 3D-printed PCL scaffold loaded with bFGF and BMSCs (PCLMF) to restore the tendon-bone interface and regulate the local inflammatory microenvironment. The PCLMF scaffolds significantly improved the biomechanical strength, histological score, and local bone mineral density at regenerated entheses at 2 weeks post-surgery and achieved optimal performance at 8 weeks. Furthermore, PCLMF scaffolds facilitated BMSC osteogenic differentiation and suppressed adipogenic differentiation both in vivo and in vitro. In addition, RNA-seq showed that PCLMF scaffolds could regulate macrophage polarization and inflammation through the MAPK pathway. The implanted scaffold demonstrated excellent biocompatibility and biosafety. Therefore, this study proposes a promising and practical strategy for enhancing tendon-bone healing in rotator cuff tears.

overall_design: This study aims to investigate the effects of 3D-printed PCL  scaffolds loaded with BMSCs and bFGF on the transcriptional levels of LPS -induced RAW264.7 cells. The goal is to evaluate whether the combination of BMSCs and BFGF can modulate the inflammatory response induced by LPS and to determine their specific impact on transcriptional regulation in RAW264.7 cells.

first: Bo

last: Tian

first: Jinmin

last: Lu

first: Xingrui

last: Huang


title: Gene expression data from pre-adipocytes 3T3-L1 treated with Tsuruazuki Extract (Tsuru)

city: TSUKUBA

first: ELDA NURAFNIE

last: IBNU RASID

state: IBARAKI

address: 1-1-1 TENNODAI

country: Japan

institute: UNIVERSITY OF TSUKUBA

summary:
Gene expression profiling of pre-adipocytes 3T3-L1 reveals anti-adipogenic potential to metabolic associated diseases through whole transcriptomic analysis.
We evaluated the effects of Tsuruazuki extract on pre-adipocytes 3T3-L1. We performed an untargeted whole-genome transcriptome analysis to explore functionality of Tsuru on 3T3-L1 cells.

overall_design: According to the manufacturer's guide, the RNA was extracted using Isogen kit (Nippon Gene Co. Ltd., Japan). Then, RNA quantity and quality were determined using the NanoDrop 2000 spectrophotometer (ThermoScientific, USA). DNA microarray analysis was conducted on control and Tsuru treated 3T3-L1 cells samples using the GeneChip WT PLUS Reagent Kit (ThermoFisher Scientific) and GeneChip™ Hybridization, Wash and Stain Kit (ThermoFisher Scientific) following the manufacturer's instructions. In brief, complementary DNA (cDNA) was synthesized from 100 ng of RNA solutions. cRNA was synthesized from in vitro transcription of cDNA and then purified and reverse transcribed. Finally, single-stranded cDNA (ss-cDNA) was synthesized, purified, fragmented, and labeled following the manufacturer's instructions. Cartridge Array Hybridization was performed using the Clariom™  S Array (Mouse; ThermoFisher Scientific) on the GeneChip™ Fluidics Station (ThermoFisher Scientific). Scanning was performed using GeneChip Scanner (ThermoFisher Scientific). The raw image data was obtained using the Transcriptome Analysis Console (TAC) software (ver. 4.0.2, ThermoFisher Scientific) and raw data were normalized following the signal space transformation robust multi-chip analysis (SST-RMA) algorithm. For differential expression analysis, a One-Way ANOVA followed by an empirical Bayes correction was performed. The detected above back-ground (DABG) cutoff was set to 0.05. The positive vs negative area under the curve (AUC) value was set at greater than or equal to 0.7. Finally, genes that passed the filter criteria of p value < 0.05 (one-way between-subjects ANOVA) and fold change > 1.1 (in linear space) were considered as differentially expressed genes (DEGs).

first: Elda

last: Nurafnie Ibnu Rasid


title: Saturation genome editing-based functional evaluation and clinical 2 classification of BRCA2 single nucleotide variants

city: Rochester

first: Fergus

last: Couch

address: 200 1st St SW

institute: MAYO CLINIC ROCHESTER

summary: Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to breast, ovarian, prostate and pancreatic cancer. However, variants of uncertain significance (VUS) (n>5000) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants. Here we report on comprehensive saturation genome editing-based functional characterization of 99% of all possible single nucleotide variants (SNVs) in the BRCA2 DNA Binding Domain hotspot for pathogenic missense variants that is encoded by exons 15 to 26. The assay was based on deep sequence analysis of HAP1 haploid cells endogenously targeted using a CRISPR/cas9 knockin approach. A total of 6959 SNVs were characterized for effects on cell survival. The assay was validated relative to nonsense and synonymous variants, ClinVar pathogenic/likely pathogenic and benign/likely benign variants and homology directed repair cell based DNA repair assay results, all of which showed >94% sensitivity and specificity. Variants were assigned posterior probabilities of pathogenicity using a VarCall two component Bayesian mixture model and were further grouped according to ACMG strength of evidence under the PS3/BS3 rule resulting in Benign Strong (n=5430), Benign Moderate (n=190), Benign Supporting (n=61), VUS (122), Pathogenic Strong (n=1021), Pathogenic Moderate (n=88), and Pathogenic Supporting (n=47). Breast cancer case-control association studies showed that pooled SNVs encoding functionally pathogenic missense variants were associated with increased risks of breast (odds ratio (OR)3.81, 95%CI: 2.88-5.07) and ovarian cancer (OR 5.93, 95%CI: 4.12-8.52). The functional data were also combined with other sources of information in the ClinGen BRCA1/2 VCEP ACMG/AMP-classification model. A total of 785 SNVs, including 261 missense SNVs, were classified as pathogenic or likely pathogenic, while 5566 SNVs, including 3786 missense SNVs, were classified as benign or likely benign. These classified variants can now be used for risk assessment and clinical care of variant carriers.

overall_design:
Saturation genome editing was used to introduce variants and the variant count on day 5 and day 14 were measured. The log2 ratio between D5 and D14 was used to measure the variant effects
Multiplexed Assays for Variant Effects (MAVE)

first: Huaizhi

first: Chunling

last: Hu

first: Jie

first: Steven

middle: N

last: Hart

first: Rohan

middle: D

last: Gnanaolivu

first: Mohamed

last: Abozaid

first: Tara

last: Rao

first: Yohannes

middle: A

last: Tecleab

first: Nicholas

last: Boddicker

first: Wenan

last: Chen

first: Marcy

middle: E

last: Richardson

middle: J


title: Transcriptional memory drives accelerated re-activation of secondary metabolite production in Aspergillus nidulans [RNA-seq]

city: Tulln

first: Harald

last: Berger

state: NOE

address: Konrad-Lorenzstraße 43

department: DAGZ

country: Austria

institute: Universität f. Bodenkultur Wien

summary: Organisms are repeatedly exposed to fluctuating environmental and nutritional conditions. Transcriptional memory has been shown to be a mechanism to cope with these fluctuations because it increases the speed and the magnitude of the cellular response to a certain re-occurring condition and therefore optimizes adaptation and fitness in a given environment. We found that genes coding for sterigmatocystin (ST) production in Aspergillus nidulans are activated stronger when cells are repeatedly exposed to nutrient starvation, compared to cells that experience this condition for the first time. We studied possible underlying mechanisms and found that persistence of the transcription factor AflR as well as a chromatin-based mechanism through histone H3 lysine 4 dimethylation (H3K4me2) are mediating the memory process. Our data indicate that AflR can undergo activation-inactivation cycles. On the other hand, an unknown mechanism mediated by a so far non-identified signal that originates from culture supernatants that were already starved once, specifically enhances the activation of starvation-dependent BGCs, including ST cluster activation.  Finally, our ChIP-Seq data show that the ST cluster harbors i) three loci of constitutive and two of inducible H3K4me2 near the aflR locus, the latter two building up H3K4me3 after sufficient time, ii) bindings sites of histone demethylase KdmB near these H3K4me2 marks. Overall, using the ST cluster as model we provide insights on transcriptional memory and BGC activation dynamics in A. nidulans.

overall_design: A. nidulans was growth for 20h on Aspergillus minimal medium (AMM) and then shifted on Salt medium (no C or N source) for 4 hours --> AMM_20h_Salt_4h A. nidulans was growth for 24h on Aspergillus minimal medium (AMM) --> AMM_24h A. nidulans was growth for 20h on Aspergillus minimal medium (AMM) , shifted on Salt medium (no C or N source) for 4 hours , shifted on AMM for 2 h --> AMM_20h_Salt_4h_AMM_2h A. nidulans was growth for 24h on Aspergillus minimal medium (AMM) , shifted on AMM for 2 h --> AMM_24h_AMM_2h A. nidulans was growth for 20h on Aspergillus minimal medium (AMM) , shifted on Salt medium (no C or N source) for 4 hours , shifted on AMM for 2 h, shifted on Salt medium (no C or N source) for 1 hour  --> AMM_20h_Salt_4h_AMM_2h_Salt_1h A. nidulans was growth for 24h on Aspergillus minimal medium (AMM) , shifted on AMM for 2 h, shifted on Salt medium (no C or N source) for 1 hour --> AMM_24h_AMM_2h_Salt_1h A. nidulans was growth for 20h on Aspergillus minimal medium (AMM) , shifted on Salt medium (no C or N source) for 4 hours , shifted on AMM for 2 h, shifted on used medium (C or N source used up by A. nidulans growth for 48h) for 1 hour  --> AMM_20h_Salt_4h_AMM_2h_Old_1h A. nidulans was growth for 24h on Aspergillus minimal medium (AMM) , shifted on AMM for 2 h, shifted on used medium (C or N source used up by A. nidulans growth for 48h) for 1 hour --> AMM_24h_AMM_2h_Old_1h A nd B denote biological replicates, 1 and 2 technical replicates

first: Franz

last: Zehetbauer

first: Florian

last: Kastner

first: Joseph

last: Strauss


title: Tracing the lineage of replicative RNAs in the process of KRAS (S17N) evolution

city: Beijing

first: Yihan

last: Lin

address: No.5 Yiheyuan Road, Haidian

institute: Peking University

summary: A dominant-negative gene therapy approach has been proposed and tested on proto-oncogene KRAS, wherein the oncogenic activity (and cell proliferation) of KRAS can be suppressed by introducing a dominant-negative KRAS allele (S17N). We employed REPLACE to conduct continuous evolution on KRAS (S17N) and examined its potential pathways for conferring resistance in this gene therapy methodology.To explore the accumulation of mutations in various RNAs during the KRAS (S17N) evolution experiment, we established a barcoded library and conducted lineage tracing of replicative RNAs carrying KRAS (S17N) throughout the evolution process.

overall_design: To investigate the process of mutation accumulation in different RNAs in the KRAS (S17N) evolution experiment, we generated a library of tens of thousands of barcodes by incorporating a 20-bp random sequence (20 Ns) downstream of the stop codon in KRAS (S17N)-P2A-PuroR gene. The library, after undergoing transformation, in vitro transcription, and electroporation, was delivered to the BHK-21 host cells. Medium was replaced with fresh medium containing 10 μg/mL puromycin 24 h post-electroporation (i.e., Day 1). After 4 d of selection (i.e., Day 5), approximately 2 million cells were transferred to a new 10-cm dish and subjected to RNA mutagenesis using molnupiravir (2 μM). When the plate reached approximately 90% confluence, the cells were consistently subcultured at a 1:5 ratio and the medium was daily replenished with 10 μg/mL puromycin and 2 μM molnupiravir treatment. The remaining cells were partially cryopreserved as backup and partially utilized for RNA extraction and mutation analysis. Cells at day 1, day 5, day 7, and day 14 were collected for total RNA isolation and reverse transcription. The KRAS (S17N)-T2A-PuroR-barcode region was amplified from both the DNA library plasmid and the aforementioned cDNA libraries using indexed primers. The purified PCR products were mixed and subjected to adapter ligation, fragment selection, primer and polymerase binding, followed by sequencing using a PacBio Sequel IIe instrument for Circular Consensus Sequencing (CCS) reads acquisition.

first: Liang

last: Ma


title: Transcriptional memory drives accelerated re-activation of secondary metabolite production in Aspergillus nidulans [ChIP-seq]

type: Genome binding/occupancy profiling by high throughput sequencing


title: Kinetics of the whole blood transcriptome in response to heat stress in pigs from two lines divergently selected for feed efficiency.

city: Castanet-Tolosan

first: Laurence

last: Liaubet

address: 24 chemin de Borde Rouge

department: Animal genetics

country: France

institute: INRAE

summary:
This study aims to use omics data to understand the genetic pathways involved in heat stress adaptation after a short and a long time exposure.
Heat stress is one of the main performance limiting factors in the pig industry and its importance is expected to increase with the consequences of climate warming. Various solutions to mitigate the effects of heat stress on pig performance and welfare have been investigated, and genetic selection appears to be a potential solution. The genetic mechanisms behind heat tolerance are still not well understood, but some differences between breeds have been shown, as well as an interaction between genetics and environment. Here, pigs divergently selected for feed efficiency were used to assess their ability to adapt.

overall_design: To achieve this, transcriptomic data from whole blood were obtained in an experiment involving 12 pigs from two RFI (residual feed intake) lines, n=6 HRFI and n=6 LRFI and was carried out in two replicates. The Low RFI (LRFI) are more efficient (less residuals) and high (HRFI) are less efficient (more residuals). The pigs were 80 days old at the start of the experiment and weighed approximately 40 kg. Blood samples were collected at different time points before (24°C) and after (30°C) exposure.  A final total of 75 samples were analyzed (38 for HRFI and 37 for LRFI). Seven time points were studied. Blood samples were collected  five days (-5) prior heat exposure. On the day of fluctuating temperature (at 10:00), blood samples were collected at 9:00 (0_9, one hour prior to the increase in temperature), and at 13:00 and 18:00 hours (0_13, 0_18). Subsequently, blood samples were collected at 1, 2, and 13 days post-exposure.

first: David

last: Renaudeau

first: Guilhem

last: Huau

first: Yann

last: Labrune

first: Jerôme

last: Lecardonnel


title: Quantification of basal RNA mutation rate in the repRNA-v4 EGFP region

summary: REPLACE was engineered from an orthogonal alphaviral RNA replication system. It generates a large, continuously diversified library of replicative RNAs through a replicase-limited mode of replication and inducible mutagenesis. We analyzed the mutation frequency at Day0 (IVT RNA), Day1, Day3, Day7, Day14, Day21 and DNA plasmid to estimate the RNA mutation rate. This data was used to guide the construction of RNA mutant libraries.

overall_design: EGFP was cloned into the repRNA-v4-derived vector, which was then transcribed in vitro into RNA and delivered into host cells via electroporation. After 24h of electroporation, cells were subjected to continuous puromycin treatment (10 µg/ml) with regular passages.We collected DNA plasmid, IVT RNA and cell samples at Day1, Day3, Day7, Day14, Day21 for sequencing.


title: LAG3 regulates the homeostasis of brain white matter microglia and affects astrocyte morphogenesis in the early postnatal period

first: Jianwei

last: Jiao

state: Beijing

address: 1 Beichen West Road

department: State Key Laboratory of Reproductive Biology

institute: Institute of Zoology, Chinese Academy of Sciences

summary: In order to further understand how microglia-derived LAG3 regulates microglia homeostasis and affects the morphogenesis of astrocytes, RNA-seq was used to analyze the transcriptome changes of microglia isolated from Lag3 conditional knockout mice and littermate-born wild-type mice in P7 period. About 7000 transcripts showed the differential expression between the microglia of the corpus callosum of Lag3fl/fl and Lag3cKO-Cx3 mice, with a fold change ≥2, and a p value < 0.05. Geneontology analysis of downregulated genes were enriched in terms related to actin recombination, ciliary recombination and others, while upregulated genes are enriched in inflammatory response and other terms. These results reflected the role of microglia Lag3 in microglia development.

overall_design: mRNA was extracted from microglia isolated from the corpus callosum of Lag3fl/fl and Lag3cKO-Cx3 mice at P7. Specifically, Agilent 2100 Bioanalyze was used to quality controlled and quantified. Then, the mRNA is converted into cDNA and bound the library. RNA-sequencing analysis was carried out using Illumina HiSeq 2500 platform in anno.txtroad Genomics.

first: Sihan

last: Li

first: Mengtian

last: Zhang

first: Chenxiao


title: ID3 Enhances PD-L1 Expression by Restructuring MYC and  Promotes Colorectal Cancer Immune Evasion [ChIP-seq]

city: Fuzhou

first: Chuanzhong

last: Huang

state: Fujian

address: No. 420 Fuma Road

institute: Fujian Cancer Hospital

summary: The inhibitor of DNA-binding protein ID3 is a vital component of immune cells and has been associated with the progression of colorectal cancer (CRC). Despite its significance, its specific role in the immune evasion strategies utilized by CRC remains unclear. RNA-seq analysis revealed that ID3 is associated with the PD-L1 immune checkpoint. We further demonstrated that ID3 modulates PD-L1 expression, suppresses the infiltration and activation of CD8+ T cells, and facilitates the immune escape of CRC cells. Additionally, we found that the knockdown of ID3 significantly enhanced the effectiveness of PD-L1 antibody treatment in combating CRC, reduced the upregulation of PD-L1 induced by the antibody, and altered the immune microenvironment within CRC. Mechanistically, our biological and structural analyses demonstrated that ID3 reconstructed the four-dimensional structure of MYC, thereby enhancing its binding affinity to the PD-L1 promoter and augmenting PD-L1 transcriptional activity. By integrating analysis of ChIP-seq, RNA-seq, and ImmPort gene sets, we found that ID3's DNA-assisted binding function was widespread and could either enhance or suppress gene transcription, not only affecting tumor immune escape through immune checkpoints, but also regulating various cytokines and immune cells involved in tumor immunity. In conclusion, our study uncovered a new mechanism by which ID3 promotes immune evasion in CRC and targeting ID3 may improve the efficacy of anti-PD-1/PD-L1 immunotherapy.

overall_design: Chromatin immunoprecipitation DNA-sequencing(ChlP-seq) for ID3 in HCT116 cells.

first: Ling

last: Wang

first: Changhua

last: Zhuo

first: Wenxin

first: Hongmei

last: Fan

first: Yilin

last: Hong

first: Yu

first: Dongmei

last: Zhou

first: Wansong

first: Lingyu

first: Jingjing

last: Zhao

first: Shuping

first: Chundong

last: Yu

first: Yunbin

last: Ye


title: IRF4 contributes to chemoresistance in IGH-BCL2 positive diffuse large B-cell lymphomas by mediating BCL2 induced SOX9 expression

city: Shanghai

first: JIAO

last: MA

address: Huangpu District

institute: Shanghai Jiao Tong University School of Medicine

summary: SOX9 was identified as a prognostic biomarker particularly in IGH-BCL2 translocated germinal center B cell (GCB) subtypes of the diffuse large B cell lymphoma (DLBCL) and plays a vital role in lymphomagenesis. However, the molecular mechanism that modulating the aberrant expression of SOX9 in this DLBCL subset remains unknown. We have demonstrated that SOX9 enhanced IGH-BCL2 positive DLBCL subset resistance to either chemotherapy or BCL2 inhibitor. Moreover, we found that inhibition of BCL2 downregulates SOX9 in IGH-BCL2 positive DLBCL subset. We further identified that IRF4 is a key regulator to mediate BCL2 induced SOX9 expression, alongside with the chip-seq confirmed that IRF4 is a key transcription factor for SOX9 in DLBCL. In addition, BCL2 promote IRF4 entry into the nucleus by enhancing its protein stability, via downregulating of proteosome ubiquitination process, and, therefore, enforce SOX9-mediated phenotypes. Finally, we showed in DLBCL cell lines and xenografted mice model that in vivo inhibition of IRF4 with hIRF4 antisense oligonucleotide (ASO) repressed lymphomagenesis and DLBCL chemoresistance. Altogether, our data support the conclusion that IRF4 plays an essential role in BCL2 induced upregulation of SOX9 expression, and targeting IRF4 may represent a promising therapeutic strategy to cure relapse and refractory DLBCL in the future.

overall_design: To identify which genes binds directly to IRF4 and the genes with IRF4-bound transcription sites

first: Jiao

last: Ma

first: Yirong

first: Zizhen

last: Xu


title: Engineering transcriptional regulators using REPLACE

first: Yihan

last: Lin

summary: Transcriptional regulators are fundamental elements in synthetic biology. We sought to use REPLACE to evolve synthetic transcriptional regulators. Through weeks of directed evolution experiments, we succesufuly modified the sensitivity of TetR and PadR to ligand molecules.

overall_design: To evolve PadR, the host cells were initially modified to achieve stable integration of a reporter gene plasmid ((OPadR)6-phCMVmini-PuroR-NLS-mCherry-hPEST-BGHpolyA-pEF1α/HTLV-mTagBFP2-NLS) into the nsP4-expressing BHK-21 cells using lentivirus. The VP64-PadR gene was cloned into the repRNA-v4-derived plasmid. After in vitro transcription, the resulting RNA was delivered into the host cells expressing nsP4 and harboring the stably integrated reporter plasmid through electroporation. Medium was replaced with fresh medium containing 5 μg/mL of puromycin and 10 µg/mL blasticidin 24 h post-electroporation. After 4 d of selection, two million cells were transferred into a new 10-cm culture dish and subjected to consecutive two-day treatment with molnupiravir (10 µM, with daily media changes) to induce RNA mutation. Upon completion of molnupiravir treatment, we adopted different selection/screening strategies according to the specific evolution objectives (i.e., one to evolve ligand-resistant mutants and the other to evolve mutants with enhanced target gene activation in the absence of ligand). To decrease or reverse ligand sensitivity, we employed an evolutionary approach by selecting replicative RNAs containing VP64-PadR with increasing puromycin concentration in the presence of the ligand (200 µM sodium ferulate). In the initial phase of selection, we gradually escalated the concentration of puromycin to inhibit excessive cell growth in the 10-cm culture dish. By enhance the puromycin concentration (up to 60 µg/ml) during mid-screening (e.g., approximately on day 7), around 99% of cells in the dish were effectively eliminated (Approximately 104 to 105 cells in the dish survived in this ‘bottleneck’ stage). This selection was then sustained for another several days. Upon completion of the evolution, cells were collected for RNA extraction and mutation analysis. To enhance the response dynamic range of PadR, we employed an evolutionary approach by selecting replicative RNAs containing VP64-PadR with increasing puromycin concentration in the absence of the ligand (sodium ferulate). We employed a similar approach to gradually enhance the puromycin concentration. However, due to excessive PadR activation activity in the absence of sodium ferulate, we encountered difficulties in eliminating cells by enhancing puromycin concentration (up to 80 µg/ml). Consequently, at the conclusion of the screening period, we utilized flow sorting to isolate approximately the top 1% mCherry-expressing cells (Approximately 105 cells in each dish) from the remaining population for RNA extraction and mutation analysis. To evolve TetR, the host cells were modified to achieve stable integration of a reporter gene plasmid (TRE3G-PuroR-NLS-mCherry-hPEST-BGHpolyA-pEF1α/HTLV-mTagBFP2-NLS) into the nsP4-expressing BHK-21 cells using lentivirus. The TetR-VP48 gene was cloned into the repRNA-v4-derived plasmid. We next employed a similar protocol used in the evolution of PadR to evolve doxycycline-resistant TetR mutants in the presence of 1 µg/ml doxycycline.

first: Liang


title: Combining multiple stressors unexpectedly blocks bacterial migration and growth

city: Urbana

first: Joseph

last: Sanfilippo

state: IL

address: 600 S Matthews Ave

institute: University of Illinois

summary: In nature, organisms experience combinations of stressors. However, laboratory studies use batch cultures, which simplify reality and focus on population-level responses to individual stressors. In recent years, bacterial stress responses have been examined with single-cell resolution using microfluidics. Here, we use a microfluidic approach to simultaneously provide a physical stressor (shear flow) and a chemical stressor (H2O2) to the human pathogen Pseudomonas aeruginosa. By treating cells with levels of flow and H2O2 that commonly co-occur in human host tissues, we discover that previous reports significantly overestimate the H2O2 levels required to block bacterial growth. Specifically, we establish that flow increases H2O2 effectiveness 50-fold, explaining why previous studies lacking flow required much higher concentrations. Using natural H2O2 levels, we identify the core H2O2 regulon, characterize OxyR-mediated dynamic regulation, and demonstrate that multiple H2O2 scavenging systems have redundant roles. By examining single-cell behavior, we serendipitously discover that the combined effects of H2O2 and flow block pilus-driven surface migration. Thus, our results counter previous studies and reveal that natural levels of H2O2 and flow synergize to restrict bacterial motility and survival. By studying two stressors at once, our research highlights the limitations of oversimplifying nature and demonstrates that physical and chemical stress can combine to yield unpredictable effects.

overall_design: We performed RNA seqiencing on P. aeruginosa cells 5 minutes after 8 µM H2O2 exposure. Cells with no H2O2 treatment served as control.

first: Anuradha

last: Sharma

first: Alexander

middle: M

last: Shuppara

first: Gilberto

middle: C

last: Padron


title: BLADE-R: streamlined RNA extraction for molecular diagnostics and high-throughput applications.

city: Charlottesville

first: Robert

last: Cornelison

state: VA

address: 345 Crispell Drive Carter-Harrison Research Building MR6 B710 Carter-Harrison Research Building MR6 B710

department: pathology

institute: UVA School of Medicine

summary: Validation of a novel BLADE-R(beads lysis and DNA elimination for RNA extraction) as comapred to established TRIzol method being suitable for RNA sequencing and gene expression analysis.

overall_design: Quality assessment of  RNA sequencing data genretared from RNA extracted through novel BLADE-R and TRIzol method from A-172 cell line.

first: Anam

last: Tajammal

first: Adelaide

last: Fierti

first: Hui

last: Li


title: Gene expression data from 3D spheroids of human amniotic epithelial stem cell (hAESC) treated with Tsuruazuki Extract (Tsuru)

first: ELDA NURAFNIE

last: IBNU RASID

summary:
Gene expression profiling reveals mesoderm lineage-specific genes were more significantly regulated with the most prominent regulated DEGs were identified in adipose tissue.
We evaluated the effects of Tsuruazuki extract on human amniotic epithelial stem cells. We performed an untargeted whole-genome transcriptome analysis to explore functionality of Tsuru in a stem cell-based tool.

overall_design: According to the manufacturer's guide, the RNA was extracted using Isogen kit (Nip-pon Gene Co. Ltd., Japan). Then, RNA quantity and quality were determined using the NanoDrop 2000 spectrophotometer (ThermoScientific, USA). DNA microarray analysis was conducted on control and Tsuru-treated hAESC samples using the GeneChip WT PLUS Reagent Kit (ThermoFisher Scientific) and GeneChip™ Hybridization, Wash and Stain Kit (ThermoFisher Scientific) following the manufacturer's instructions. In brief, complementary DNA (cDNA) was synthesized from 100 ng of RNA solutions. cRNA was synthesized from in vitro transcription of cDNA and then purified and reverse transcribed. Finally, single-stranded cDNA (ss-cDNA) was synthesized, purified, fragmented, and labeled following the manufacturer's instructions. Cartridge Array Hybridization was performed using the Clariom S array (Human; ThermoFisher Scientific) on the GeneChip™ Fluidics Station (ThermoFisher Scientific). Scanning was performed using GeneChip Scanner (ThermoFisher Scientific). The raw image data was obtained using the Transcriptome Analysis Console (TAC) software (ver. 4.0.2, ThermoFisher Scientific) and raw data were normalized following the signal space transformation robust multi-chip analysis (SST-RMA) algorithm. For differential expression analysis, a One-Way ANOVA followed by an empirical Bayes correction was performed. The detected above back-ground (DABG) cutoff was set to 0.05. The positive vs negative area under the curve (AUC) value was set at greater than or equal to 0.7. Finally, genes that passed the filter criteria of p value < 0.05 (one-way between-subjects ANOVA) and fold change > 1.1 (in linear space) were considered as differentially expressed genes (DEGs).

first: Elda

last: Nurafnie Ibnu Rasid


title: Methylation profiling of plasma cell-free DNA in pediatric brain tumor patients

city: Chicago

first: Shejuan

last: An

address: 303 East Superior Street

institute: Northwestern University

type: Methylation profiling by high throughput sequencing

summary: Using cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq), we analyzed methylation profiles in 93 plasma samples from 77 pediatric brain tumor patients and 16 non-neoplastic patients. Binomial GLMnet classifiers of tumor and tumor subtypes were built in training sets, and performance was evaluated in test sets. The methylation profiles from plasma cfMeDIP-seq discriminated tumor from non-tumor patients with 0.83 accuracy (precision = 0.93, sensitivity = 0.86, and specificity = 0.67). Among major tumor subtypes vs. nontumors, circumscribed astrocytic glioma showed an accuracy of 0.86 (precision = 0.88, sensitivity = 0.88, and specificity = 0.83), and glioneuronal tumors had an accuracy of 0.83 (precision, sensitivity, and specificity = 0.83). Circumscribed astrocytic glioma and glioneuronal tumors could be discriminated from other tumor subtypes with 0.79 and 0.82 accuracy, respectively.

overall_design: We analyzed methylation profiles in 93 plasma samples from 77 pediatric brain tumor patients and 16 non-neoplastic patients. Binomial GLMnet classifiers of tumor and tumor subtypes were built in training sets, and performance was evaluated in test sets.

first: Craig

last: Horbinski

first: Kathleen

last: McCortney

first: Jordain

last: Walshon

first: Kaethe

last: Leonard

first: Matthew

last: McCord

first: Michael

last: DeCuypere


title: Acute IL-6 exposure triggers canonical IL6Ra signaling in hiPSC microglia, but not neural progenitor cells

city: London

first: Amalie

middle: Charlotte Maria

last: Couch

address: 5 Cutcombe Road

department: Basic and Clinical Neuroscience

country: United Kingdom

institute: King's College London

summary:
Background: Prenatal exposure to elevated interleukin (IL)-6 levels is associated with increased risk for psychiatric disorders with a putative neurodevelopmental origin, such as schizophrenia (SZ), autism spectrum condition (ASC) and bipolar disorder (BD). Although rodent models provide causal evidence for this association, we lack a detailed understanding of the cellular and molecular mechanisms in human model systems. To close this gap, we characterized the response of human induced pluripotent stem cell (hiPSC-)derived microglia-like cells (MGL) and neural progenitor cells (NPCs) to IL-6 in monoculture.
Results: We observed that human forebrain NPCs did not respond to acute IL-6 exposure in monoculture at both protein and transcript levels due to the absence of IL6R expression and soluble (s)IL6Ra secretion. By contrast, acute IL-6 exposure resulted in STAT3 phosphorylation and increased IL6, JMJD3 and IL10 expression in MGL, confirming activation of canonical IL6Ra signaling. Bulk RNAseq identified 156 up-regulated genes (FDR < 0.05) in MGL following acute IL-6 exposure, including IRF8, REL, HSPA1A/B and OXTR, which significantly overlapped with an up-regulated gene set from human post-mortem brain tissue from individuals with schizophrenia. Acute IL-6 stimulation significantly increased MGL motility, consistent with gene ontology pathways highlighted from the RNAseq data and replicating rodent model indications that IRF8 regulates microglial motility. Finally, IL-6 induces MGLs to secrete CCL1, CXCL1, MIP-1α/β, IL-8, IL-13, IL-16, IL-18, MIF and Serpin-E1 after 3h and 24h.
Conclusion: Our data provide evidence for cell specific effects of acute IL-6 exposure in a human model system, ultimately suggesting that microglia-NPC co-culture models are required to study how IL-6 influences human cortical neural progenitor cell development in vitro.

overall_design: To characterize the response of human induced pluripotent stem cell (hiPSC-)derived microglia-like cells (MGL) in monoculture, we exposed N=3 donor lines (M3_CTR_36S, 127_CTM_01 and 014_CTM_02) on day 14 of MGL differentiation to 3h of IL-6 stimulation (100ng/ml) and carried out bulk-RNAseq analysis to profile the MGL's transcriptomic response.

first: Anthony

last: Vernon


title: Escaping from a dominant‐negative KRAS using REPLACE

first: Yihan

last: Lin

summary: A dominant-negative gene therapy approach has been proposed and tested on proto-oncogene KRAS, wherein the oncogenic activity (and cell proliferation) of KRAS can be suppressed by introducing a dominant-negative KRAS allele (S17N). We employed REPLACE to conduct continuous evolution on KRAS (S17N) and examined its potential pathways for conferring resistance in this gene therapy methodology.

overall_design: KRAS (S17N) cDNA was cloned into the repRNA-v4-derived vector. The vector was in vitro transcribed into RNA and the resulting RNAs were electroporated into aforementioned host cells. Medium was replaced with fresh medium containing 10 μg/mL puromycin 24 h post-electroporation (i.e., Day 1). After 4 d of selection (i.e., Day 5), approximately 2 million cells were transferred to a new 10-cm dish and subjected to RNA mutagenesis using molnupiravir (2 μM). When the plate reached approximately 90% confluence, the cells were consistently subcultured at a 1:5 ratio and the medium was daily replenished with 10 μg/mL puromycin and 2 μM molnupiravir treatment. The remaining cells were partially cryopreserved as backup and partially utilized for RNA extraction and mutation analysis. After another 9 d of culture, cells were harvested for total RNA extraction, sequencing, and mutation analysis. As a control, analogous experiments were conducted utilizing wild-type KRAS.

first: Liang

last: Ma


title: Regulation of aquaporin-2 abundance by TAZ  in the kidney collecting duct cells

city: Daegu

first: HongSeok

last: Choi

address: 680, Gukchaebosang-ro, Jung-gu

department: School of medicine

country: South Korea

institute: Kyungpook national university

summary: Background: The transcriptional coactivator with a PDZ-binding motif (TAZ), a downstream effector of the Hippo signaling pathway, regulates the expression of target genes by acting as a transcription cofactor. TAZ knockout (KO) mice exhibit polycystic kidneys and polyuria. This study aimed to investigate the role of TAZ in vasopressin-induced AQP2 regulation. Methods: 1) TAZ knockdown mediated by siRNA in mpkCCDc11 cells; 2) qRT-PCR, semiquantitative immunoblotting, and immunocytochemistry of AQP2; and 3) Next Generation Sequencing (NGS) in mpkCCDc11 cells, a mouse collecting duct cell line. Results: Endogenous expression of AQP2 was induced in mpkCCDc11 cells by treatment with dDAVP (10-9 M) treatment. Treatment with dDAVP (10-9 M) for 24 h increased AQP2 mRNA (12,608 ± 177% of control) and AQP2 protein levels (287 ± 15%). On the contrary, the increase induced by dDAVP in AQP2 mRNA (9,240 ± 241% of the control) and protein levels (215 ± 23%) was significantly reduced in TAZ-KD cells. TonEBP protein levels remained unchanged in TAZ-KD cells. NGS identified several potential AQP2 transcription factors (TF), including Klf6, Irf3, Cebpb, and Nr4a1, selected based on previous in silico database analysis. Among these, Nr4a1 was chosen for further investigation due to its significantly reduced expression of mRNA in TAZ-KD cells, as confirmed by qRT-PCR. Conclusion: TAZ appears to affect dDAVP-induced AQP2 trafficking through mechanisms not mediated by the cAMP/PKA pathway, suggesting the involvement of other non-canonical pathways. TAZ may also regulate AQP2 abundance, potentially through interaction with several transcription factors, including Nr4a1.

overall_design: To investigate the impact of TAZ on regulation of aquaporin-2 in kidney collecting duct cells , TAZ knockdown was induced in mpkCCDc11 cells, a mouse cortical kidney collecting duct cell line, by treating them with TAZ-siRNA and vasopressin, followed by observation of changes. Subsequently, we conducted RNA-sequencing using three samples from each group treated with control-siRNA,TAZ-siRNA and vasopressin, respectively, to analyze transcriptomics. Changes in genes involved in aquaporin-2 transcription were analyzed based on NGS data.

first: Hongseok

first: Hyoju

last: Jang

first: Tae-hwan

last: Kwon


title: Transcriptomic profiling of blood mTreg and mTeff from healthy control (HC) and multiple sclerosis (MS)

city: New Haven

first: Tomokazu

middle: S

last: Sumida

state: CT

address: 300 George St,, 3rd floor room349

department: Neurology

institute: Neurology, Yale School of Medicine

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

overall_design: Refer to individual Series


title: Transcriptomic and epigenetic profiling of blood mTreg and mTeff from healthy control (HC) and multiple sclerosis (MS) [ATAC-Seq]

summary: Autoimmune diseases, among the most common disorders of young adults, are mediated by genetic and environmental factors. While CD4+Foxp3+ regulatory T cells (Tregs) play a central role in preventing autoimmunity, the molecular mechanism underlying their dysfunction is unknown. Here, we performed comprehensive transcriptional and epigenetic profiling of Tregs in multiple sclerosis (MS) to identify central transcriptional programs regulating human autoimmunity.

overall_design: CD4+CD25hiCD127loCD45RO+ memory Tregs and CD4+CD25low/negCD127hiCD45RO+ memory Tconvs are sorted by FACS and subjected to ATAC-seq

first: David

last: Hafler


title: Unravelling Role of miRNA-mRNA Interactome in Pathophysiology of Arrhythmogenic Cardiomyopathy [mRNA-seq]

city: Cádiz

first: Fernando

last: Bonet

address: Plaza Falla 9

country: Spain

institute: University of Cádiz

summary: Arrhythmogenic cardiomyopathy is an inherited entity characterized by irregular cell-cell adhesion, cardiomyocyte death, fibro-fatty replacement of ventricular myocytes, leading to malignant ventricular arrythmias, contractile dysfunction and sudden cardiac death. Pathogenic variants in genes that encode desmosome are the predominant cause of arrhythmogenic cardiomyopathy. Moreover, signalling pathways such as Wnt/ß-catenin and transforming growth factor-β have been involved in the disease progression. However, still little is known about the molecular pathophysiological mechanisms that underlie arrhythmogenic cardiomyopathy pathogenesis. We used mRNA and small RNA sequencing to analyse the transcriptome of health and arrhythmogenic cardiomyopathy autopsied human hearts. Our results showed 697 differentially expressed genes, and eight differentially expressed miRNAs. Functional enrichment revealed mitochondrial respiratory-related pathways, impaired response to oxidative stress, apoptotic signalling pathway, inflammatory response-related and extracellular matrix response pathways. Furthermore, analysis of miRNA-mRNA interactome identified eleven negatively correlated miRNA-target pairs for arrhythmogenic cardiomyopathy. Our finding revealed novel arrhythmogenic cardiomyopathy-related miRNAs with important regulatory function in disease pathogenesis highlighting their value as potential key targets for therapeutic approaches.

overall_design: To investigate the molecular pathophysiological mechanisms underlying arrhythmogenic cardiomyopathy. By employing mRNA and small RNA sequencing, we aimed to analyze the transcriptome of healthy and arrhythmogenic cardiomyopathy-affected human hearts, identifying differentially expressed genes and miRNAs. Additionally, we sought to elucidate the functional pathways and miRNA-mRNA interactions involved in the disease, with the goal of discovering novel miRNAs with regulatory functions that could serve as potential therapeutic targets for arrhythmogenic cardiomyopathy.

first: Oscar

last: Campuzano

first: José

last: Córdoba-Caballero

first: Mireia

last: Alcalde

first: Georgia

last: Sarquella-Brugada

first: Aitana

last: Braza-Boïls

first: Ramon

last: Brugada

first: Francisco

last: Hernández-Torres

first: Maribel

last: Quezada-Feijoo

first: Mónica

last: Sánchez

first: Alipio

last: Mangas

first: Juan

last: Ranea

first: Rocío

last: Toro


title: ID3 Enhances PD-L1 Expression by Restructuring MYC and  Promotes Colorectal Cancer Immune Evasion [RNA-seq]

first: Chuanzhong

last: Huang

overall_design: To investigate the role of the inhibitor of DNA-binding protein ID3 in immune escape of colorectal cancer, we established ID3-knockout colorectal cancer cell line HCT116 using CRISPR/Cas9 technology to study the gene expression regulated by ID3. We then performed gene expression profiling analysis using data obtained from RNA-seq of sgID3 and sgCtrl. Comparative gene expression profiling of RNA-seq data is sgID3_1, sgID3_2, sgCtrl_1 and sgCtrl_2.

first: Ling

last: Wang

first: Changhua

last: Zhuo

first: Wenxin

last: Chen

first: Hongmei

last: Fan

first: Yilin

last: Hong

first: Yu

last: Zhang

first: Dongmei

last: Zhou

first: Wansong

last: Lin

first: Lingyu

first: Jingjing

last: Zhao

first: Shuping

first: Chundong

last: Yu

first: Yunbin

last: Ye


title: Blue‐shifting green fluorescent proteins using REPLACE

first: Yihan

summary: In the directed evolution experiments of EGFP or StayGold, the objective was to blue-shift the excitation light spectrum of GFP towards shorter wavelengths, resulting in an enhancement of fluorescence intensity upon 405 nm laser excitation. This facilitates the advancement of novel fluorescent proteins. These experiments represent a category of experiments that utilize REPLACE to achieve discontinuous directed evolution through FACS sorting (screening).

overall_design: EGFP or StayGold was cloned into the repRNA-v4-derived vector. The vector was in vitro transcribed into RNA and delivered into host cells via electroporation. After 24 hours of electroporation, cells were subjected to continuous puromycin treatment (10 µg/ml) with regular passages. After another 5-7 days, a relatively stable and homogeneous population of polyclonal cells carrying replicative RNA with expression of EGFP or StayGold was generated (i.e., Parent). Approximately 1-2×106 cells were passaged into a new 10-cm culture dish and treated with molnupiravir (10 µM) for two consecutive days with daily media changes. Then, they were divided equally into five separate 10-cm dishes and subjected to flow cytometry sorting after an additional cultivation period of approximately 3-4 days. The sorting process involved drawing a triangular region along the diagonal and applying a gate to approximately 0.5% positive cells of the cell population. About 104 cells were sorted and cultured as positive population (i.e., Post_1st). After about 7 days, these cells are expected to proliferate to a quantity of around 107. Similarly, these cells were exposed to a two-day treatment with molnupiravir (10 µM) and subsequently transferred onto five new 10-cm dishes for cultivation over a period of 3-4 days. The positive population was then sorted using flow cytometry at a ratio of 0.5%. The sorted cells were cultured to a count of approximately 106 and utilized for cryopreservation and total RNA isolation (i.e., Post_2nd). The extracted RNA was reverse transcribed into cDNA and then utilized for mutation analysis and mutant identification.

first: Liang

last: Ma


title: Tracing the lineage of replicative RNAs carrying MEK1 in the evolution of drug resistance

summary: Drug resistance poses a significant clinical challenge, and comprehending the mechanisms underlying this resistance can facilitate the design of novel inhibitors and advance cancer treatment. The REPLACE system was employed to examine resistance mutations in MEK1 during the administration of 3 allosteric inhibitors. To explore the accumulation of mutations in various RNAs during the MEK1 evolution experiment, we established a barcoded library and conducted lineage tracing of replicative RNAs carrying MEK1 throughout the drug resistance evolution process.

overall_design: To investigate the process of mutation accumulation in different RNAs in the MEK1 evolution experiment, we generated a library of tens of thousands of barcodes by incorporating a 20-bp random sequence (20 Ns) downstream of the stop codon in MEK1-P2A-PuroR gene. The library, after undergoing transformation, in vitro transcription, and electroporation, was delivered to the BHK-21 host cells. Medium was replaced with fresh medium containing 5 μg/mL puromycin 24 h post-electroporation (i.e., Day 1). After 4 days of selection by puromycin (i.e., on Day 5), approximately 2 million of cells were transferred to a new 10-cm dish and subjected to RNA mutagenesis using a concentration of 2 μM molnupiravir. On Day 7,therapeutic drug (2-5 μM cobimetinib) was added into the medium for selection. Puromycin and molnupiravir were also added to the medium simultaneously. One weeks later (i.e., Day 14), survived cells were collected. Cells at day 1, day 5, day 7, and day 14 were collected for total RNA isolation and reverse transcription. The MEK1-T2A-PuroR-barcode region was amplified from both the DNA library plasmid and the aforementioned cDNA libraries using indexed primers. The purified PCR products were mixed and subjected to adapter ligation, fragment selection, primer and polymerase binding, followed by sequencing using a PacBio Sequel IIe instrument for Circular Consensus Sequencing (CCS) reads acquisition. Simultaneously, RNAs from day 1, day 5, day 7, and day 14 were also subjected to FastGNS sequencing.


title: Evolution of drug resistance enabled by replicative RNAs carrying MEK1

summary: Drug resistance poses a significant clinical challenge, and comprehending the mechanisms underlying this resistance can facilitate the design of novel inhibitors and advance cancer treatment. The REPLACE system was employed to examine resistance mutations in MEK1 during the administration of 3 allosteric inhibitors (i.e., cobimetinib, trametinib, and selumetinib). These experiments represent a category of experiments that utilize REPLACE to achieve continuous evolution (or adaptation) by linking the activity of evolutionary targets with the survival of mammalian cells.

overall_design: MEK1 cDNA from H. sapiens was cloned into the repRNA-v4-derived vector. The vector was in vitro transcribed into RNA and the resulting RNAs were electroporated into aforementioned host cells expressing nsP4. Medium was replaced with fresh medium containing 5 μg/mL puromycin 24 h post-electroporation (i.e., Day 1). After 4 days of selection by puromycin (i.e., on Day 5), approximately 2 million of cells were transferred to a new 10-cm dish and subjected to RNA mutagenesis using a concentration of 2 μM molnupiravir. On Day 7, the cells in each dish were approaching 90% confluence (~ 107 cells per dish). Therapeutic drug (i.e., 2-5 μM cobimetinib, 5-10 μM trametinib, or 50-100 μM selumetinib) was added into the medium for selection. Puromycin and molnupiravir were also added to the medium simultaneously. One week later (i.e., Day 14), approximately 105 cells successfully survived and adapted to the environment with inhibitor. The cells were collected for total RNA extraction, sequencing, and mutation analysis.


title: Quantification of induced RNA mutation by nucleoside analogs in the repRNA-v4 EGFP region

summary: REPLACE was engineered from an orthogonal alphaviral RNA replication system. It generates a large, continuously diversified library of replicative RNAs through a replicase-limited mode of replication and inducible mutagenesis. We analyzed the variation of RNA mutations induced by two nucleoside analogues over time and at different concentrations. This data was used to guide the construction of RNA mutant libraries.

overall_design: EGFP was cloned into the repRNA-v4-derived vector, which was then transcribed in vitro into RNA and delivered into host cells via electroporation. After 24h of electroporation, cells were subjected to continuous puromycin treatment (10 µg/ml) with regular passages. After an additional 5-7 days, a relatively stable and homogeneous population of cells carrying replicative RNA expressing EGFP was established. These cells were then treated with nucleoside analogues at specified concentrations and durations. Total RNA was extracted from the cells and subjected to RNA sequencing to analyze the mutation information.


title: Identification of a novel signature based on immune microenvironment to predict prognosis，immunotherapeutic efficacy and immunogenicity in lung adenocarcinoma

city: zhongshan

first: yongyan

last: pei

address: Long life water, wuguishan street, zhongshan city

department: School of Pharmaceutical and Chemical Engineering

institute: Guangdong Pharmaceutical University

summary: Abstract: Immunotherapy has emerged as a crucial treatment approach for lung adenocarcinoma (LUAD), yet a notable percentage of patients do not exhibit the anticipated prognosis and therapeutic response. In this study, we embarked on a comprehensive analysis of immune infiltration-related genes in LUAD by integrating transcriptome sequencing data and TCGA dataset. And an immune risk prognosis model was developed, which accurately forecasts the prognosis and predicts the immunotherapy efficacy in LUAD. Patients with LUAD were categorized into high-risk and low-risk groups based on the model's risk value. The high-risk group exhibited a poorer prognosis, higher tumor mutation burden (TMB), greater genomic heterogeneity and lower immune scores. In contrast, the low-risk group manifested characteristics conducive to immunotherapy, including a more favorable prognosis, heightened sensitivity to immunotherapy and reduced likelihood of immune escape. The robustness of the model's predictive capability was corroborated using the GEO dataset, Nomogram and an independent immunotherapy cohort. Furthermore, the model risk value was found to be significantly associated with the prognosis of LUAD patients, genomic heterogeneity, immune cell infiltration, immune regulation and immunotherapy outcomes. Additionally, we characterized the significance of the model's feature factors using single-cell clustering and pseudo-temporal analysis, revealing that the model's feature genes are predominantly located in tumor-infiltrating T cells, particularly in the exhausted and effector subtypes. TNFRSF11A emerged as a key gene in the model, promoting LUAD cell proliferation, migration, and apoptosis, and involvement in LUAD M6A methylation and PD-L1-mediated immune escape. TNFRSF11A was mainly expressed in T cell clusters and represented a potential immune system regulatory factor associated with T cell exhaustion, fostering malignant progression in LUAD cells. Our study offers new mechanistic insights and therapeutic targets for the biological behaviors and immune suppression in LUAD.

overall_design: Three LUAD surgical samples and Three corresponding LUAD-adjacent cancer samples were collected from hospital samples for transcriptome sequencing to identify differentially expressed lncRNAs and mRNAs in LUAD.LncRNA and mRNA profiles of 3 LUAD surgical samples and 3 corresponding LUAD paracancer samples

first: Panli

last: Peng

first: Wenyou

first: Xukai

last: Li

first: Yongyan

last: Pei

first: Junhong

last: Lv


title: Meningeal immune signaling supports emotional adaptation following threat

city: Chongqing

first: Bin

last: Wu

address: 136 Zhongshan Er Road

department: Pediatric Research Institute

institute: Children's Hospital of Chongqing Medical University

summary: Social creatures must attend to threat signals from conspecifics and respond appropriately, both behaviorally and physiologically. In this work, we show a threat-sensitive immune signaling that orchestrates psychological processes and is amenable to social modulation. Repeated encounters with socially-cued threats triggered neutrophil priming preferentially in males. Meningeal niche-specific neutrophil activity was correlated with attenuated defensive responses to cues. The neutrophil-specific membrane protein CD177 responded to threat-predicting social cues, and its genetic ablation abrogated male behavioral phenotypes. Neutrophil CD177 signaling facilitated optimal meningeal IFN-γ production, which blunted neural response to threatening stimuli by enhancing intrinsic GABAergic inhibition within the prelimbic cortex. Initiation of meningeal neutrophil-mediated IFN-γ signaling was sensitized by negative emotional states and governed by socially dependent androgen release. This male-biased hormone/neutrophil regulatory axis is seemingly conserved in humans. Our findings provide insights into how immune responses influence behavioral responses to threats, suggesting a possible neuroimmune basis of emotional regulation.

overall_design: To confirm the sex differences in the state of meningeal neutrophils (MNs) following social threat exposure, we isolated neutrophils from the dura mater tissues of male and female mice after exposure for RNA sequencing.

first: Ling

last: Meng

first: Zhifang

last: Dong


title: Mouse kidney ischemia-reperfusion injury time course

city: Indianapolis

first: Takashi

last: Hato

state: IN

address: 950 W. Walnut St. R2 202

department: Medicine

institute: Indiana University

summary: Time course experiments involving bilateral renal ischemia reperfusion injury (IRI) in C57BL/6J mice (0 hr control, 20 min bilateral ischemia without reperfusion, 4, 16, 24, 36, 48, and 72 hrs post IRI). This dataset also includes IRI at 48 hrs and 72 hrs in Azin1 A-to-I locked and Azin1 A-to-I uneditable mice.

overall_design: Ischemia-reperfusion injury (IRI) was performed under isoflurane anesthesia. Prior to surgery, mice were given extended-release buprenorphine at a dose of 3.25 mg/kg. The mice were subjected to a 20-minute bilateral renal pedicle clamp followed by reperfusion. Kidney tissues were harvested at the following time points: control without IRI, 20 min after ischemia without reperfusion, 4, 16, 24, 36, 48, or 72 hrs after IRI. A heating pad was used to ensure their rectal temperature remained above 36 °C throughout the surgical procedure. No antibiotics or fluid resuscitation were administered.


title: Girk3 deletion increases osteoblast maturation and bone mass accrual in adult male mice

first: Alexandra

last: Krivonos

state: MN

address: 200 1st St SW

department: Quantitative Health Sciences

institute: Mayo Clinic

summary: Osteoporosis and other metabolic bone diseases are prevalent in the aging population. While bone has the capacity to regenerate throughout life, bone formation rates decline with age and contribute to reduced bone density and strength. Identifying mechanisms and pathways that increase bone accrual in adults could prevent fractures and accelerate healing. G protein-gated inwardly rectifying K+ (GIRK) channels are key effectors of G protein-coupled receptor signaling. Girk3 was recently shown to regulate endochondral ossification. Here, we demonstrate that deletion of Girk3 increases bone mass after 18 weeks of age. Male 24-week-old Girk3-/- mice have greater trabecular bone mineral density and bone volume fraction than WT mice. Osteoblast activity is moderately increased in 24-week-old Girk3-/- mice compared to WT mice. In vitro, both calvarial osteoblasts and bone marrow stromal cells from Girk3-/- mice are more osteogenic than WT cells and have altered expression of genes that regulate the wingless-type MMTV integration site (Wnt) family. Wnt inhibition via Dickkopf-1 (Dkk1) or β-catenin inhibition via XAV939 prevents the enhanced mineralization, but not proliferation, in Girk3-/- BMSCs and slows these processes in WT cells. Finally, selective ablation of Girk3 from cells expressing Cre from the 2.3kb-Col1a1 promoter, including osteoblasts and osteocytes, is sufficient to increase bone mass and bone strength in male mice at 24 weeks of age. Taken together, these data demonstrate that Girk3 regulates progenitor cell proliferation, osteoblast differentiation, and bone mass accrual in adult male mice.

overall_design: Femurs were isolated from 24 week old WT (n=4) and Girk3-/- (n=4) male mice. The diaphyses were cut off, marrow was flushed, and the bones were flushed with PBS to isolate diaphyseal bone. Bone was homogenized in Trizol, then total RNA was extracted and sent for bulk RNA Sequencing.

first: Samantha

last: Weaver

first: Jennifer

last: Westendorf


title: Interleukin-1β Induces Human Endothelial Surface Expression and Trans-presentation of Interleukin-15 by Relieving let-7c-3p Suppression of Protein Translation (bulk RNA-Seq)

city: West Haven

first: Francesc

last: Lopez

department: Department of Genetics

institute: Yale University

summary: Expression of interleukin-15 (IL-15) on the surface of human graft endothelial cells (ECs) bound to the IL-15 receptor a (IL-15Ra) subunit can increase the activation of cytotoxic T lymphocytes (CTLs), potentiating allograft rejection. Our previous work showed that surface expression of this protein complex could be induced by alloantibody-mediated complement activation through increased IL-1b synthesis, secretion, and autocrine/paracrine IL-1-mediated activation of NF-kB. Here we report that cultured human ECs express 8 differently spliced IL-15 transcripts. Remarkably, IL-1β does not alter the expression level of any IL-15 transcript but induces surface expression independently of RNA Polymerase II-mediated transcription while requiring new protein translation. Mechanistically, IL-1β causes an NF-kB-mediated reduction in the level of microRNA Let-7c-3p, thereby relieving a block of translation of IL-15 surface protein. Let7c-3p antimir can induce EC surface expression of IL-15/IL-15Ra in the absence of complement activation or of IL-1, enabling IL-15 trans-presentation to boost CD8 T cell activation. Because of the complexity we have uncovered in IL-15 regulation, we recommend caution in interpreting increased total IL-15 mRNA or protein levels as a surrogate for trans-presentation.

overall_design: Cultured human endothelial colony forming cells were treated with IL1β or TNF or no treatment for 6 hours following 48 hours of IFNγ pre-treatment or no pre-treatment. Cells were lysed and RNA purified with Qiagen miRneasy.

first: Clancy

middle: W

last: Mullan

first: Luanna

last: Summer

last: Lopez-Giraldez

first: Zuzana

last: Tobiasova

first: Thomas

last: Manes

first: Shruthri

last: Yasothan

first: Guiyu

last: Song

first: Daniel

last: Jane-Wit

first: W

last: Saltzman

first: Jordan

last: Pober


title: Interleukin-1? Induces Human Endothelial Surface Expression and Trans-presentation of Interleukin-15 by Relieving let-7c-3p Suppression of Protein Translation

type: Non-coding RNA profiling by high throughput sequencing


title: Transcriptomic profiling reveals potential protective mechanisms of agarwood extract on benzo[a]pyrene-impaired neuronal differentiation in SH-SY5Y cells

city: Bangkok

first: Anchalee

last: Prasansuklab

address: Sabbasastravicaya Building (11th floor), Soi Chulalongkorn 62, Phyathai Road, Pathumwan

country: Thailand

institute: College of Public Health Sciences, Chulalongkorn University

summary: Benzo[a]pyrene (B[a]P) has been recognized to inhibit neurodifferentiation and to be a cause of neurodegenerative diseases. Agarwood or Aquilaria crassna (AC), a useful plant for health-promoting properties, has the potential to counteract B[a]P by promoting neuronal growth and survival. In this study, the protective effect of AC leaf ethanolic extract (ACEE) on B[a]P-induced impaired neuronal differentiation was investigated. We employed transcriptomic analysis and identified the canonical pathway, the biological network, and differentially expressed genes (DEGs) that changed their expression levels consistently in response to neuronal differentiation and neurogenesis. Several genes, such as CXCR4, ENPP2, GAP43, GFRA2, NELL2, NFASC, NSG2, NGB, BASP1, and NEUROD1, mediated neuroprotective effect of ACEE in B[a]P-exposed SH-SY5Y cells. Notably, the biological network of these genes and Western blot confirmed that neuroglobin (NGB) was an important key in this study. Moreover, ACEE protected the neurons through the PI3K/Akt and ERK-signaling pathways. ACEE-identified phytochemicals, clionasterol and lupenone, were the possible constituents that acted as potent agonists for the CXCR4, GFRA, and RXR receptors. Therefore, ACEE could be a potential alternative medicine to prevent B[a]P-impaired neuronal differentiation and neurodegenerative diseases.

overall_design: To investigate how B[a]P exposure and the treatment of ACEE altered the transcriptome profiles of cultured RA-differentiated SH-SY5Y cells, we conducted separate RNA-Seq analyses of RA-differentiated SH-SY5Y cells treated with 1 µM B[a]P and/or co-treated with 25 µg/mL ACEE in comparison to untreated control cells.

first: Nattaporn

last: Pattarachotanant

first: Suporn

last: Sukjamnong

first: Panthakarn

last: Rangsinth

first: Kamonwan

last: Chaikhong

first: Chanin

last: Sillapachaiyaporn

first: George

middle: P

last: Leung

first: Valerie

last: Hu

first: Tewarit

last: Sarachana

first: Siriporn

last: Chuchawankul

first: Tewin

last: Tencomnao


title: Loss of DUSP1 expression creates a cold tumor microenvironment in cutaneous squamous cell carcinoma

city: Madrid

first: Ana

last: Sastre-Perona

state: Madrid

address: Calle Paseo de la Castellana 261, INGEMM, Laboratorio de Epigenética, Hospital Universitario La Paz

institute: IdiPAZ

summary: Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent cancer. In most of the cases, surgical removal of the tumor is a curative treatment but 3-5% of the tumors will progress or produce metastasis having a poor prognosis. Here we demonstrated the tumor suppressive role of DUSP1, a dual phosphatase that negatively regulates ERK activity. We demonstrated that DUSP1 constrains ERK signaling during inflammation and wound healing to sustain homeostasis by transiently decreasing its levels, but its permanent downregulation promotes progression to squamous cell carcinoma. Using mouse and human models we demonstrated that DUSP1 loss enhances ERK signaling, and tumor growth. RNAseq analysis revealed that DUSP1 null tumors are enriched in cancer stem cell gene signatures, but surprisingly, are also depleted from CD4, CD8, macrophages and DC cells, and DUSP1 null cancer cells express lower levels of chemo and cytokines, been immune excluded. Our data suggest that DUSP1 acts restricting cancer cell proliferation and promoting immune recruitment, presenting promising diagnostic and therapeutic potential.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Compared lesions produced by DMBA/TPA treatment in WT or Dusp1KO mice to identify differentially expressed genes. Compared control A431 cells isolated from SCC xenographs with DUSP1 KO cells to identify differentially expressed genes.

first: Leandro

last: Sastre


title: Differential microvascular endothelial cell responses in the retina in diabetes compared to the heart and kidneys, a spatial transcriptomic analysis.

first: Subrata

last: Chakrabarti

state: ON

address: Richmond St

department: Pathology

country: Canada

institute: Western University

summary: Endothelial cells and high glucose-induced endothelial dysfunction are the common origin of chronic diabetic complications such as retinopathy, nephropathy, and cardiomyopathy. Yet their common origins, the vascular manifestations of such complications are different. We examined the basal heterogeneity between microvascular endothelial cells (MECs) from the retina, kidneys, and heart, as well as their differential responses to hyperglycemia in diabetes. To this extent, we used a spatial transcriptomic approach to investigate gene expression differences across retinal, renal, and cardiac MECs in diabetic and non-diabetic mouse models. We further validated MEC heterogeneity in vitro using human retinal and cardiac MECs. We found that MECs from different target organs of major diabetic complications were transcriptomically distinct at the basal state and respond differently to hyperglycemia.

overall_design: Retinal, renal, and cardiac microvscular endothelial cells from 3 diabetic and 3 non-diabetic mice were analyzed using spatial transcriptomics

first: Eric

last: Wang

first: Biao

last: Feng

first: Shali

last: Chen


title: Single cell sequencing reveals the immunosuppressive trajectory in the tumor microenvironment of human giant cell tumor of bone

city: Guangzhou

first: Wei

last: Luo

address: No.396, Tongfu Middle Road, Haizhu District, Guangzhou city

institute: Guangzhou Red Cross Hospital

summary: GCTB is a type of bone tumor. we used scRNA-seq analysis to investigate the tumour microenvironment (TME) in GCTB.

overall_design: The study collected tissue samples of GCTB from a single patient who had "Resection of giant cell tumor of distal femur+left knee joint replacement surgery"

first: Yiming

last: Liu

first: Xiguan

last: Yao

first: Yude

last: Xu

first: Libing

last: Dai

first: Qiao

first: Peigeng

first: Weichao

last: Yang

first: Yi

first: Haixiong

last: Miao

first: Jiake

first: Dongping

last: Ye


title: Human enteric nervous system progenitor transplantation restores functional responses in Hirschsprung Disease patient-derived tissue

city: Vienna

first: Igor

last: Adameyko

address: Spitalgass 4

department: Neuroimmunology Department

institute: Medical University of Vienna

summary: Objective Hirschsprung disease (HSCR) is a severe congenital disorder affecting 1:5000 live births. HSCR results from failure of enteric nervous system (ENS) progenitors to fully colonise the gastrointestinal tract during embryonic development. This leads to aganglionosis in the distal bowel, resulting in disrupted motor activity and impaired peristalsis. Currently, the only viable treatment option is surgical resection of the aganglionic bowel. However, patients frequently suffer debilitating, lifelong symptoms, with multiple surgical procedures often necessary. Hence, alternative treatment options are crucial. An attractive strategy involves the transplantation of ENS progenitors generated from human pluripotent stem cells (hPSCs).  Design ENS progenitors were generated from hPSCs using an accelerated protocol and characterised, in detail, through a combination of single cell RNA-sequencing, protein expression analysis and calcium imaging. We tested ENS progenitors’ capacity to integrate and restore functional responses in HSCR colon, after ex vivo transplantation to organotypically cultured patient-derived colonic tissue, using organ bath contractility.  Results We found that our protocol consistently gives rise to high yields of cell populations exhibiting transcriptional and functional hallmarks of early ENS progenitors. Following transplantation, hPSC-derived ENS progenitors integrate, migrate and form neurons within explanted human HSCR colon samples. Importantly, the transplanted HSCR tissue displayed increased basal contractile activity and increased responses to electrical stimulation compared to control tissue.  Conclusion Our findings demonstrate, for the first time, the potential of hPSC-derived ENS progenitors to repopulate and restore functional responses in human HSCR patient colonic tissue.

overall_design: ENS progenitors generated from Human embryonic stem cells were collected at day 0 (SHE-AT-g001), day 4 (SHE-AT-g006) and day 6 (SHE-AT-g007) of differentation and cryopreserved.  Cells were analysed by single cell RNA sequencing by single cell discoveries (Utrecht, The Netherlands)

first: Benjamin

last: Jevans

first: Fay

last: Cooper

first: Yuliia

last: Fatieieva

first: Antigoni

last: Gogolou

first: Yi-Ning

last: Kang

first: Restuadi

last: Restuadi

first: Pieter

last: Vanden Berghe

first: Nikhil

last: Thapar

first: Peter

last: Andrews

first: Paolo

last: De Coppi

first: Anestis

last: Tsakiridis

first: Conor

last: J McCann


title: Rapid and robust isolation of microglia and vascular cells from brain subregions for integrative single-cell analyses

city: Stockholm

first: Klas

last: Blomgren

address: Solnavägen 9, 171 65 Solna

country: Sweden

institute: Karolinska institutet

summary: Cell isolation protocols from brain tissue constitute prolonged ex vivo processing durations, rendering them suboptimal for transcriptomic studies. Εspecially, yielding sufficient quantities of microglia or vascular cells requires the use of large tissue volumes and the addition of an enrichment step. In this study, we developed a simple, rapid, and reproducible cell isolation method for generation of single-cell suspensions from micro-dissected brain regions, providing high numbers of microglia and vascular cells, without requiring an additional encrihment step. Cells isolated using this method are suitable for molecular profiling studies using 10× Genomics Chromium single-cell RNA sequencing with high reproducibility. Our method is valuable for longitudinal unbiased molecular profiling of microglia and vascular cells within different brain regions, spanning multiple time points across physiological development or disease progression.

overall_design: Female WT C57Bl/6J (Charles River, Sulzfeld, Germany, stock #000664) were deeply anesthetized with sodium pentobarbital (100 mg/kg, ABCUR AB #444362, Sweden) and transcardially perfused with 20 ml of ice-cold 1× phosphate buffered saline without Ca2+ and Mg2+ (PBS; pH 7.4; Gibco/Life Technologies #10010056). Brain cerebral cortices were isolated from three three-week-old and hippocampi were isolated from three five-week-old mice. Cells were isolated per each cortical or hippocampal preparation. Tissues from all 3 experiments were handled in the same way for the single-cell suspension. Isolated cells were processed for single-cell RNA sequencing at the eukaryotic single-cell genomics facility (ESCG) at Karolinska Institutet, Sweden using the 10x Genomics Chromium method (version 3.0.1) and sequenced using Illumina NovaSeq 600 with an S2 flow cell. The Cellranger 5.0.1 pipeline was used to align the raw sequencing reads to the mouse reference genome, Mus musculus version mm10, and generate the unique molecular identifier (UMI) count matrix. Each preparation included 3 technical replicates and each replicate was processed independently before integrating them together.

first: Efthalia

last: Preka

first: Alejandro

last: Lastra Romero

first: Ahmed

last: Osman


title: G9a/GLP Inhibition Promotes T Cell Development from Pluripotent Stem Cells

city: Boston

first: Mohamad Ali

last: Najia

state: MA

address: 1 Blackfan Circle

department: Hematology/Oncology

institute: Boston Children's Hospital

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

overall_design: Refer to individual Series


title: Phlpp1 regulates endochondral ossification via the chondrocyte phospho-proteome

city: Rochester

first: Alexandra

last: Krivonos

summary: Appendicular growth and bone mass acquisition are controlled by a variety of growth factors, hormones, and mechanical forces in a dynamic process called endochondral ossification. Chondrocytes in the growth plate must proliferate and undergo hypertrophy to drive growth plate expansion and lay the template for bone. Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 and 2 (Phlpp1 and Phlpp2) are protein phosphatases that regulate intracellular signaling cascades through posttranslational modification of AKT, PKC, and S6K, among others. Phlpp1 controls chondrocyte proliferation and survival and germline deletion of Phlpp1 suppresses bone lengthening. Here, we demonstrate that Phlpp2 does not regulate endochondral ossification. Phlpp2-/- mice are phenotypically indistinguishable from their wildtype (WT) littermates, with similar bone length, bone mass, and growth plate dynamics. By contrast, Phlpp1/2-/- mice are phenotypically indistinguishable from Phlpp1-/- mice. Deletion of Phlpp1 or Phlpp2 had moderate effects on the chondrocyte transcriptome and proteome compared to WT control cells. By contrast, Phlpp1-/- and Phlpp1/2-/- chondrocytes had significantly altered phospho-proteomes compared to WT and Phlpp2-/- chondrocytes. Data integration via multiomics analysis revealed that RAF-MEK-ERK signaling was altered in cells lacking Phlpp1. In conclusion, these data demonstrate that Phlpp1, but not Phlpp2, regulates endochondral ossification via the chondrocyte phospho-proteome.

overall_design: Chondrocytes were isolated from five-day-old female wildtype (WT), Phlpp1-/-, Phlpp2-/-, and Phlpp1/2-/- mice (n=3 per genotype) and RNA was submitted for bulk RNA-Sequencing.

first: Samantha

middle: R

last: Weaver

first: Jennifer

last: Westendorf


title: G9a/GLP Inhibition Promotes T Cell Development from Pluripotent Stem Cells [ATAC-Seq]

summary: Elucidating mechanisms of T cell development can guide in vitro T cell differentiation from Pluripotent Stem Cells (iPSCs) and inform off-the-shelf T cell-based immunotherapies. Using a stroma-free human iPSC-T cell differentiation platform, we screened for epigenetic modulators that govern T cell specification and identified the H3K9-directed histone methyltransferases G9a/GLP as repressors of T cell fate. We show that G9a/GLP controls the cell fate decision between myeloid and lymphoid lineages in hematopoietic stem and progenitor cells (HSPCs). Inhibition of G9a/GLP promotes the production of lymphoid cells during zebrafish embryonic hematopoiesis, demonstrating the evolutionary conservation of G9a/GLP function. Importantly, chemical-induced epigenetic reprogramming via G9a/GLP inhibition facilitates the generation of robust iPSC-T cells that bear transcriptional similarity to peripheral blood αβ T cells. When engineered to express Chimeric Antigen Receptors, the epigenetically engineered iPSC-T cells exhibit enhanced antitumor activity both in vitro and in a xenograft mouse model.

overall_design: iPSC-derived T cells were treated with G9a/GLP inhibitor, UNC0224

first: Mohamad

middle: T


title: Interleukin-1ßces Human Endothelial Surface Expression and Trans-presentation of Interleukin-15 by Relieving let-7c-3p Suppression of Protein Translation (small RNA-Seq)

first: Francesc

last: Lopez

first: Clancy

last: Mullan

first: Luanna

last: Summer

last: Lopez-Giraldez

first: Zuzana

last: Tobiasova

first: Thomas

last: Manes

first: Shruthri

last: Yasothan

first: Guiyu

last: Song

first: Daniel

last: Jane-Wit

first: W

last: Saltzman

first: Jordan

last: Pober


title: G9a/GLP Inhibition Promotes T Cell Development from Pluripotent Stem Cells [RNA-Seq]


title: Reciprocal regulation of extracellular matrix proteins by EWS-FLI1 and AP-1 in Ewing sarcoma cells

city: Iowa City

first: Emma

last: Croushore

state: Iowa

address: 25 S Grand Ave

department: Pediatric Hematology/Oncology

institute: University of Iowa

summary: Ribonucleotide reductase (RNR) is the rate limiting enzyme in the synthesis of deoxyribonucleotides and the target of multiple chemotherapy drugs, including gemcitabine and hydroxyurea. In previous work, we identified that inhibition of RNR in Ewing sarcoma tumors upregulates the expression levels of multiple members of the activator protein-1 (AP-1) transcription factor family, including c-Jun and c-Fos, and downregulates the expression of c-Myc. However, the broader functions and downstream targets of AP-1, which are highly context- and cell-dependent, are unknown in Ewing sarcoma tumors. Consequently, in this work, we used genetically defined models, transcriptome profiling, and gene set enrichment analysis to identify that AP-1 and EWS-FLI1, the driver oncogene in most Ewing sarcoma tumors, reciprocally regulate the expression of multiple extracellular matrix proteins, including fibronectins, integrins, and collagens. AP-1 expression in Ewing sarcoma cells also drives, coincident with these perturbations in gene and protein expression, changes in cell morphology and phenotype. Furthermore, we also identified that the EWS-FLI1 oncoprotein dysregulates the expression of multiple AP-1 proteins, aligning with previous reports demonstrating genetic and physical interactions between EWS-FLI1 and AP-1. Overall, these results provide novel insight into the distinct, EWS-FLI1-dependent features of Ewing sarcoma tumors and identify a novel, reciprocal regulation of extracellular matrix components by EWS-FLI1 and AP-1.

overall_design: We performed RNA-seq in Ewing sarcoma cell lines with and without simultaneous upregulation of the AP-1 proteins c-Jun and c-Fos. AP-1 upregulation was induced by 72 hour addition of doxycycline to cell culture media.

first: Croushore

last: Emma E

first: Gordon

last: David J


title: In vitro exposure of Atlantic salmon to inorganic arsenic (AsIII) and arsenosugar

city: Bergen

first: Kai

middle: Kristoffer

last: Lie

state: Hordaland

address: Pobox 2029 Nordnes

department: Feed Safety

country: Norway

institute: Institute of Marine research

summary: Marine ingredients can contain relatively high levels of arsenic. The main aim of this study is to assess the hepatic toxicity of arsenic species in Atlantic salmon (Salmo salar) in vitro. Primary hepatocytes were isolated from six male Atlantic salmon and exposed for 48 h to establish dose-response curves for arsenite (AsIII) (0.1-3 μM) and for arsenosugar (AsSug) (5-250 μM). Regarding arsenite, results obtained indicated that the dose between 0.1 and 1 represented a marked threshold for effect. Very few genes were differentially expressed (N=5, p-adjust < 0.1) following AsIII 0.1 µM treatment compared to AsIII 1µM (N=2338 p-adjust < 0.01). Similar was observed for AsSug exposure where exposure to 50 µM resulted in 195 DEGs (p-adjust < 0.1) vs 6899 (p-adjust < 0.01) following AsSug 250µM exposure

overall_design: Primary hepatocytes were isolated from Atlantic salmon end exposed to different levels of arsenite and arsenosugar in vitro.

first: Marta

last: Silva

middle: K

first: Liv

middle: I

last: Søfteland


title: In vitro exposure of Atlantic salmon to inorganic arsenite (AsIII), arsenate and arsenobetaine

overall_design: Primary hepatocytes were isolated from Atlantic salmon end exposed to different levels of arsenite and arsenite and arsenobetain in vitro.


title: ionizing radiation and SRSF1 knockdown in triple-negative breast cancer cells

city: Oxford

first: Majd

last: Abdulghani

state: oxfordshire

address: Old Road Campus Research Building

institute: University of Oxford

summary: The goal of this study was to investigate the impact of 10 Gy ionizing radiation (IR) and/or SRSF1 depletion on isoform switching in the triple-negative breast cancer cell line, MDA-MB-231.

overall_design: 4 conditions, with three biological replicates each: siControl + 0 Gy, siSRSF1 + 0 Gy, siControl + 10 Gy, siSRSF1 + 10 Gy

first: Hala

last: Estephan

first: Robert

last: Parker

first: Sam

last: Palmer

first: Aaron

last: Jeffries

first: Nicola

last: Ternette

first: Timothy

last: Humphrey


title: Modulation of Neurodegeneration by CSFR1 Inhibition in an a-synuclein model of DLB/PD

city: Bethesda

first: Changyoun

last: Kim

state: Maryland

address: 35 Convent Dr. Rm 1A-1002

department: Molecular Neuropathology Unit

institute: National Institute on Aging, National Institutes of Health

summary: we treated a widely used and extensively studied synucleinopathy model of transgenic mice (Line 61) with PLX3397 and analyzed their neuropathology, behavioral deficits, and microglial transcriptomes.

overall_design: Gene expression profiling analysis of RNA-seq data following CSFR1 inhibition in mouse brain microglia.


title: High throughput screens identify long non-coding RNA LOUP contributions to NFkB signaling and monocyte differentation.

city: Santa Cruz

first: Susan

last: Carpenter

state: CA

address: Red Hill Rd, Biomedical Sciences

department: MCD

institute: UC Santa Cruz

summary: Long non-coding RNAs (lncRNAs) account for the largest portion of RNA from the transcriptome and yet most of their functions remain unknown. Here we performed a high throughput CRISPRi reporter-based screen to identify lncRNAs that regulate TLR4-NFkB signaling in human monocytes and those that contribute to PMA-induced differentiation . We successfully identified numerous non-coding and protein-coding genes that can positively or negatively regulate these pathways. To understand the functional roles of lncRNAs in TLR4/NFkB signaling, we chose to further study one top candidate from our screens, LOUP (lncRNA originating from upstream regulatory element of SPI1 [also known as PU.1]. It’s been previously shown by Trinh et al. that the LOUP transcript directly mediates interactions between a nearby enhancer element of SPI1 and the SPI1 promoter. SPI1 is a transcription factor that drives myeloid cell fate, is highly expressed in monocytes, and regulates transcription of inflammatory genes.  Here we’ve demonstrated that while LOUP expression can enhance SPI1 expression in monocytes, knockdown of LOUP also leads to a broad upregulation of NFkB targeted genes, both at baseline and upon TLR4-NFkB activation, and that this mechanism is independent of LOUP’s regulatory effects on SPI1. We found that LOUP harbors three small open reading frames (sORFs) capable of being translated, and that two sORFs in particular, are responsible for LOUP’s ability to negatively regulate TLR4/NFkB signaling. This work emphasizes the value of high-throughput screening to rapidly identify functional lncRNAs in the innate immune system.

overall_design:
For TLR4-NFkB screening, library infected and selected THP1-NFKB-dCas9 were stimulated with 200 ng/ml of LPS for 24 h to induce expression of GFP (NF-kB responsive) across three clonal lines: A, B, and C. Prior to sorting, cells were collected in FACS buffer (1XPBS, 1%FBS, 5mM EDTA). Stimulated cells were analyzed by flow cytometry alongside unstimulated cells to ensure the mean fluorescence intensity (MFI) of stimulated cells was > 10-fold compared to unstimulated cells. All flow cytometry experiments and screening were conducted on a BD FACSAria II. GFP was excited using a 488-nm laser and detected using a 525/50-nm filter. Sorting was conducted using 4-way purity into 2 tubes and a 100-mm nozzle. Cells were gated by forward (FSC-A) and side scatter (SSC-A) for live cells, then for single cells using FSC-A/FSC-H. Lastly, we evaluated GFP expression (SSC versus GFP), FACS sorted and collected the top/bottom 20% into separate tubes. At least 100 cells/sgRNA (100X coverage) for each sorted population were collected and cryo-preserved in 90% FBS, 10% DMSO for later processing. DNA was extracted and sgRNA guide sequences were sequenced and counted. Unsorted +LPS samples were collected for each clonal line as well (A,B,C), thus each clonal line has GFP_Hi, GFP_Lo, and Unsorted frations
For PMA differentiation screening, the same library was infected and selected THP1-NFKB-dCas9 were stimulated with 2nM PMA on day 8 and 5nM PMA on day 9 to induce differentiation. On day 11 differentiated macrophages were collected and DNA was extracted and sgRNAs sequences were sequenced and counted. Comparisons were made against those present at Day 3 without PMA treatment. The same clonal lines (A,B,C) were used and all samples were used in the analysis with the exception of Clone B +PMA Day 11 because cell coverage fell below 500X during screening.

first: Haley

last: Halasz

first: Eric

last: Malekos

first: Sergio

last: Covarrubias


title: Highly concentrated trehalose prevents IL-4/IL-13-induced skin barrier impairment by suppressing IL-33 expression and increasing Nrf2 activation in epidermal keartinocytes

city: Ube

first: Yoichi

last: Mizukami

state: Yamaguchi

department: Science Research Center

institute: Yamaguchi university

summary: Skin barrier dysfunction initiates or deteriorates various cutaneous problems, such as atopic dermatitis (AD). In high concentrations, the nonreducing disaccharide α-d-glucopyranosyl α-d-glucopyranoside (trehalose) can induce transient senescence-like state in fibroblasts and promote wound repair. Here we have investigated the direct effect of a high concentration of trehalose on primary human keratinocytes (KCs) and demonstrated its specific role in the skin barrier. RNA-seq analysis revealed that trehalose regulates many skin-barrier-associated genes in T helper 2 (Th2) cytokines interleukin (IL)-4/IL-13-treated and -untreated KCs. Using monolayer-cultured post-confluent normal human KCs and living skin equivalent (LSE), we identified IL-4/IL-13-downregulated differentiation markers and various epidermal antimicrobial proteins, all of which were significantly upregulated or restored by trehalose (60 mg/mL). Trehalose inhibited IL-33 expression and reduced nuclear IL-33 by activating MEK5- extracellular signal-regulated kinase (ERK)-5 and suppressing MEK1/2-ERK pathways. It also increased nuclear factor erythroid 2-related factor 2 (Nrf2)-heme oxygenase 1 (HMOX1) activation via c-Jun N-terminal kinase (JNK), thus, neutralizing IL-4/IL-13-mediated oxidative stress. Trehalose prevented IL-4/IL-13-mediated signal transducer and activator of transcription (STAT)3 and STAT6 activation and restored various skin barrier molecules reduced by IL-4/IL-13 via IL-33 downregulation and Nrf2-HMOX1 activation. This study demonstrates that trehalose might play salubrious roles as skin barrier repair in AD.

overall_design: RNAseq analysis of human primary keratinocyte treated with IL-4/IL-13 and/or trehalose.

first: Xiuju

last: Dai

first: Kenji

last: Watanabe

first: Teruko

last: Tsuda

first: Ken

last: Shiraishi

first: Hideki

last: Mori

first: Masamoto

last: Murakami

first: Yasuhiro

last: Fujisawa

first: Jun

last: Muto


title: The Ciliary GTPase ARL13B maintains Ductule Physiology through the Primary and Motile Cilia in Male Reproduction

city: Québec

first: Charles

last: Joly Beauparlant

state: QC

address: 2705 Bd Laurier

institute: Université Laval

summary: ARL13B is a small regulatory GTPase that controls ciliary membrane composition in both motile cilia and non-motile primary cilia. In this study, we investigated the role of ARL13B in the efferent ductules, tubules of the male reproductive tract essential to male fertility in which primary and motile cilia co-exist. We used a genetically engineered mouse model to deleteArl13bin efferent ductule epithelial cells, resulting in compromised primary and motile cilia architecture and functions. This deletion led to disturbances in reabsorptive/secretory processes and triggered an inflammatory response. The observed male reproductive phenotype showed significant variability linked to partial infertility, highlighting the importance of ARL13B in maintaining a proper physiological balance in these small ducts. These results emphasize the dual role of both motile and primary cilia functions in regulating efferent duct homeostasis, offering deeper insights into how cilia related diseases affect the male reproductive system.

overall_design: To investigate the function of Arl13b in efferent ductules physiology and male fertility we established a conditionnal Knock Out mice of Arl13b using the Cre lox system to specifically ablated Arl13b following the expression of cre recombinase under the promotor of Villin. We then performed gene expression profiling analysis using efferent ductules of 5 differents mice for each control and cKO.

first: Augière

last: Céline

first: Belleannée

last: Clémence


title: Bulk RNA sequencing and analysis of sorted murine lung endothelial cells from WT, DNMAML, and Notch1f/f mice

city: Chicago

first: Kostandin

middle: V

last: Pajcini

address: 909 S Wolcott Ave.

department: Pharmacology and Regenerative Medicine

institute: University of Illinois at Chicago

summary: We assessed the transcriptional profiles of sorted lung endothelial cells from WT, DNMAML, and Notch1f/f mouse models. The goal of the study is to determine the signaling pathways downstream of Notch signaling that are involved in the regulation of pulmonary vascular permeability

overall_design: mRNA expression profiles from sorted murine lung endothelial cells from WT, DNMAML, and Notch1f/f mice.

middle: Y

last: Paik


title: SRSF1 knockdown in HEK293T cells with ionizing radiation

first: Majd

last: Abdulghani

summary: The goal of the study is to identify differentially expressed isoforms in response to SRSF1 knockdown and/or ionizing radiation in HEK293T cells

overall_design: There are four conditions: siSRSF1 + 10 Gy ionizing radiation (IR), siControl + 10 Gy IR, siSRSF1 + 0 Gy IR, siControl + 0 Gy IR. siSRSF1 means SRSF1 was knocked down using an siRNA oligo targeting all known isoforms of SRSF1. siControl means the cells were transfected with a pool of non-targeting oligos as control. 10 Gy means cells were exposed to 10 Gy of IR, while 0 Gy are mock-trated samples. There are five biological replicates of each condition. The samples were collected 48 hours after IR or mock IR.

first: Tim

last: Humphrey


title: Calcitonin gene-related peptide promotes reparative function of interleukin-4 educated human macrophages via COX1/PGD2 signalling

city: Calgary

first: Paul

last: Gordon

state: AB

address: 3330 Hospital Drive NW

institute: University of Calgary

summary: Interleukin-4 activated human macrophages (M(IL4) promote epithelial wound healing and exert an anti-colitic effect. Blood monocyte-derived M(IL4)s from healthy donors and individuals with Crohn’s disease had increased mRNA expression of the calcitonin gene-related peptide (CGRP) receptor chain, RAMP1, suggesting neural modulation of the M(IL4)s function. Thus, human (MIL4)s were treated with CGRP and the cells' phagocytic, epithelial wound repair and anti-colitic functions were assessed. M(IL4,CGRP)s had increased mannose receptor (CD206) and FcgRIIa (CD32a) mRNA expression, a subtle, but significant increase in phagocytosis, and decreased production of CXCL1, CCL3, CCL4, CCL5 and CXCL9 following exposure to E. coli. When delivered systemically (106 cells, ip.) to oxazolone-treated rag1-/- mice, M(IL4,CGRP) had an anti-colitic effect superior to M(IL4)s from the same healthy blood donor. The human colon CaCo2 epithelial cell line in an in vitro wounding model revealed that conditioned medium (CM) from M(IL4,CGRP) had increased amounts of TGFb and increased wound-healing capacity compared to matched M(IL4)-CM. Moreover, M(IL4,CGRP)s displayed increased cyclooxygenase (COX)-1 and prostaglandin D2, and CM from M(IL4,CGRP)s treated with indomethacin or SC-560 to inhibit COX1 activity failed to promote repair of wounded CaCo2 cell monolayers. These data confirm the human M(IL4)s’ anti-colitic effect that could be enhanced by the local availability of CGRP, which may be dependent, at least in part via macrophage COX1/PDG2 activity. Thus, neuronal input is revealed as an important local modifier capable of boosting the anti-colitic effect of autologous M(IL4) transfer to treat enteric inflammation

overall_design: 4 samples of blood derived macrophages treated with IL-4 and CGRP were compared to 4 samples of IL4-treated blood derived macrophages.

first: D

last: McKay

first: B

last: Callejas


title: Ribosome profiling sequencing (Ribo-seq) in Huh7 control and SMYD5 KO cells upon normal condition or Torin1 treatment

city: Shanghai

first: Chenxi

last: He

address: 131 Dong'an Rd, Mingdao Bldg, Rm 512

institute: Fudan University

summary: To understand how RPL40 K22me3 transferred by SMYD5 affected ribosome function on normal culture condition or after mTOR inhibition, we conducted ribosome profiling sequencing (Ribo-seq)  in Huh7 control (NC) and SMYD5 KO cells.

overall_design: Huh7 control (NC) and SMYD5 KO cells were culturedHuh7 cells were cultured in DMEM supplemented with 10% FBS and 1% P/S or treated by 50 nM Torin1 for 12 hrs before collecting. Ribo-seq was performed as ARTseq Ribosome Profiling Kit’s instructions with modifications. About 10^7 cells were pre-treated by 100 μg/ml cycloheximide (MCE) for 10 min at 37℃, then washed and collected by ice-cold PBS containing 100 μg/ml cycloheximide. Cells were lysed by 120 μl Mammalian Polysome Buffer (10 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, 1% Trion X-100 with 1x protease inhibitor cocktail (Roche)) for 10 min. After centrifugation at 13,000 g for 10min at 4℃. 10 ul lysate was kept for input and purified by RNA Clean & Concentrator-5 kit (RCC-5 R1016, zymo), followed by rRNA removal with Ribo-off rRNA Depletion (N406, Vazyme) and library preparation with VAHTS Universal RNA-seq Library Prep Kit (NR605, Vazyme). Add about 30U RNase I (EN0601, Thermo) to 100ul lysate and digest for 45 min at room temperature. Digestion was stopped by the addition of 4 μl of Superase-In (AM2696, Thermo). Meanwhile, MicroSpin S-400 HR columns (27514001, Cytiva) were equilibrated with 3 ml of Mammalian Polysome Buffer by gravity flow and emptied by centrifugation at 600g for 4 min. We then immediately loaded 100 μl of the digested lysate on the column and eluted the column by centrifugation at 600g for 2 min. RNA was extracted by RCC-5 and separated on 15% denaturing urea-PAGE gel. After SYBR gold (S11494, Thermo) staining, the size ranges from 25 nt to 40 nt was cut out and recovered by small-RNA PAGE Recovery Kit (R1070, zymo). The eluted RNA were mix with Superase-In, T4 PNK and Buf A (EK0031, Thermo) at 37 ℃ for 15 min and supplemented by 1 mM ATP (R0441 ) for another 30 min before extraction by RCC-5 and generation library by Small RNA Library Prep Kit (NR811, Vazyme). The libraries were sequenced by NovaSeq 6000 (conducted by Nanjing Gaoxin Precision Medicine Technology Co., Ltd).

first: Ling

last: Ge

first: Xiang

last: Li

first: Fei

last: Lan


title: DNA methylation analysis of Conyza canadensis.L in the native and non-native range.

city: Srinagar

first: Mudasir

last: Zahoor

state: Jammu and Kashmir

address: Hazratbal, Srinagar

department: CIRI

country: India

institute: University of Kashmir

summary: To understand widespread differences in the DNA methylation patterns of Conyza canadensis leaf samples from its native and non-native ranges. Using Whole Genome Bisulfite Sequencing, we found average read coverages in high mapped reads across native and non-native samples of  Conyza canadensis. Using R bioconductor package, we found enrichment score of methylated sites in both native and non-native samples. while analyzing CG, CHG and CHH methylation, we found relatively low CG and CHG methylation across transcriptional units in natives over non-natives. However, differentially methylated regions were found to be 53% hypomethylated and 41% hypermethylated in non-natives on genic regions.

overall_design: We selected two native samples (native rep_1, native rep_2) and two non-native samples (non-native rep_1, non-native rep_2). We then performed Whole Genome Bisulfite Sequencing on both native and non-native samples and analyzed data using R software. Here, no reference or control sample has been taken into consideration. Native samples (native rep_1, native rep_2) signify samples taken from their native habitat while non-native samples  (non-native rep_1, non-native rep_2) signify samples taken from non-native habitat.

first: Ambreen

last: Shah

first: Ikhlak

last: Ahmed

first: Ishfaq

last: Pandith

first: Zafar

last: Reshi

first: Mohammad

last: Altaf