title: A conserved structured non-coding RNA coordinates growth and virulence in Clostridioides difficile. [dRNA-Seq ModT]

status: Public on Oct 31 2024

city: Würzburg

first: Johannes

last: Suzer

state: Bavaria

address: Josef Schneider Straße D15

department: Institute of Molecular Infection Biology Würzburg

country: Germany

institute: Universität Würzburg

type: Expression profiling by high throughput sequencing

summary: Bacterial non-coding RNAs fulfill a variety of cellular functions, for example as catalysts, as structural components in multiprotein complexes or as regulators of gene expression at the transcriptional and post-transcriptional level. Some RNAs display exceptionally broad conservation across bacterial phyla and are involved in fundamental and unique cellular functions. Hence, the characterization of new RNA families with deep sequence and/or structure conservation has the potential to reveal new molecular and biological RNA functions. Here, we characterize the Clostridioides difficile ‘raiA motif’ RNA, which is conserved in approx. 2,500 bacterial species from the phyla Bacillota and Actinomycetota. We show that its transcript abundance and stability in exponentially growing bacteria rivals that of ribosomal RNAs. Deletion of the ‘raiA motif’ RNA is associated with delayed transition into stationary phase, and changes in stationary phase pathways such as spore formation, hence we renamed it ModT (modulator of transition phase). Mechanistically, we show that ModT-mediated changes in cellular cyclic di-GMP levels are linked to the pronounced sporulation defect in the modT mutant. ModT is produced in two isoforms that are largely identical in their secondary structure, but differ in their in vivo half-lives as well as their capacity to complement phenotypes associated with modT deletion. Importantly, we show that expression profiles and isoform patterns of ModT are conserved in C. perfringens and P. sordellii, and that these orthologs can functionally replace ModT in C. difficile. In summary, our findings indicate that ModT fulfills a conserved biochemical function in regulating growth transitions in bacteria, and provide a crucial step towards delineating the molecular function of its highly conserved tertiary structure.

overall_design: To identify cellular functions, we deleted the first 215 nt of modT (ΔmodT), corresponding to the conserved ModT consensus motif, from C. difficile 630 by homologous recombination and performed comparative transcriptome analysis of Wildtype and ΔmodT strains grown in TY medium supplemented with glucose. We collected samples in the exponential phase (3.5 hours post inoculation) and during the transition phase (9 hours post inoculation), when the knockout strain begins to outgrow the wild type.

first: Tina

last: Lence

last: Sulzer

first: Kilian

last: Andress

first: Anne-Sophie

last: Gribling-Burrer

first: Vanessa

last: Lamm-Schmidt

first: Lars

last: Barquist

first: Redmond

middle: P

last: Smyth

first: Franziska

last: Faber


title: High-fat diet during mouse pregnancy impairs fetal heart development

city: Shanghai

first: Kaixin

last: Ge

address: 180 Fenglin Rd

department: Department of Cardiovascular Surgery

country: China

institute: Zhongshan Hospital, Fudan University

summary: Maternal obesity is associated with detrimental outcomes in offspring. However, the specific effects of gestational exposure to a high-fat diet (HFD) on fetal heart development remain poorly understood. In the study, we investigated that maternal overnutrition accounted for defective development in offspring, and cell proliferation was restricted severely in neonatal organ. Starting with this clue, we found decreased cell cycle and similar cross area of cardiomyocytes in offspring exposed to a maternal HFD. Moreover, RNA sequencing analysis detected that HFD diminished fatty acid metabolism, enhanced inflammation response and up-regulated substantially the pathway TP53 regulates transcription of cell cycle genes in postnatal day 0 (P0) cardiomyocytes. Further, normal lactation after delivery not only recovered from growth retardation but also maintained normal cardiac function in offspring. These data demonstrated a potential TP53 pathway-dependent mechanism through which maternal HFD contributed to offspring cardiac defects.

overall_design: To investigate the mechanisms through which transcriptional pathway in HFD heart might lead to defective proliferation in cardiomyocytes, we isolated cardiomyocytes from all groups of hearts at P0 followed by RNA-seq.

first: Defang

last: Deng

first: Jufeng

last: Meng

first: Jinjun

last: Cao

first: Chen

last: Liu

first: Hui

last: Zhang


title: Bone marrow endosteum houses Hedgehog-susceptible Dlx5-expressing osteoblast precursor cells

city: Houston

first: Shion

last: Orikasa

state: TX

address: 7500 Cambridge St

country: USA

institute: University of Texas Health Science Center at Houston School of Dentistry

summary: A single cell transcriptional profile of osteoblasts and bone marrow stromal cells of the murine femur at postnatal day 49 (P49), lineage-marked by Dlx5-creER at P21

overall_design: GFP+ and tdTomato+ cells were FACS-isolated from Col1a1-GFP; Dlx5-creER; R26R-tdTomato femur bone marrow at P49 (tamoxifen at P21). 73,000 cells were loaded onto 10X Genomics Chromium Single Cell Expression Solution version 2 platform. cDNA was sequenced by Illumina NovaSeq 6000

first: Noriaki

last: Ono

first: Yuki

last: Matsushita


title: MGC-803 Cells: Control vs. MGC-803 Cells MYBL2 Overexpressed

city: Bengbu

first: Jingya

last: Chen

address: Donghai Road

institute: Bengbu Medical University

summary: MYB proto-oncogene like 2 (MYBL2) has been reported to be involved in the occurrence and development of various tumors, however, its role in gastric cancer (GC) remains elusive.  In this study, the effects of MYBL2 on gene transcription and protein expression were analyzed by RNA sequencing and Western blot assays, respectively.

overall_design: In order to further explore the mechanism underlying the effect of MYBL2 overexpression on the occurrence and development of GC, we performed an RNA-seq analysis of the transcriptomes of MYBL2 overexpressing in MGC-803 cells

first: Huazhang

last: Wu


title: Chromatin repression by PRC2 results in reduced gene expression driving key features of CD8 T cell exhaustion

city: Philadelphia

first: Gregory

last: Donahue

state: PA

address: 3400 Civic Center Blvd, Bldg 421

department: Cell & Developmental Biology

institute: The University of Pennsylvania

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Development of an orthotopic medulloblastoma zebrafish model for rapid drug testing

city: Stockholm

first: Niek

last: van Bree

address: Solnavägen 9

department: Microbiology, Tumor and Cell biology (MTC)

country: Sweden

institute: Karolinska Institutet

summary: Medulloblastoma (MB) is one of the most common malignant brain tumor in children. Current preclinical in vivo model systems for MB have increased our understanding of molecular mechanisms regulating MB development; however, they may not be suitable for high-throughput screening efforts. We demonstrate here that transplantation of ten different MB cell lines or patient-derived cells into the blastula stage of zebrafish embryos leads to orthotopic tumor growth that can be observed within 24 hours after transplantation. Importantly, the homing of transplanted cells to the hindbrain region and the aggressiveness of tumor growth are enhanced by pre-culturing cells in a neural stem cell-like medium. The change in culture conditions rewires the transcriptome towards a more migratory and neuronal phenotype, including the expression of guidance molecules SEMA3A and EFNB1, both of which correlate with lower overall survival in MB patients. Furthermore, we highlight that the orthotopic zebrafish MB model has the potential to be used for rapid drug testing.

overall_design: To investigate transcriptional changes in UW228-3 and D425 Med medulloblastoma cells cultured in neural stem cell media compared to standard culture media.

first: Margareta

middle: T

last: Wilhelm


title: PARG inhibitor sensitivity correlates with accumulation of single strand DNA gaps in preclinical models of ovarian cancer

city: Boston

first: Huy

last: Nguyen

state: MA

address: 450 Brookline Ave

department: Radiation Oncology

institute: Dana-Farber Cancer Institute

summary: Poly (ADP-ribose) glycohydrolase (PARG) is a dePARylating enzyme which promotes DNA repair by removal of poly (ADP-ribose) (PAR) from PARP1-parylated proteins.  Loss or inhibition of PARG results in replication stress and sensitizes cancer cells to DNA damaging agents, suggesting that PARG inhibitors may provide a therapeutic option for cancer therapy. Indeed, PARG inhibitors (PARGi) are now undergoing clinical development for patients having tumors with homologous recombination deficiency (HRD), such as ovarian and breast cancer patients with germline or somatic BRCA1/2-mutations. Biomarkers of PARGi response will be required for the optimal development of these important new drugs. PARP inhibitors kill BRCA-deficient cancer cells by increasing ssDNA gaps during replication. Here, we report that, similar to PARP inhibitors, PARGi treatment can also cause an accumulation of ssGAPs in sensitive cells. PARGi exposure increased accumulation of S-Phase specific PAR, a marker for Okazaki fragment processing (OFP) defects on lagging strands, in sensitive cells but not in resistant cells.  PARGi also caused accumulation of PAR at the replication fork and increased pRPA, a marker for replication stress, at the replication fork in sensitive cells. Additionally, PARGi exhibited monotherapy activity in some HR-deficient, as well as HR-proficient,  patient-derived organoids of ovarian cancer, and drug sensitivity directly correlated with the accumulation of ssDNA gaps. Taken together, PARGi treatment results in toxic accumulation of PAR at replication forks resulting in ssDNA gaps due to OFP defects during replication. Regardless of the BRCA-status, the induction of ssDNA gaps in preclinical models of ovarian cancer cells correlates with PARGi sensitivity. Patient-derived organoids will be a useful model system for testing PARGi sensitivity and functional biomarkers

overall_design: RMUGS, the most PARGi sensitive cell line, was exposed to a gradually increasing concentration of PARGi until the cells started to grow in the presence of PARGi(4 months) at a similar  rate to the parental line without PARGi . The cells with acquired resistance to PARGi, namely RMUGS-R cells, exhibited an approximately 10 fold higher IC50 for growth inhibition by PARGi compared to the sensitive parental cells.For RNA seq, the resistant cells were  maintained in a medium without the drug for 2 weeks. The experiment was performed in triplicates. Total RNAs were extracted and sequenced .  Differential gene expression analysis was performed by DESeq2 and limma, comparing the mRNA expression levels of PARGi-resistant cells to those of the parent cells.

first: Ramya

last: Ravindranathan

first: Kalindi

last: Parmar

first: Alan

middle: D

last: D’Andrea


title: Epitope-specific T cell responses after SARS-CoV-2 vaccination [scRNA-dextramer_s2]

city: Erlangen

first: Kilian

last: Schober

address: Maximiliansplatz 2

department: Mikrobiologisches Institut – Klinische Mikrobiologie, Immunologie und Hygiene

institute: Universitätsklinikum Erlangen

summary: Clonal expansion is a hallmark of adaptive immunity, and appears to be driven by high antigen-specific receptor avidity as shown by research using murine in vivo models. In humans, however, the functionality of antigen-specific T cell clonotypes that are recruited into primary and recall immune responses remains surprisingly elusive. In this regard, the vaccination program during the SARS-CoV-2 pandemic represented a unique research opportunity by provision of highly standardized cohorts of healthy human individuals receiving immunizations against a previously unseen antigen. Here, we analyzed 30 HLA-typed individuals before, short-term and long-term after three mRNA vaccinations against SARS-CoV-2. We performed an in-depth characterization of the magnitude, phenotype, clonal composition and functionality of antigen-specific CD8 T cell responses by ELISPOT, flow cytometry and single-cell RNA sequencing, including CITE seq of 130 surface antigens and 16 T cell epitope specificities. 89 T cell receptors (TCRs) covering three complete epitope-specific repertoires were re-expressed by CRISPR/Cas9-mediated orthotopic TCR replacement and tested for their avidity. TCR repertoires underwent continuous tailoring, with high TCR avidity being linked to both clonal persistence and expansion. However, epitope-specific repertoires also maintained diversity by concomitant contraction and new emergence of functional T cell clones over time. These data on the phenotype and clonal selection within human antigen-specific T cell responses instruct our understanding of human T cell biology and may guide the development of enhanced or novel vaccines.

overall_design: Antigen-specific T cells (as well as unspecific T cells as a control) were identified by DNA-baroded pMHC multimers (Dextramers) including the SARS-CoV-2 spike specificity LTD as well as 15 additional SARS-CoV-2 spike epitopes. Dextramer+ and - cells were sorted by FACS and subjected to 5' scRNAseq on a 10x Genomics platform including CITEseq for Dextramers and TotalSeq-C anti-human hashtag antibodies; different samples from different donors were labelled with hasthag antibodies

first: Katharina

last: Kocher

first: Felix

last: Drost


title: RNA-seq analysis using A549 cells with FXR knockdown and control A549 cells with or without ionizing radiation treatment.

city: Beijing

first: Li

last: Xinyan

address: No.17, Panjiayuan Nanli, Chaoyang District, Beijing

institute: Cancer Hospital Chinese Academy of Medical Sciences

summary: In this study, we explored the role of FXR in the response to ionizing radiation (IR) in A549 human lung cancer cells. FXR was stably knocked down in A549 cells using shRNA, and four experimental groups were established: control A549 cells (non-irradiated), control A549 cells (irradiated), FXR knockdown A549 cells (non-irradiated), and FXR knockdown A549 cells (irradiated). RNA sequencing (RNA-seq) was performed to identify gene expression changes associated with FXR knockdown and irradiation. This dataset provides insights into the molecular mechanisms by which FXR influences the cellular response to radiation and its potential impact on cancer therapy.

overall_design: The experiment was designed to investigate the impact of FXR knockdown on the gene expression profile of A549 cells following ionizing radiation. A549 cells were stably transfected with shRNA targeting FXR or a control shRNA. The experiment included four groups: (1) control A549 cells without irradiation, (2) control A549 cells exposed to irradiation, (3) FXR knockdown A549 cells without irradiation, and (4) FXR knockdown A549 cells exposed to irradiation.Each group included three biological replicates, totaling 12 samples. RNA was extracted from all 12 samples and subjected to RNA sequencing to compare the transcriptomic changes induced by FXR knockdown and ionizing radiation. This design allows for the analysis of the specific role of FXR in the cellular response to radiation.

first: Xinyan

last: Li

first: Min

last: Deng


title: single-cell RNA-seq analysis of 2 ovary tissues as part of creating a ovary cell atlas

city: Oxford

first: Cecilia

last: Lindren

address: Old Road Campus

department: Big Data Institute

country: United Kingdom

institute: University of Oxford

summary: We aimed to create an ovary cell atlas using publicly available RNA-seq data. To complement the data we sequenced 2 ovary tissue samples from patients undergoing elective surgery at the JR hospital in Oxford. We applied single-nucei sequencing using the 10x Chromium technology.

overall_design: 2 independent ovary samples from 2 different patients


title: Chromatin repression by PRC2 results in reduced gene expression driving key features of CD8 T cell exhaustion [RNA-seq]

summary: Exhausted CD8 T cells result from chronic antigen stimulation and have reduced ability to clear disease. The epigenetic regulation of the dysfunctional phenotype of exhausted CD8 T cells is a promising avenue for therapies aimed at reversing or preventing CD8 T cell exhaustion. Here, utilizing an in vitro model, we show global increase of the repressive histone modification H3K27me3 in CD8 T cell exhaustion as well as increased gene expression of the subunits of PRC2, the complex responsible for H3K27me3 deposition. H3K27me3 correlated with decreased gene expression and localized to naïve/memory T cell genes in CD8 T cell exhaustion. PRC2 inhibition increased expression of many of these naïve/memory genes while reducing expression of key exhaustion genes. Further, we identified potential enhancers and transcription factors predicted to promote expression of the PRC2 subunits. Our study highlights the importance of the repressive epigenetic landscape of exhausted CD8 T cells as well as a novel role for PRC2 in T cell biology.

overall_design: RNA-seq derived from acute or chronic exhausted CD8+ T cells, treated with EED226 (PRC2 inhibitor) or DMSO; 5 reps ACUTE DMSO, 3 reps ACUTE EED226, 7 reps CHRONIC DMSO, 5 reps CHRONIC EED226.

first: Parisa

middle: F

last: Samareh

first: Paula

last: Agudelo-Garcia

first: Zhen

last: Zhang

first: Michael

last: Gilbert

first: Mariel

last: Mendoza

first: Hua

last: Huang

first: Jennifer

middle: E

first: Amy

last: Baxter

first: Zeyu

first: Kate

middle: A

last: Alexander

first: E

middle: J

last: Wherry

first: Charly

middle: R

last: Good

first: Shelley

middle: L

last: Berger


title: Chromatin repression by PRC2 results in reduced gene expression driving key features of CD8 T cell exhaustion [Cut & Run]

first: Gregory

last: Donahue

overall_design: CUT&RUN data for H3K27ac or H3K27me3, derived from acute or chronic exhausted CD8+ T cells; H3K27ac ACUTE 4 reps, H3K27ac CHRONIC 4 reps, H3K27me3 ACUTE 3 reps, H3K27me3 CHRONIC 3 reps

last: Wu

last: Chen


title: A conserved structured non-coding RNA coordinates growth and virulence in Clostridioides difficile. [DMS-MaP]

first: Johannes

last: Suzer

summary: Bacterial non-coding RNAs fulfill a variety of cellular functions, for example as catalysts of chemical reactions, as structural components in multiprotein complexes or as regulators of gene expression at the transcriptional and post-transcriptional level. Some RNAs, such as ribosomal RNAs, display exceptionally broad conservation across bacterial phyla and are involved in fundamental and unique cellular functions. Hence, the characterization of new RNA families with deep sequence and/or structure conservation has the potential to reveal new molecular and biological RNA functions. Here, we characterize the Clostridioides difficile ‘raiA motif’ RNA, which we rename ModT (modulator of transition phase), a member of an RNA family that is conserved in approx. 2,500 physiologically diverse bacterial species belonging to the phyla Bacillota and Actinomycetota. We show that ModT transcript abundance and stability in exponentially growing bacteria rivals that of ribosomal RNAs. Deletion of modT is associated with delayed transition into stationary phase, and changes in stationary phase pathways such as spore formation and toxin production. Mechanistically, we show that ModT-mediated changes in cellular cyclic di-GMP levels are linked to a pronounced sporulation defect in the modT mutant. ModT is produced in two isoforms that are largely identical in their secondary structure, but differ in their in vivo half-lives as well as their capacity to complement the sporulation phenotype of the modT mutant. Importantly, we show that expression profiles and isoform patterns of ModT are conserved in C. perfringens and P. sordellii, and that these orthologs can functionally replace ModT in C. difficile. In summary, we provide functional evidence that ModT fulfills a conserved biochemical function in bacteria that is likely enabled by its complex tertiary structure.

overall_design: To gain insights into the secondary structure and structure dynamics of ModT, we performed Dimethyl Sulfate Mutational profiling (DMS-MaP) experiments in vitro with in vitro transcribed ModT folded in a physiological salt buffer, in vivo under native conditions in C. difficile cultures natively expressing ModT at different growth stages, and in deproteinated cell lysates obtained from the same cultures. Each condition was treated with Dimethyl Sulfate (DMS) as nucleotide-modifying reagent and Ethanol as a control. ModT was reverse transcribed with an error-prone reverse transcriptase introducing mutations at modified A and C sites, which were determined by next generation sequencing. The conditionally varying modification frequencies at each site were used to infer a normalized reactivity score to the A or C nucleotides in ModT, which was evaluated to draw conclusions on the secondary structure.

first: Tina

last: Lence

last: Sulzer

first: Kilian

last: Andress

first: Anne-Sophie

last: Gribling-Burrer

first: Vanessa

last: Lamm-Schmidt

first: Redmond

last: Smyth

first: Lars

last: Barquist

first: Franziska

last: Faber


title: TRIB3 inhibition by palbociclib sensitizes prostate cancer to ferroptosis via SOX2-mediated SLC7A11 downregulation

first: chenyu

last: liu

address: No.10, West Toujiao, Right Anmenwai, Fengtai District

institute: Capital Medical University

summary: Palbociclib is a CDK4/6 inhibitor approved for the treatment of breast cancer by suppressing cell proliferation. However, monotherapy with palbociclib was discouraging in prostate cancer, calling for a mechanism-based effective therapy. In this study, we reported in prostate cancer that palbociclib is a potent sensitizer of ferroptosis, which is worked out by downregulating the expression of TRIB3, a gene highly expressed in prostate cancer. Specifically, TRIB3 knockdown augmented the response of prostate cancer cells to ferroptosis inducers, whereas, TRIB3 overexpression rescued prostate cancer cells from palbociclib-induced ferroptosis. Mechanistically, TRIB3 inhibition by palbociclib resulted in downregulation of SOX2, which subsequently led to compromised expression of SLC7A11, a cystine/glutamate antiporter that counteracts ferroptosis. Functionally, a combined treatment of palbociclib with ferroptosis inducer significantly suppressed prostate cancer growth in a xenograft tumor model. Together, these results uncover an essential role of TRIB3/SOX2/SLC7A11 axis in palbociclib-induced ferroptosis, suggesting palbociclib a promising targeted therapy in combine with ferroptosis induction for the treatment of prostate cancer.

overall_design: Palbociclib were purchased from Selleck (S1116) and dissolved in sterile deionized water. LNCaP cells were treated with 0.5μM palbociclib for one week. Cell cycle assays were choose to identifity the successment of cell models. Total RNA was isolated from LNCaP control (Crtl) and LNCaP palbociclib-treated cells (pal), The RNA sequencing was performed with 27 Illumina HiSeq 6000 as instructed by the manufacturer (lc-bio, Hangzhou). Fastp 28 software (https://github.com/OpenGene/fastp) were used to remove the reads that 6 1 contained adaptor contamination, low quality bases and undetermined bases with 2 default parameter. Then sequence quality was also verified using fastp. We used 3 HISAT2 (https://ccb.jhu.edu/software/hisat2) to map reads to the reference genome of 4 Homo sapiens GRCh38). The mapped reads of each sample were assembled using 5 StringTie (https://ccb.jhu.edu/software/stringtie) with default parameters. Then, all 6 transcriptomes from all samples were merged to reconstruct a comprehensive 7 transcriptome using gffcompare (https://github.com/gpertea/gffcompare/). After the 8 final transcriptome was generated, StringTie and was used to estimate the expression 9 levels of all transcripts. StringTie was used to perform expression level for mRNAs by 10 calculating FPKM (FPKM = [total_exon_fragments / mapped_reads(millions) × 11 exon_length(kB)]). The differentially expressed mRNAs were selected with fold 12 change > 2 or fold change < 0.5 and with parametric F-test comparing nested linear 13 models (p value < 0.05) by R package edgeR 14 (https://bioconductor.org/packages/release/bioc/html/edgeR.html).

first: Yangyi

last: Zhang

first: Chenyu

last: Liu

first: Yalan

last: Yang

first: He

last: Ren

first: Tianyi

first: Yinuo

last: Huang

first: Shinan

first: Qiang

last: Sun

first: Hongyan


title: An essential role for Cmtr2 in mammalian embryonic development [RNA-Seq]

first: Alena

last: Yermalovich

address: 450 Brookline Avenue

summary: CMTR2 is an mRNA cap methyltransferase with poorly understood physiological functions.  It catalyzes 2'-O-ribose methylation of the second transcribed nucleotide of mRNAs, potentially serving to mark RNAs as “self” to evade the cellular innate immune response. Here we analyze the consequences of Cmtr2 deficiency in mice. We discover that constitutive deletion of Cmtr2 results in mouse embryos that die during mid-gestation, exhibiting defects in embryo size, placental malformation and yolk sac vascularization. Endothelial cell deletion of Cmtr2 in mice results in vascular and hematopoietic defects, and perinatal lethality. Detailed characterization of the constitutive Cmtr2 KO phenotype shows an activation of the p53 pathway and decreased proliferation, but no evidence of interferon pathway activation. In summary, our study reveals the essential roles of Cmtr2 in mammalian cells beyond its immunoregulatory function.

overall_design: To examine the impact on gene expression in the Cmtr2 constitutive knockout embryos, we analyzed RNA sequences from E8.5 embryos that appeared morphologically similar to the wild-type littermate controls.  We also sequenced embryos and yolk sacs isolated 1 day later at E9.5 where the Cmtr2-/- embryos are smaller than the littermate controls. E8.5 group contains samples from two different litters; E9.5 embryo and yolk sac groups contain samples from four different litters.

first: Zarin

last: Mohsenin

first: Mitzy

last: Cowdin

first: Bruno

last: Giotti

first: Akansha

last: Gupta

first: Alice

last: Feng

first: Lior

last: Golomb

first: Douglas

middle: B

last: Wheeler

first: Kelly

last: Xu

first: Alexander

last: Tsankov

first: Ondine

last: Cleaver

first: Matthew

last: Meyerson


title: Natural killer cells cooperate with neutrophils to suppress pathological angiogenesis in neovascular age-related macular degeneration

city: Tianjin

first: Xue

last: Dong

state: TIANJIN

address: Qixiangtai Road

department: Department of Pharmacology

institute: Tianjin Medical University

summary: Neovascular age-related macular degeneration (nvAMD) is the leading cause of blindness in the elderly population associated with focal inflammation, however, understanding of the precise immune components governing this process is still limited. Here, we identified natural killer (NK) cells as a prominent lymphocyte population infiltrating the perivascular space of choroidal neovascularization (CNV) lesions. O-link analysis and single-cell RNA sequencing approaches identified CCR5-expressing NK cells is essential for further NK cell recruitment and extravasation in CNV site. NK cells suppress pathological angiogenesis via through  clearance of senescent vasculatures, a process that requires neutrophil extracellular traps (NETs). Mechanistically, the release of NETs results  from NKG2D-dependent NK cell activation and production of IFN-γ at CNV lesions. Notably , age is the strongest risk factor for AMD to an aged immune system; our human and mouse data showed that aged NK cells exhibited compromised protective effects. Additionally, expansion of NK cells by IL-2 complex improved CNV formation. Collectively, our results revealed a previously unrecognized role of NK cells in suppressing nvAMD progression, suggesting these cells as a potential target for future immune therapies for patients  with nvAMD.

overall_design: To characterize the expression pattern of NK cell signature genes in the laser-induced photocoagulation mouse model of AMD (laser-induced CNV model), we performed RNA-sequencing of adult mouse retina-choroid complexes 3,7, and 14 days after laser burn and age-matched normal controls.To investigate the mechanisms underlying the protective effects of NK cells, we performed transcriptome analysis of the retina-choroid complex from Rag2-/- and Rag2-/-γc-/- mice three days after laser injury.

first: Yinting

last: Song


title: An essential role for Cmtr2 in mammalian embryonic development [scRNA-Seq]

overall_design: To identify potential targets of Cmtr2, we performed and analyzed single-nucleus RNA sequencing (snRNA-seq) from four Cmtr2+/+ and five Cmtr2-/-  E9.5 embryos from 5 different litters.

middle: V


title: Cell-type-specific translational regulation by human DUS enzymes [tRNA DESeq]

city: Princeton

first: Nathan

last: Yu

state: New Jersey

address: Frick Chemistry Laboratory

department: Chemistry

institute: Princeton University

type: Non-coding RNA profiling by high throughput sequencing

summary: Dihydrouridine is an abundant and evolutionary conserved modified nucleoside present on tRNA, but characterization and functional studies of individual modification sites and associated DUS writer enzymes in mammals is lacking. Here we use a chemical probing strategy, RNABPP-PS, to identify 5-chlorouridine as a general activity-based probe for human DUS enzymes. We map D modifications using mechanism-based RNA-protein crosslinking and also through chemical reactivity and mutational profiling to reveal the landscape of D modification sites on human tRNAs. Further, we knockout individual DUS genes in two model human cell lines to investigate their regulation of tRNA expression levels and codon-specific translation elongation. We show that whereas D modifications are present across most tRNA species, loss of D only perturbs the translational function of a subset of tRNAs in a cell-type-specific manner. Our work provides powerful chemical strategies for investigating D and DUS enzymes in diverse biological systems and provides insight into the role of a ubiquitous tRNA modification in translational regulation.

overall_design: DESeq of 24 samples from HEK293T cells, 3 replicates of DUS1L KO #1, 3 replicates of DUS1L KO #2, 3 replicates of DUS2L KO #1, 3 replicates of DUS2L KO #2, 3 replicates of DUS3L KO #1, 3 replicates of DUS3L KO #2, and 6 replicates of WT HEK293T cells after RNA extraction, tRNA isolation, and spike-ins.

first: Wei

last: Dai

first: Ang

last: Li

first: Muhan

last: He

first: Ralph

last: Kleiner


title: The collaboration of GSDMD and GSDME in folic acid (FA)-induced renal injury

city: Fuzhou

first: Zhimin

last: Chen

address: Chazhong Road NO.20

institute: the First Affiliated Hospital, Fujian Medical University, Fuzhou

summary: Acute kidney injury(AKI) is associated with an increased risk of chronic kidney disease(CKD). There is still a lack of effective prevention for AKI-CKD transition. In the present study, we simultaneously explored the contribution of GSDMD and GSDME in folic acid (FA)-induced nephropathy, a model mimicking the essential components of the AKI-CKD transition. We found that blockage of both GSDMD and GSDME-mediated pyroptosis could have accumulative protection against FA-induced AKI-CKD transition. GSDME-mediated pyroptosis played a crucial role in tubular cell damage. GSDMD could exert important functions in infiltration of inflammatory cells and NETs formation. Pyroptotic tubular cells triggered NETs generation and macrophage polarization. NETs promoted macrophage-to-myofibroblast transition. Our results illustrated the orchestration of GSDMD and GSDME in AKI-CKD transition, providing new insights into the molecular mechanism for the clinical dilemma.

overall_design: scRNA-seq profiling of control kidney and folic acid (FA)-induced nephropathy kidney.

first: Yanfang


title: Gene expression profile at single cell level of a rare seen and young tracheal adenoid cystic carcinoma

first: Li

last: Zhou

address: Room 701, Block B, Building 3, Yard 8,Haiying Road, Fengtai District

institute: ERT

summary: tracheal adenoid cystic carcinoma is one kind of non-small-cell lung cancer with limit treatment options. We used single cell RNA sequencing (scRNA-seq) to analyze the molecular characteristic of tracheal adenoid cystic carcinoma, aiming to recognized this disease deeply and provide potential treatment plan in the future.

overall_design: Single cells obtained from the tumor tissue, the adjacent tissues, and blood were analyzed by RNA seq

first: Bo

last: Yang

first: Huchao

first: Yi

last: Liu


title: mRNA sequencing analysis during mouse trophoblast stem cells differentiation into trophoblast giant cells

first: Junpeng

address: 145 Guangyuan Rd, Xuhui District

department: School of Medicine, Shanghai Jiao Tong University

institute: The International Peace Maternity and Child Health Hospital

summary:
We performed RNA-seq to determine the differential expression genes from undifferentiated trophoblast stem cells (TSCs) and 7-days differentiated trophoblast giant cells (TGCs).
We performed RNA-seq to determine the differential expression genes from undifferentiated trophoblast stem cells (TSCs) and 7-days differentiated trophoblast giant cells (TGCs). mTSCs were nurtured in a stem cell medium  specifically designed for their growth. The differentiation of mTSCs into TGCs were enabled using a specialized differentiation medium for 7-days. This medium was essentially SCM without the components of FGF4 and heparin.

overall_design: Undifferentiated mouse Trophoblast Stem Cells (mTSCs) and 7-days differentiated Trophoblast Giant Cells (TGCs)  were selected for RNA-Seq, in triplicate. Then, RNA extraction was performed using TRIzol® Reagent (Invitrogen). And The library preparation was constructed by NEBNext Ultra RNA Library Prep Kit for Illumina (NEB).Final libraries were quantified by Agilent 2100 Bioanalyzer then deeply sequenced on the Illumina HiSeq 2500 platform.


title: Large scale analysis of leaf gene expression upon treatment of apple grafted scions with acibenzolar-S-methyl or water at 20 or 35°C

city: Angers

first: Alexandre

last: Degrave

address: André Lenôtre

country: France

institute: Agrocampus Ouest

summary: Investigation of whole genome gene expression level changes in the youngest expanded leaves of GDDH13 grafted scions either sprayed with reverse osmosis water or acibenzolar-S-methyl (ASM) either exposed to control temperature (21°C/day ; 18°C/night) or three successive heat days (35°C for 4 h during the day ; 18°C/night). The morning of the third heat day, treatment was performed by spraying to runoff (with a pressurized hand sprayer) with the commercial product Bion 50 WG (Syngenta, Basel, Switzerland; 50% of ASM) prepared in reverse osmosis water at a final concentration of 0.4 g/L. All plants were then returned to control thermal conditions (21°C/day ; 18°C/night) for 2 days. The youngest developped leaf of each plant was sampled 72 hours after the treatment.

overall_design: A total of 8 samples were associatied as pairs to perform 4 competitive hybridisations, including dye switchs. Biological replicates are from 2 independantly grown apple plant series. These comparisons allowed to determine the impact of ASM on apple gene expression at control temperature, its impact at high temperature (35°C), as well as the impact of high temperature on water-treated plants or ASM-treated plants.

first: Erwan

last: Chavonet

first: Muriel

last: Bahut

first: Sandra

last: Pelletier

first: Matthieu

last: Gaucher

first: Marie-Noëlle

last: Brisset


title: Dynamic enhancer interactome promotes senescence and aging [Hi-ChIP II]

first: Gregory

last: Donahue

summary: Gene expression programs are regulated by enhancers which act in a context-specific manner, and can reside at great distances from their target genes. Extensive three-dimensional (3D) genome reorganization occurs in senescence, but how enhancer interactomes are reconfigured during this process is just beginning to be understood. Here we generated high-resolution contact maps of active enhancers and their target genes, assessed chromatin accessibility, and established one-dimensional maps of various histone modifications and transcription factors to comprehensively understand the regulation of enhancer configuration during senescence. Hyper-connected enhancer communities/cliques formed around genes that are highly expressed and within essential gene pathways in each cell state. In addition, motif analysis indicates the involvement of specific transcription factors in hyper-connected regulatory elements in each condition; importantly, MafK, a bZIP family transcription factor, was upregulated in senescence, and reduced expression of MafK ameliorated the senescence phenotypes. Because the accumulation of senescent cells is a key feature of aging, we further investigated enhancer connectomes in the liver of young and aged mice. Hyper-connected enhancer communities were identified during aging, which regulate essential genes that maintain cell differentiation and homeostasis. These findings reveal that hyper-connected enhancer communities correlate with high gene expression in senescence and aging and provide potential hotspots for therapeutic intervention in aging and age-associated diseases.

overall_design: Two biological replicates each of H3K27ac Hi-ChIP in young and aged mouse hepatocytes. Data are in three batches of technical repeats per replicate for both conditions.

first: Lu

last: Wang

first: Greg

first: Claire

last: Chung

first: Chen

last: Zhang

first: Aaron

last: Havas

first: Xue

last: Lei

first: Hideki

last: Tanizawa

first: Osamu

last: Iwasaki

first: Caiyue

last: Xu

first: Wenliang

first: Khoa

last: Tran

first: Charly

last: Good

first: Robert

last: Faryabi

first: Golnaz

last: Vahedi

first: Ken-Ichi

last: Noma

first: Peter

last: Adams

first: Shelley

last: Berger


title: Cell-type-specific translational regulation by human DUS enzymes [iCLIP]

first: Nathan

last: Yu

overall_design: iCLIP sequencing samples of 5-ClUrd-treated 3xFLAG-DUS1L cells, 2 biological replicates with 2 untreated replicates and iCLIP sequencing samples of 5-ClUrd-treated 3xFLAG-DUS2L cells, 2 biological replicates with 2 untreated replicates

first: Wei

last: Dai

first: Ang

last: Li

first: Muhan

last: He

first: Ralph

last: Kleiner


title: Argonaute3-SF3B3 complex controls pre-mRNA splicing to restrain Type-2 immunity [miRNA-seq]

city: South San Francisco

first: Jason

last: Hackney

state: CA

address: 1 DNA Way

department: Department of Bioinformatics and Computational Biology

institute: Genentech, Inc.

summary: Argonaute (AGO) proteins execute Micro-RNA (miRNA) mediated gene silencing. However it is currently debated whether all 4 mammalian AGO proteins (AGO1, AGO2, AGO3 and AGO4) are required for miRNA activity. To address this, we generated a mouse with deleted Ago1, Ago3 and Ago4 genes (Ago134), and found that these Argonaute proteins are completely redundant for miRNA biogenesis, miRNA homeostasis or miRNA function, a role that is carried out exclusively by AGO2. Instead, AGO1/3/4 are required to curtail the expansion of Type-2 adaptive immunity in mice via regulation of precursor-mRNA (pre-mRNA) splicing in CD4+ TH lymphocytes. Gain- and loss-of-function experiments demonstrate that nuclear AGO3 interacts directly with SF3B3, a component of the U2 spliceosome complex, to aid global mRNA splicing. Our work uncouples AGO1, AGO3 and AGO4 from miRNA-mediated RNAi, discovers a new AGO3:SF3B3 complex in the cellular nucleus, and reveals an underappreciated mechanism by which AGO proteins prevent hyper-inflammatory disease.

overall_design: Comparison of miRNAs from wild type, AGO2 knockout and AGO1/AGO3/AGO4 knockout CD4 T cells. Mouse splenocytes were isolated and naïve CD4+ T cells were obtained via beads purificationa and FACS sorting. Naïve CD4+ T cells were activated and polarized towards Th2 subtype, and total RNA collected at day 0, day 7 and day 14 of in vitro culture. Total RNA was subject to miRNA Seq using TrueSeq Illumina kit. Paired-end sequencing of high depth reads in naïve and activated T cells (nT and TH2 7 days) from littermate WT and AGO134-KO mice, and CD4-Cre;AGO2flox/flox and CD4-Cre mice.

first: Riccardo

last: Guidi

first: Christopher

last: Wedeles

first: Joseph

last: Guillory

first: Zora

last: Modrusan


title: Argonaute3-SF3B3 complex controls pre-mRNA splicing to restrain Type-2 immunity [RNA-seq: cultured cells]

overall_design: Comparison of wild type and AGO1/AGO3/AGO4 knockout Th2 cells at 7 days and 15 days of culture. Mouse splenocytes were isolated and naïve CD4+ T cells were obtained via beads purification and FACS sorting. Naïve CD4+ T cells were activated and polarized towards Th2 subtype, and total RNA collected at day 0, day 7 and day 14 of in vitro culture. Total RNA was subject to bulk RNA Seq using TrueSeq Illumina kit. Paired-end sequencing of high depth reads in naïve and activated T cells (nT and TH2 7 days) from littermate WT and AGO134-KO mice.


title: High Rates of Placental Inflammation in Samples Collected by the MOMI Consortium

city: SAN FRANCISCO

first: Joshua

last: Robinson

address: 513 Parnassus Ave HSE16

department: Obstretrics, Gynecology and Reproductive Sciences

institute: University of California, San Francisco

summary: OBJECTIVES: The Multi-Omics for Mothers and Infants (MOMI) consortium aims to improve birth outcomes. We analyzed full thickness placental samples (e.g., basal plate, placenta/chorionic villi and the chorionic plate) collected by the 5 MOMI sites: The Alliance for Maternal and Newborn Health Improvement (AMANHI) Bangladesh, AMANHI Pakistan, AMANHI Tanzania, The Global Alliance to Prevent Prematurity and Stillbirth (GAPPS) Bangladesh and GAPPS Zambia.  STUDY DESIGN. The teams collected biopsies from 294 preterm (gestation <37 weeks) and 291 term (gestation ≥37 weeks) births. They were formalin-fixed and paraffin embedded. Tissue sections from these samples were stained with hematoxylin and eosin and subjected to morphological analyses. Other placental biopsies (n = 35 preterm, 21 term) were collected in RNAlater, which enabled bulk transcriptomics.  RESULTS. The morphological analyses revealed a surprisingly high rate of inflammation involving the basal plate, placenta/chorionic villi and the chorionic plate. The rate in chorionic villus samples ranged from 25% (Pakistan site) to 60% (Zambia site) of cases. Leukocyte infiltration in this location vs. the basal plate or chorion plate correlated with preterm birth. The transcriptomic analyses identified 267 genes as differentially expressed (DE) between placentas from preterm vs. term births (123 upregulated, 144 downregulated). Mapping the DE genes onto single cell RNA-seq data from human placentas suggested that all the component cell types, either singly or in subsets, contributed to the observed dysregulation. Consistent with the histopathological findings, (Gene Ontology) GO analyses highlighted leukocyte infiltration/activation and inflammatory responses in both the fetal and maternal compartments.   CONCLUSION. Together these data suggested geographic- and/or population-based differences in placental inflammation and the triggers of preterm birth.

overall_design: Placental biopsies (n = 35 preterm, 21 term) were profiled for global RNA expression.

first: Susan

last: Fisher

first: Matthew

last: Gormley

first: Michael

last: McMaster

first: Brian

last: DeVeale


title: RCC2 Promotes Prostate Cancer Cell Proliferation and Migration Through Hh/GLI1 Signaling Pathway and Cancer Stem-like Cells

first: Bao

last: Zhang

address: No.15, Yuquan Road

institute: Aerospace Center Hospital

summary: The goal of this study is to explore the function of RCC 2 on PCa development

overall_design: Then RNA-sequencing analysis were performed and signaling pathway were identified.

first: Shenghan

last: Wang

first: Zhentao

last: Lei

last: Liu


title: Neuronal Ash1L Gates Skin Psoriasis Via Activity-dependent Release of miR-let-7b

first: Wan-Jie

last: Du

address: 131 Dong'an Road

department: State Key Laboratory of Medical Neurobiology and MOE Frontier Center for Brain Science, Institutes of Brain Science

institute: Fudan University

summary: To investigate the deficiency of a histone methyltransferase, Ash1L, in primary sensory neurons in the regulation of neurite hyperinnervation and neuronal excitability, we established Ash1l haploinsufficiency mice. We then performed gene expression profiling analysis using data obtained from RNA-seq of dorsal root ganglions from Ash1l+/- and its littermate wild type mice.

overall_design: Differential gene expression profiling analysis of RNA-seq data of dorsal root ganglions (DRGs) from Ash1l+/- and its littermate wild type mice

first: Huan

last: Yang

first: Qingjian

last: Han


title: Circulating miRNAs and Machine Learning for Lateralizing Primary Aldosteronism

city: Budapest

first: Bálint

last: Vékony

address: Korányi Sándor utca 2/a

department: Department of Internal Medicine and Oncology

country: Hungary

institute: Semmelweis University

summary: Primary aldosteronism (PA) is the most prevalent cause of secondary hypertension. Its two main clinical forms are unilateral adenoma (UPA) and bilateral hyperplasia (BAH), which require markedly different medical treatments, so differentiating between the two is of utmost clinical importance. The current gold standard method for this is adrenal vein sampling (AVS), the application of which is hindered by limited availability and high skill requirements. Our goal was to identify circulating microRNAs – or their combinations – which enable differentiation between the two most prevalent aetiologies of PA from a peripheral blood sample. MicroRNA specific sequencing was performed on an Illumina platform, using EDTA coagulated blood samples taken during AVS, from 18 patients (10 uni-, and 8 bilateral). First, plasma samples from both adrenal veins were evaluated. Bioinformatical analysis applying the DeSeq2 algorithm was used to evaluate the differences in expression; and a neural network model, tasked to identify the most fit individual and groups of microRNAs for differentiation was used. The microRNAs comprising the five best performing models were then validated using reverse transcription real-time PCR.

overall_design: 36 EDTA coagulated plasma samples taken during adrenal vein sampling were analised. First, microRNA were isolated from the samples, then a microRNA specific next generation sequencing was performed on an Illumina MiSeq platform.

first: Gábor

last: Nyirő

first: Zoltán

last: Herold

first: János

last: Fekete

first: Filippo

last: Ceccato

first: Sven

last: Gruber

first: Lydia

last: Kürzinger

first: Mirko

last: Parasiliti-Caprino

first: Nikolette

last: Szücs

last: Szeredás

first: Siti

middle: K

last: Mohammed Nazri

first: Vanessa

last: Fell

first: Mohammed

last: Basiony

first: Magdolna

last: Dank

first: Elena

last: Aisha Azizan

first: Irina

last: Bancos

first: Felix

last: Beuschlein

first: Péter

last: Igaz


title: Endothelial cell activation sustains estradiol-independent long-term sterile inflammation and promotes endometrial hyperplasia

city: Nanjing

first: Jie

last: Pan

address: xianlin street 1800

department: School of Life sciences

institute: Nanjing University

summary: In this study, we elucidate that increased expression of SHP2 in endothelial cells drives the inflammatory activation of endothelial cell through dephosphorylation of its substrate RIPK1 at Y380 site and thus further makes endothelial dysfunction by influencing the transcript factors AP-1 and its target gene IGF1 and inflammatory related genes. The inflammatory activated endothelial cells recruited and activated macrophages, which enhances the inflammatory activation of endothelial cells in a positive feedback manner. Increased growth factor IGF1 in tissue leads to abnormal gland epithelial cell proliferation. Therefore, all the results indicate the importance of tissue endothelial cells and long-term estradiol independent sterile tissue inflammation as regulators of the endometrium hyperplasia. Targeting tissue inflammation through SHP2 inhibition or other anti-inflammation strategies may be potential interventions of EH.

overall_design: To investigate the role of SHP2 in regulating the gene expression in HUVEC after estradiol treatment, we treated HUVECs with estradiol or estradiol plus SHP2 inhibitor SHP099. We also knockdown SHP2 in HUVECs using virus to compare the same gene expression with that treated with SHP099 in the condition of estradiol.

first: Yang

last: Sun


title: Effects of Denervation on Muscle Function

first: Zhiqiang

last: Huang

state: Jiangsu

address: Hankou 22

department: Medical School

summary: Innervation of skeletal muscle fibers plays a crucial role in the maintenance of muscle tone and normal functioning, but little is known to date about denervated muscle atrophy and its underlying mechanisms. To this end, we performed RNA sequencing of skeletal muscle from sciatic nerve-excised C57BL/6 and BALB/c mice to investigate the underlying mechanisms of denervated skeletal muscle atrophy.

overall_design: We investigated the important role of the muscle membrane protein α7nAChR in the maintenance of muscle function by denervation.

first: Xiaolu

last: Jin

first: Hongwei

last: Wang


title: Sodium Butyrate Ameliorates Renal Tubular Lipid Accumulation Through the PP2A-TFEB axis in Diabetic Nephropathy

first: Jiayin

last: Tao

address: Chaoyang Beisanhuandong Street 11

institute: Beijing University of Chinese Medicine

summary: Background: Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease worldwide with limited treatment options. The intricate pathogenesis of dysregulated lipid metabolism leading to the development of DKD remains obscure. Lipophagy, which refers to the autophagic degradation of intracellular lipid droplets, has been found to be impaired in DKD, resulting in renal tubule dysfunction and ectopic lipid deposition (ELD). The lipotoxity in renal tubules is closely related to the pathogenesis of DKD. Therefore, it is crucial to develop effective and safe agents that can restore autophagic flux and lipophagy in renal tubules for potential preventive interventions. Purpose: This study seeks to investigate the preventive effect of sodium butyrate (NaB) on ELD in the progression of DKD and elucidate the underlying mechanisms involved.Methods: The beneficial effects of NaB were investigated in glucolipotoxicity (GLT)-stimulated HK-2 cells and the streptozotocin (STZ) injection combined with a high-fat diet induced DKD model mice. Additionally, we managed to silence transcription factor EB (TFEB) in HK-2 cells and establish the renal specific silence model by retrograde ureteral infusion of AAV9-shTFEB in mice. We investigated the expression profile of RNAs in HK-2 cells through. The mechanism of NaB is demonstrated by RNA sequencing (RNA-seq), pulldown MS assay, SPR experiments etc.Results: Our findings revealed that serum butyrate level is reduced in DKD patients and is statistically correlated with urinary protein excretion and eGFR. Sodium butyrate (NaB) dose-dependently improved lipid deposition in HK-2 cells and renal tubular epithelial cells of DKD model mice. This effect may be attributed to the promotion of TFEB dephosphorylation, as NaB can directly interact with PPP2R1A to activate PP2A phosphorylation activity. Therefore, NaB may play a role in improving renal lipid deposition in DKD through the PP2A-TFEB pathway. Conclusion: NaB acts as a PP2A-TFEB axis activator that restores lipophagy to ameliorate ELD and renal dysfunction in DKD.

overall_design:
To investigate the function of NaB in the regulation of renal lipid accumulation, we administrated GLT into the HK-2 cell culture to simulate high sugar and fat environment in DN.
We then performed gene expression profiling analysis using data obtained from RNA-seq of GLT stimulated HK-2 cells with or without NaB treatment.

first: Sinan

last: Ai

first: Jingwei

last: Zhou

first: Weijing

last: Liu

first: Danting

last: Li

first: Zhen

first: Liang

last: Peng


title: Cell-type-specific translational regulation by human DUS enzymes

first: Nathan

last: Yu

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

overall_design: Refer to individual Series


title: Effects of OGA inhibitor PUGNAc on nascent RNA expression in vivo

city: Bethesda

first: Brian

last: Lewis

state: Maryland

address: 9000 Rockville Pike, Building 10 Room 2N105

department: CCR/Laboratory of Pathology

institute: NCI

summary: We used 4sU incorporation to detect changes in nascent RNA expression in response to the O-GlcNAc aminidase inhibitor PUGNAc.  We found that the distribution of 5' promoter proximal RNAs increased nearly as much as those using the known CDK9 inhibitor flavopiridol.  The similar distributions between the known elongation inhibitor flavopiridol and the OGA inhibitor suggested that the PUGNAc blocked RNA pol II elongation in vivo.

overall_design: Hela cells were treated with inhibitors in two separate experiments

first: David

last: Levens


title: Carrier Subclass

city: La Jolla

first: Louise

middle: C

last: Laurent

address: SCRM, 2880 Torrey Pines Scenic Drive

department: Obstetrics, Gynecology, and Reproductive Sciences

institute: UCSD

summary: To evaluate performance of immunomagnetic separation, differential ultracentrifugation and size exculsion chromatography in the isolation of different extracellular RNA carriers from biofluids.

overall_design: Serum, plasma, placental explant supernatant and cell culture supernatants were collected. Extracellular RNA carriers were isolated using various methods including immunomagnetic separation, differential ultracentrifugation and size exclusion chromatography and analysed using small RNA seq

first: Srimeenakshi

last: Srinivasan

first: Peter

last: Hoff

first: Abbas

last: Hakim

first: Willi

last: Cheung

first: Priyadarshini

last: Pantham

first: Jeff

last: Franklin

first: Steve

last: Gould

first: Robert

last: Raffai

first: Anil

last: Sood

first: Saumya

last: Das


title: Cell-type-specific translational regulation by human DUS enzymes [tRNA NaBH4]

summary: Dihydrouridine is an abundant and evolutionary conserved modified nucleoside present on tRNA, but characterization and functional studies of individual modification sites and associated DUS writer enzymes in mammals is lacking. Here we use a chemical probing strategy, RNABPP-PS, to identify 5-chlorouridine as a general activity-based probe for human DUS enzymes. We map D modifications using mechanism-based RNA-protein crosslinking and also through chemical reactivity and mutational profiling to reveal the landscape of D modification sites on human tRNAs. Further, we knockout individual DUS genes in two model human cell lines to investigate their regulation of tRNA expression levels and codon-specific translation elongation. We show that whereas D modifications are present across most tRNA species, loss of D only perturbs the translational function of a subset of tRNAs in a cell-type-specific manner. Our work provides powerful chemical strategies for investigating D and DUS enzymes in diverse biological systems and provides insight into the role of a ubiquitous tRNA modification in translational regulation.

overall_design: Sequencing of NaBH4 treated samples for mutational analysis of HEK293T WT, 3 biological replicates treated, 3 control; HEK293T DUS3L KO, 3 biological replicates treated, 3 control; HeLa WT, 2 biological replicates treated, 2 control.

first: Wei

last: Dai

first: Ang

first: Muhan

last: He

first: Ralph

last: Kleiner


title: Epitope-specific T cell responses after SARS-CoV-2 vaccination [scRNA_dextramer_mixed_time]

first: Kilian

last: Schober

address: Maximiliansplatz 2

institute: Universitätsklinikum Erlangen

summary: Clonal expansion is a hallmark of adaptive immunity, and appears to be driven by high antigen-specific receptor avidity as shown by research using murine in vivo models. In humans, however, the functionality of antigen-specific T cell clonotypes that are recruited into primary and recall immune responses remains surprisingly elusive. In this regard, the vaccination program during the SARS-CoV-2 pandemic represented a unique research opportunity by provision of highly standardized cohorts of healthy human individuals receiving immunizations against a previously unseen antigen. Here, we analyzed 30 HLA-typed individuals before, short-term and long-term after three mRNA vaccinations against SARS-CoV-2. We performed an in-depth characterization of the magnitude, phenotype, clonal composition and functionality of antigen-specific CD8 T cell responses by ELISPOT, flow cytometry and single-cell RNA sequencing, including CITE seq of 130 surface antigens and 16 T cell epitope specificities. 89 T cell receptors (TCRs) covering three complete epitope-specific repertoires were re-expressed by CRISPR/Cas9-mediated orthotopic TCR replacement and tested for their avidity. TCR repertoires underwent continuous tailoring, with high TCR avidity being linked to both clonal persistence and expansion. However, epitope-specific repertoires also maintained diversity by concomitant contraction and new emergence of functional T cell clones over time. These data on the phenotype and clonal selection within human antigen-specific T cell responses instruct our understanding of human T cell biology and may guide the development of enhanced or novel vaccines.

overall_design: Antigen-specific T cells (as well as unspecific T cells as a control) were identified by DNA-baroded pMHC multimers (Dextramers) including the SARS-CoV-2 spike specificity LTD as well as 15 additional SARS-CoV-2 spike epitopes. Dextramer+ and - cells were sorted by FACS and subjected to 5' scRNAseq on a 10x Genomics platform including CITEseq for Dextramers and TotalSeq-C anti-human hashtag antibodies; different samples from different donors were labelled with hasthag antibodies

first: Katharina

last: Kocher

first: Felix

last: Drost


title: miRNAs and their targets regulate plant architecture in Brassica napus

city: Wuhan

first: li

last: chen

address: Huazhong Agricultural University

institute: National Key Laboratory of Crop Genetic Improvement

summary:
Background: MicroRNAs (miRNAs), a class of non-coding small RNAs, are crucial to the regulation of various developmental processes. Plant architecture is a collection of genetically controlled agronomic traits that determine crop production and mechanized harvesting. Although several genes had been found to regulate plant architecture, the mechanisms whereby miRNAs regulate plant architecture in the rapeseed Brassica napus remain unknown.
Results: In this study, we characterized a rod-like rapeseed mutant with an ideal plant architecture that substantially enhanced its breeding potential. To explore miRNAs that contribute to the rapeseed plant architecture, backcross progenies that developed into small plants (rod-like) and tall plants (normal) were used for study. Four small RNA (sRNA) libraries and two degradome libraries from the shoot apex of normal and rod-like plants were sequenced. A total of 925 non-redundant B. napus miRNA precursors were identified, representing 315 precursors for 74 known miRNAs and 610 precursors for 327 novel miRNAs. Expression analysis revealed that 10 known miRNAs and 7 novel miRNAs were differentially expressed between the normal and rod-like plants. In addition, 408 targets were identified through degradome sequencing and 14 targets were further validated via RNA ligase-mediated 5′ rapid amplification of cDNA ends.  Furthermore, the functions of miR319 and its target gene TCP4 were studied and provided a novel insight into how miR319 regulates plant architecture.
Conclusions: Correlation analysis between differentially expressed miRNAs and their targets demonstrated that nutrition and metal deprivation, energy supply deficiency, senescence and TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCPs) contributed to the premature termination of shoot development in rod-like mutant. The work further elucidates the mechanism of miRNAs participate in the regulation of plant architecture.

overall_design: small RNA and degradome sequencing in the shoot apex of rod-like and normal B. napus

first: Li

last: Chen

first: Jinxiong

last: Shen


title: Deciphering the Transcriptomic Landscape of Type 2 Diabetes: Insights from Bulk RNA Sequencing and Single-Cell Analysis [RNA-seq]

status: Public on Oct 30 2024

city: Saint-Petersburg

first: Aleksandr

last: Tkachenko

address: Mendeleyevskaya line, 3

country: Russia

institute: D. O. Ott Research Institute of Obstetrics and Gynecology

summary: Type 2 diabetes (T2D) is a chronic metabolic disorder characterized by insulin resistance and relative insulin deficiency. It is a significant public health concern worldwide, with an estimated prevalence of over 422 million individuals affected globally. This number is projected to rise, making diabetes one of the leading causes of morbidity and mortality. It is associated with numerous severe microvascular and macrovascular complications, including retinopathy, nephropathy, cardiovascular diseases, and neuropathy, which substantially impact patients' quality of life and healthcare systems. The global prevalence of T2D necessitates a deeper understanding of its underlying mechanisms to develop more effective prevention and treatment strategies. Recent advancements in transcriptomics and single-cell technologies have revolutionized our ability to investigate the cellular and molecular landscapes of T2DM. Transcriptomic analysis provides insights into gene expression patterns in T2D, revealing pathways involved in insulin signaling, metabolic regulation, and inflammation. The importance of inflammation driven by immune cells in the pathogenesis of T2D has been repeatedly demonstrated. This makes research focused on the analysis of gene expression in whole blood cells especially relevant. Transcriptomic markers of T2D have been investigated in studies utilizing expression arrays and RNA-seq. While expression arrays allow for the analysis of a gene set limited by the design of microarrays, RNA-seq allows investigating the entire transcriptome. A study focused on the analysis of transcriptomic markers of T2D in whole blood using RNA-seq demonstrated differential expression of genes associated with inflammation, insulin resistance, and mitochondrial dysfunction. However, the heterogeneity of immune cell populations poses significant challenges in understanding the disease's complexity. Single-cell RNA sequencing (scRNA-seq) enables researchers to dissect this cellular diversity, allowing for the identification of distinct cell types and their specific contributions to T2DM progression. By integrating transcriptomic data with single-cell analyses, we can elucidate the dynamic interactions between various cell types and their roles in T2DM. In this study, we aimed to analyze transcriptomic markers of T2D in whole blood cells and conduct single-cell sequencing on isolated PBMCs from T2D patients and healthy controls in order to advance our understanding of the processes involved in the pathogenesis of T2D and to highlight the significance of transcriptomic and single-cell research in T2D studies.

overall_design: To investigate transcriptomic changes in patients with type 2 diabetes we compared single cell RNA sequencing and bulk RNA sequencing data from affected individuals with data from healthy controls

first: Alexander

first: Ziravard

middle: N

last: Tonyan

first: Yulia

last: Nasykhova

first: Yuri

last: Barbitoff

first: Iaroslav

last: Renev

first: Maria

middle: M

last: Danilova

first: Anastasiia

last: Mikhailova

first: Olga

last: Glavnova

first: Sergey

last: Chepanov

last: Selkov

first: Nikita

last: Golovkin

first: Margarita

last: Vlasova

first: Andrey

middle: S

last: Glotov


title: Deciphering the Transcriptomic Landscape of Type 2 Diabetes: Insights from Bulk RNA Sequencing and Single-Cell Analysis [scRNA-seq]


title: Strigolactones are involved in enhancing iron uptake in maize

city: Amherst

first: Elsbeth

last: Walker

address: 611 North Pleasant Street

department: Biology

institute: University of Massachusetts Amherst

summary: A whole transcriptome (RNA-seq) study of maize root and shoots under iron sufficient, deficient and resupply conditions was carried out to determine the genes that are iron-regulated in the roots and shoots.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for wild type W22 maize plants under three conditions (+Fe, -Fe and Fe resupply) in roots and shoots

first: Stavroula

last: Fili


title: Distinct modes of non-canonical initiation regulate selective host versus viral mRNA translation during poxvirus-induced shutoff

city: Chicago

first: Chorong

last: Park

address: 303 E Chicago Ave

institute: Northwestern Univeristy

summary: Many viruses potently inhibit host protein synthesis while employing unconventional strategies to sustain their own translation, but how and why certain cellular mRNAs continue to be translated remains unclear. Here, we show that during shutoff by Vaccinia Virus (VacV) several host mRNAs increase in polysome occupancy but few, topped by JUN, result in increased protein abundance. Translation of viral mRNAs depended on the small ribosomal protein, Receptor for Activated C Kinase 1 (RACK1) and the eukaryotic Initiation Factor, eIF3. Cryo-electron microscopy (cryo-EM) further showed that eIF3 bound to virus-modified RACK1-containing 40S subunits that exhibit altered head rotation during infection. By contrast, RACK1 and eIF3 were dispensable for JUN translation. Moreover, structurally distinct 5’ untranslated regions (5’UTRs) in viral versus JUN mRNAs conferred differential eIF3 dependencies. Altogether, we reveal how distinct modes of non-canonical initiation support the production of both host and viral proteins that facilitate poxvirus replication despite infection-induced shutoff.

overall_design: To investigate how vaccinia virus infection alters host mRNA expression, we used HAP1 cell lines and RACK1 knock out cell lines with or without VacV infection then, performed ribosome profiling and subsequently samples were subjected for polyA selected RNA sequencing.

first: Aaron

last: Ferrell

first: Peter

first: Derek

last: Walsh

first: Nathan

last: Meade


title: Analysis of miRNA expression upon stimulation of LPS activated THP-1 macrophages with Fasciola hepatica Excretory-Secretory Products (Fh-ES)

city: Warsaw

first: Piotr

last: Bąska

state: Mazowieckie

address: Ciszewskiego 8

department: Department of Preclinical Sciences

country: Poland

institute: Warsaw University of Life Sciences WULS – SGGW

summary: LPS activated THP-1 macrophages were stimulated with Fh-ES followed by analyses of miRNA  expression change. The analyses showed no change of miRNA expression  change (p<0.05, FDR correction).

overall_design: Changes of miRNA expression were performed using miRNA microarray technique. The analyses were performed on eight replicates of control samples (LPS activated THP-1 macrophages) and eight replicates of experimental samples (LPS activated THP-1 macrophages stimulated with Fh-ES).


title: Expression profiles of human cumulus cells from patients with PCOS and non-PCOS

city: Zhongshan

first: Yuezhou

last: Chen

address: East 2 Sunwen Road

department: Center for Reproductive Medicine

institute: Zhongshan City People's Hospital

summary: To reveal mRNAs expression differences in cumulus cells between polycystic ovary syndrome (PCOS) and non-PCOS women. mRNAs expression profile of the cumulus cell samples with PCOS and non-PCOS were determined by  Illumina Novaseq 6000.

overall_design: 40 pooled RNAs from CC samples (23 PCOS and 17 non-PCOS pooled RNAs) were separately analyzed on Illumina Novaseq 6000

first: Minyu

last: Xie

first: Siyun

last: Wu

first: Zehua

last: Deng

first: Yan

last: Tang

first: Yiqing

last: Guan

first: Yun

last: Ye

first: Qiandong

last: He

first: Lei

last: Li


title: Argonaute3-SF3B3 complex controls pre-mRNA splicing to restrain Type-2 immunity [RNA-seq: dissected tissues]

first: Jason

last: Hackney

address: 1 DNA Way

summary: Argonaute (AGO) proteins execute Micro-RNA (miRNA) mediated gene silencing. However it is currently debated whether all 4 mammalian AGO proteins (AGO1, AGO2, AGO3 and AGO4) are required for miRNA activity. To address this, we generated a mouse with deleted Ago1, Ago3 and Ago4 genes (Ago134), and found that these Argonaute proteins are completely redundant for miRNA biogenesis, miRNA homeostasis or miRNA function, a role that is carried out exclusively by AGO2. Instead, AGO1/3/4 are required to curtail the expansion of Type-2 adaptive immunity in mice via regulation of precursor-mRNA (pre-mRNA) splicing in CD4+ TH lymphocytes. Gain- and loss-of-function experiments demonstrate that nuclear AGO3 interacts directly with SF3B3, a component of the U2 spliceosome complex, to aid global mRNA splicing. Our work uncouples AGO1, AGO3 and AGO4 from miRNA-mediated RNAi, discovers a new AGO3:SF3B3 complex in the cellular nucleus, and reveals an underappreciated mechanism by which AGO proteins prevent hyper-inflammatory disease.

overall_design: Comparison of gene expression from tissues isolated from either wild-type or AGO1/AGO3/AGO4 knockout mice.

first: Riccardo

last: Guidi

first: Christopher

last: Wedeles

first: Joseph

last: Guillory

first: Zora

last: Modrusan


title: Targeting CD93 on Monocytes revitalizes Anti-Tumor Immunity by Enhancing the Function and Infiltration of CD8+ T Cells

city: Guangzhou

first: Da

last: Jiang

state: Guangdong

address: No. 135 West Xingang Road Guangzhou City

department: School of Life Sciences

institute: Sun Yat-sen University

summary: In this study, we found that monocytes in the peritumoral tissues of HCC significantly increased levels of CD93 expression, and these CD93+ monocytes collocated with CD8+ T cells, whose density was much higher in peri-tumor than intra-tumor areas. In vitro experiments showed that glycolytic switch mediated tumor-induced CD93 up-regulation in monocytes via the Erk signaling pathway. CD93 on the one hand could enhance PD-L1 expression through the AKT-GSK3β axis, while on the other hand induce monocytes to produce versican, a type of matrix component which interacted with hyaluronan and collagens to inhibit CD8+ T cell migration. Consistently, levels of CD93+ monocytes positively correlated with the density of peritumoral CD8+ T cells while negatively correlated with that of intratumoral CD8+ T cells. Targeting CD93 on monocytes not only increased the infiltration and activation of CD8+ T cells, but also enhanced tumor sensitivity to anti-PD-1 treatment in mice in vivo.

overall_design: Levels of CD93 expression on monocytes from paired nontumor, peri-tumor and tumor tissues of human hepatocellular carcinoma (HCC) were evaluated. The underlying mechanisms mediating effects of CD93+ monocytes on the inhibition and tumor exclusion of CD8+ T cells were studied through both in vitro and in vivo experiments.

first: Ai-Qi

last: Huang

first: Bai-Xi

last: Zhu

first: Limin

last: Zheng


title: Deciphering the landscape of GATA-mediated transcriptional regulation in gastric cancer

city: Larissa

first: Antonis

last: Giakountis

address: Biopolis, Mezourlo

department: Biochemistry and Biotechnology

country: Greece

institute: University of Thessaly

summary: Gastric cancer (GC) is an asymptomatic malignancy in early stages, with a limited and cost-ineffective diagnostic toolbox that globally contributes to severe mortality rates on an annual basis. Ectopic expression of the lineage survival transcription factors (LS-TFs) GATA 4 and 6 promotes stomach oncogenesis, at the stage of premalignant gastric lesions. However, LS-TFs play important physiological roles that hinder their direct therapeutic targeting, therefore their downstream target genes are of particular interest for developing cancer-specific molecular biomarkers or therapeutic agents. In this work we couple inducible knock-down systems with chromatin immunoprecipitation and RNA-seq to thoroughly detect and further characterize direct targets of GATA-mediated transcriptional regulation in gastric cancer cells. Our experimental and computational strategy provides evidence that these transcription factors regulate the expression of several coding and non-coding RNAs that in turn mediate for their cancer-promoting phenotypes. Finally, the prognostic and diagnostic potential of selected target transcripts is evaluated in patient biopsies, emphasizing on gastrointestinal tumors.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for AGS cells following inducible GATA4 and 6 knock-down

first: Giakountis

last: Antonis


title: The deubiquitinase OTUD1 attenuates periodontal inflammation by restricting neutrophil protein transport

city: Beijing

first: Jia

last: Song

state: China

address: Peking University School of Medicine, 38 Xueyuan Road, Haidian District, Beijing

institute: Peking University

summary: We report that OTUD1 acts as an immune regulator in periodontitis. OTUD1 inhibits neutrophils migration to inflammatory site via blocking surface protein presentation. Our data reveals that activation of OTUD1 may be a potential strategy for periodontitis treatment.

overall_design: Study of  gingiva infiltration immune cells from WT or Otud1-/- mice in the context of experimental periodontitis using 10x scRNA-seq technologies.

first: Xuehui

last: Zhang

first: Dan

last: Lu


title: Screening for blood-borne biomarkers of adiponectin action using the receptor agonist ALY688

city: TORONTO

first: Gary

last: Sweeney

state: ON

address: 4700 Keele street

country: Canada

institute: York University

summary: The employment of adiponectin-based therapeutic strategies emerges as a compelling approach in the management of diverse metabolic pathologies and the attenuation of inflammatory processes. This has precipitated the development of adiponectin receptor agonists. However, the effective surveillance of pharmacodynamic actions within the context of clinical applications remains challenging. Therefore, in this study we attempted to identify and translate peripheral biomarkers to monitor adiponectin actions in blood samples, by using a small, 10 amino acid peptide, adiponectin receptor agonist, ALY688. RNA-sequencing analysis of whole blood validated the anti-inflammatory effect of ALY688 in a sublethal LPS mouse model treated with or without ALY688. Furthermore, RNAseq analysis identified a cluster of genes, including Tgfb2, Rorc, and Il17rb, that was significantly increased in the group administered ALY688 in comparison to vehicle treated group. This cluster of differentially expressed genes was validated by real-time quantitative PCR and further verified in human peripheral blood mononuclear cells treated with ALY688 ex vivo. Collectively, these data established anti-inflammatory effects of ALY688 and identified a gene cluster signature that can be assessed in blood samples to rapidly and easily biomarker pharmacodynamic action of adiponectin or its mimetics.

overall_design: Plasma of mice injected with or without ALY688 was performed RNA sequencing to indentify biomarkers for monitoring ALY688 action. Besides, to validate the anti-inflammatory effects of ALY688, we conducted RNA-sequencing analysis of whole blood in a sublethal LPS mouse model, comparing outcomes between mice treated with ALY688 and those left untreated.


title: Effect of an LXR agonist on the growth of MMTV-PyMT mammary tumors

city: Washington

first: Lu

last: Jin

address: 3970 Reservoir Rd NW

department: Oncology

institute: Georgetown University

summary: Our findings indicate that LXR activation reduces tumor development and increases survival

overall_design: MMTV-PyMT mice were adminstered an LXR agonist by the oral route continuously beginning at 6 wk of age to measure the effect of LXR activation on tumor progression.

first: Robert

last: Glazer

first: Changyan

last: Zhou


title: MRI and gene signatures in facioscapulohumeral dystrophy muscle: implications for clinical trial design and mechanisms of disease progression

city: Seattle

first: Stephen

last: Tapscott

state: WASHINGTON

address: 1100 Fairview N. Ave

department: Human Biology

institute: Fred Hutch Cancer Research Center

summary: Identifying the aberrant expression of DUX4 in skeletal muscle as the cause of facioscapulohumeral dystrophy (FSHD) has led to rational therapeutic development and clinical trials. Several studies support the use of MRI characteristics and the expression of DUX4-regulated genes in muscle biopsies as biomarkers of FSHD disease activity and progression. We performed lower-extremity MRI and muscle biopsies in the mid-portion of the tibialis anterior (TA) muscles bilaterally in FSHD subjects and validated our prior reports of the strong association between MRI characteristics and expression of genes regulated by DUX4 and other gene categories associated with FSHD disease activity. We further show that measurements of normalized fat content in the entire TA muscle strongly predict molecular signatures in the mid-portion of the TA, indicating that regional biopsies can accurately measure progression in the whole muscle and providing a strong basis for inclusion of MRI and molecular biomarkers in clinical trial design. An unanticipated finding was the strong correlations of molecular signatures in the bilateral comparisons, including markers of B-cells and other immune cell populations, suggesting that a systemic immune cell infiltration of skeletal muscle might have a role in disease progression.

overall_design: A study cohort comprising 34 FSHD subjects, 16 female and 18 male, aged 21 to 69 (mean 47.1±14SD) was recruited at three sites of Seattle Paul D. Wellstone Muscular Dystrophy Specialized Research Center, including University of Washington Medical Center, University of Rochester Medical Center, and Kansas University Medical Center. All participants underwent initial functional testing, including lower extremity dorsiflexor strength measurement, lower extremity MRI, and needle muscle biopsy of both left and right tibialis anterior (TA) muscles.  Muscle biopsies were processed for histology, RNA extraction, and sequencing. DNA was also extracted for bisulfite sequencing (BSS). Out of 68 attempted muscle biopsies, 64 yielded sufficient RNA for sequencing and met the criteria and an RNA integrity number > 4.

first: Chao-Jen

last: Wong

first: Seth

last: Friedman

first: Sean

last: Bennett

first: Takako

middle: I

last: Jones

first: Peter

first: Dennis

middle: W

last: Shaw

first: Silvia

last: Blemker

first: Richard

last: Lemmers

first: Olivia

last: DuCharme

first: Silvère

last: van der Maarel

first: Leo

middle: H

last: Wang

first: Rabi

last: Tawil

first: Jeff

last: Statland


title: Single-cell transcriptomics and TCR repertoire sequencing of T cells of NOD mice

city: Boston

first: MD ZOHORUL

last: ISLAM

address: 1 Joslin Pl Rm 4503, Kostic Lab

department: Section on Pathophysiology and Molecular Pharmacology

institute: Joslin Diabetes Center

summary: T cells primarily drive the autoimmune destruction of pancreatic beta cells in Type 1 diabetes (T1D). However, the profound yet uncharacterized diversity of the T cell populations in vivo has hindered obtaining a clear picture of the T cell changes that occur longitudinally as T1D onset happens. Here we profiled the transcriptome and TCR repertoire of T cells at single-cell resolution from longitudinally collected peripheral blood and islets of Non-Obese Diabetic (NOD) mice using single-cell RNA sequencing technology. We detected clonal expansion and characterized the transcriptional landscape of "islets-matching" T cell clones in the blood and "blood-matching" T cell clones in islets of diabetic NOD mice using the TCR as a molecular barcode. The clonally matching cells show enriched interferon-gamma response pathways than non-matching cells in blood and islets. In addition, we identified a set of transcriptional markers associated with the matching status of the T cell clones. This study provides a single-cell level transcriptome and TCR repertoire atlas of T cells in NOD mice. The results show that TCR and transcriptional signature can be used combinedly to develop biomarker panels for tracking and investigating islets infiltrating T cells.

overall_design: Paired single-cell RNA sequencing (scRNA-seq) and TCR repertoire sequencing on T cells collected from peripheral blood and pancreatic islets of NOD mice were conducted. Six paired islets and blood samples from diabetic mice and four blood samples from non-diabetic mice were used for single-cell RNA-seq and V(D)J repertoire sequencing.

first: Md

middle: Z

last: Islam

first: Sam

last: Zimmerman

first: Jacob

last: Luber

first: Jose

last: Ordovas-Montanes

first: Aleksandar

middle: D

last: Kostic

first: Michael

last: Robben


title: PD-1+CD8+ T Cell-Mediated Hepatocyte Pyroptosis Promotes Progression of Murine Autoimmune Liver Disease

first: Jie

last: Long

address: No.106 Zhongshan 2nd Road

institute: Guangdong Provincial People's Hospital

summary: The specific mechanisms underlying effector pathways in autoimmune liver disease remain enigmatic and therefore constructing appropriate murine models to investigate disease pathogenesis becomes critical. A spontaneous severe murine model of autoimmune liver disease has been previously established in dnTGFβRII Aire-/- mice, exhibiting disease phenotypes that resemble both human primary biliary cholangitis (PBC) and autoimmune hepatitis (AIH). The data suggest that auto-reactive liver-specific CD8+ T cells are the primary pathogenic cells in liver injury. In this study, these data are advanced through the use of both single-cell sequencing and extensive in vitro analysis. The results identify a specific expanded pathogenic subset of PD-1+CD8+ T cells in the liver, exhibiting strong functional activity and cytotoxicity against target cells. Depletion of PD-1+CD8+ T cells using CAR-T cells effectively alleviates the disease. GSDMD-mediated pyroptosis is found to be aberrantly activated in the livers of model mice, and treatment with a GSDMD-specific inhibitor significantly inhibits disease progression. In vitro experiments reveal that PD-1+CD8+ T cells can induce the pyroptosis of hepatocytes through elevated production of granzyme B and perforin-1. These results provide a novel explanation for the cytotoxic activity of pathogenic liver PD-1+CD8+ T cells in autoimmune liver diseases and offer potential therapeutic targets.

overall_design:
CD45+ cells were sorted using the FACS Aria II cell sorter and dispensed into individual microwell plates. The transcriptome of CD45+ lymphocytes from dnTGFβRII, Aire-/-, dnTGFβRII Aire+/- and dnTGFβRII Aire-/- mice (each sample mixed with hepatic cells from 2-3 mice) was split to construct the single cell TCR library and the targeted single cell mRNA library, which was amplified by multiplexed polymerase chain reaction  and sequenced using an Illumina NovaSeq platform. To explore the function state of hepatic cells in different groups, we extracted the total RNA of liver tissues or 5×10^5 hepatic CD8+ T cells from dnTGFβRII Aire-/- mice and their control littermates, then we build the cDNA library and performed the bulk RNA sequencing.
To explore the function state of hepatic cells in different groups, we extracted the total RNA of liver tissues from dnTGFβRII Aire-/- mice with/without PD-1 targeting CAR-T cells and wild type mice, then we build the cDNA library and performed the bulk RNA sequencing.

first: Si-Yu

last: Yang


title: Transmembrane serine protease 2 and proteolytic activation of the epithelial sodium channel in mouse kidney: Late effects of aldosterone

city: Erlangen

first: Alexandr

last: Ilyaskin

address: Waldstr. 6

department: Institute of Cellular and Molecular Physiology

summary: The appropriate regulation of the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron is required for sodium homeostasis and thereby for the long-term regulation of arterial blood pressure. A unique feature of ENaC is its activation by proteolytic cleavage. However, the relevant proteases involved in proteolytic ENaC activation in the kidney remain elusive. Here, we examined a potential role of transmembrane serine protease 2 (TMPRSS2). A mouse cortical collecting duct cell line (mCCDcl1) was used as a model system. We performed RNA-Seq transcriptome analysis of mCCDcl1 cells, which confirmed coexpression of Tmprss2 and all three ENaC subunits (Scnn1a, Scnn1b, Scnn1g) in this cell line. Importantly, high baseline expression of TMPRSS2 was detected in these cells without a stimulatory effect of aldosterone on its function or transcription. TMPRSS2 knockout in mCCDcl1 cells compromised γ-ENaC cleavage and reduced baseline and aldosterone-stimulated transepithelial short-circuit currents, which could be rescued by chymotrypsin. A compensatory transcriptional upregulation of other proteases was not observed. From these findings we conclude that TMPRSS2 is likely to contribute to proteolytic ENaC activation in the kidney.

overall_design: Late effects of aldosterone exposure (24 h) on expression of TMPRSS2 and other serine proteases in wild-type mCCDcl1 cells were investigated. RNA-seq analysis was performed with aldosterone-treated cells (7 samples) versus vehicle-treated cells (7 samples).

first: Florian

last: Sure

first: Sara

last: Afonso

first: Marko

last: Bertog

first: Christoph

last: Korbmacher


title: Human microRNA-122b-5p regulates chikungunya virus replication in macrophages by directly targeting viral 3'-UTR and cellular histone deacetylase 4

city: New Delhi

first: Sujatha

last: Sunil

state: Delhi

address: Aruna Asaf Ali Road

department: Vector Borne Disease Group

country: India

institute: International Centre for genetic engineering and Biotechnology

summary: We found that Histone Deacetylase 4 is the target of human miR-122b-5p using transcriptome approach and report thst HDAC4 is downregulated in CHIKV infected human macrophages through global transcriptome profiling

overall_design: 6 samples (each contains 3 triplicates) = 18 samples

first: Priyanshu

last: Srivastava

first: Sakshi

last: Chaudhary


title: OGD-Induced Lactylation of H3K9 Contributes to M1 polarization and Inflammation of microglia Through  TNF pathway

city: 成都

first: Lu

last: He

address: WUHAN

institute: TONGJI

summary: Hypoxia-induced M1 polarization of microglia and the resultant inflammatory response are pivotal mechanisms in the development of hypoxic-ischemic encephalopathy (HIE). Recent studies have identified lactylation of histones as a novel epigenetic modification, in which lactate groups are added to lysine residues on histones. Lactate, a product of cellular respiration, accumulates in the cytoplasm when cells experience hypoxia due to the conversion of pyruvate by lactate dehydrogenase. Therefore, histone lactylation may be involved in the pathogenesis of HIE. This study aims to investigate the role and mechanism of histone lactylation in hypoxia-induced M1 polarization of microglia and the associated inflammation, with the goal of providing new insights for the research and treatment of HIE.

overall_design: In this study, we examined the effects of oxygen-glucose deprivation (OGD) on microglial polarization using both an HIE animal model and a cell model. The levels of histone lactylation at different lysine residues were detected by Western blot. Microglial polarization and inflammatory cytokines induced by OGD were experimentally verified by immunofluorescence, qPCR, and Western blot. Finally, RNA sequencing, ChIP-qPCR and siRNA was performed to elucidate the potential mechanisms by which H3K9 lactylation regulates M1 polarization of microglia.


city: Geneva

first: Raquel

last: ROUCO GARCIA

address: Rue Michel-Servet 1

department: Department of Genetic Medicine and Development

country: Switzerland

institute: University of Geneva

summary: Cis-regulatory elements control gene expression in time and space and their disruption can lead to pathologies. Reporter assays allow the functional validation of enhancers and other regulatory elements, and transgenic mice provide a powerful tool to study gene regulation in vivo. However, these experiments are time-consuming and inefficient. Here, we increase the throughput of transgenic reporter assays by using a piggyBac transposon-based system, and use it to decode the regulatory landscape of atrial fibrillation, a prevalent cardiac arrhythmia. We systematically interrogated ten human atrial fibrillation associated loci, finding five new cardiac-specific enhancers. We also connected novel genes to atrial fibrillation through genome editing and three-dimensional chromatin structure analysis. Of note, we identified a bivalent regulatory element in the second intron of the CAV1 gene at the 7q31 locus, differentially acting upon four genes. Our approach also detected negative regulatory elements such as an ubiquitous silencer in the 16q22 locus that regulates ZFHX3 and can outcompete heart enhancers. Our study characterizes the function of new genetic elements that might be of relevance for the better understanding of gene regulation in cardiac arrhythmias. Thus, we have established a new framework for the efficient dissection of the genetic contribution to common human diseases.

overall_design: 4C-seq experiments were perfomed on adult mouse atria to study the genomic interactions of the candidate enhancer regions. Two biological replicates per viewpoint were produced, sent to sequence and analysed independently.

first: Jesus

last: Victorino

first: Isabel

last: Rollan

last: Rouco

first: Javier

last: Adan

first: Miguel

last: Manzanares


title: PAX6-WNK2 axis governs corneal epithelial homeostasis

first: Li

middle: Qiong

last: Zhu

address: No.7 Jinsui road

summary: PURPOSE. Limbal stem/progenitor cells (LSC) continuously proliferate and differentiate to replenish the corneal epithelium and play a vital role in corneal function and normal vision. Previous study revealed that Paired box 6 (PAX6) is a master transcription factor involved in determining the fate of corneal epithelial cells (CEC). However, the molecular events downstream of PAX6 remain largely unknown. In this study, we aimed to clarify the regulation network of PAX6 in driving CEC differentiation. METHODS. An air-liquid culture system was used to differentiate LSC into mature CEC. Specific targeting PAX6 short-hairpin shRNAs were used to knock down PAX6 in LSC. RNA-seq was used to analyze shPAX6-transfected CEC and CEC differentiation-associated genes to identify the potential downstream targets of PAX6. RNA-seq analysis, quantitative real-time PCR, and immunofluorescence staining were performed to clarify the function of WNK lysine deficient protein kinase 2 (WNK2), a downstream target of PAX6, and its relationship with corneal diseases. RESULTS. WNK2 expression increased during CEC differentiation and decreased upon PAX6 depletion. The distribution of WNK2 was specifically limited to the central corneal epithelium and suprabasal layer of the limbus. Knockdown of WNK2 impaired the expression of CEC-specific markers (KRT12, ALDH3A1, and CLU), disrupted the corneal differentiation process, and activated the terms of keratinization, inflammation, and cell proliferation, consistent with PAX6-depleted CEC and published microbial keratitis. Thus, aberrant expression of WNK2 was linked to corneal ulcers. CONCLUSIONS. As a downstream target of PAX6, WNK2 plays an essential role in corneal epithelial cell differentiation and maintenance of corneal homeostasis.

overall_design:
To elucidate the regulatory mechanisms of PAX6 in driving corneal epithelial cells (CEC) differentiation, human primary limbal stem/progenitor cells (LSC) were cultured. A serum-free air-liquid interface system was established to obtain mature CEC in vitro. Short-hairpin shRNAs were used to knock down the expression of PAX6 or WNK2 in LSC, which were then differentiated into CEC, with scramble shRNA serving as control for each group.
RNA samples frpm LSC, CEC, scramble-transfected CEC, shPAX6-transfected CEC, and shWNK2-transfected CEC were extracted and subjected to RNA-seq analysis. Differential gene expression analysis was performed to compare the gene expression: LSC versus CEC, scramble-transfected CEC versus shPAX6-transfected CEC, and scramble-transfected CEC versus shWNK2-transfected CEC.

first: Liqiong

first: Chaoqun

last: Chen

first: Siqi

last: Wu

first: Huizhen

last: Guo

first: Lingyu

last: Li

first: Wang

first: Dongmei

last: Liu

first: Yu

last: Zhan

first: Xinyue

last: Du

first: Jiafeng

first: Jieying

last: Tan

first: Ying

last: Huang

first: Kunlun

last: Mo

first: Xihong

last: Lan

first: Jin

last: Yuan

first: Hong

last: Ouyang

first: Xiangjun

first: Jianping

last: Ji


title: Reprogramming of normal fibroblasts into ovarian cancer-associated fibroblasts via non-vesicular paracrine signaling induces an activated fibroblast phenotype

city: Sioux Falls

first: Pilar

last: de la Puente

state: South Dakota

address: 2301 E 60th St N

institute: Sanford Research

summary: Cancer-associated fibroblasts (CAFs) are key contributors to ovarian cancer (OC) progression and therapeutic resistance through dysregulation of the extracellular matrix (ECM). CAFs are a heterogenous population derived from different cell types through activation and reprogramming. Current studies rely on uncharacterized heterogenous primary CAFs or normal fibroblasts that fail to recapitulate CAF-like tumor behavior. Here, we present that conditioned media from ovarian cancer lines leads to an increase in the activated state of fibroblasts demonstrated by functional assays and up-regulation of known CAF-related genes and ECM pathways. Phenotypic and functional characterization demonstrated that the conditioned CAFs expressed a CAF-like phenotype, strengthened proliferation, secretory, contractility, and ECM remodeling properties when compared to resting normal fibroblasts, consistent with an activated fibroblast status. Moreover, conditioned CAFs significantly enhanced drug resistance and tumor progression. Critically, the conditioned CAFs resemble a transcriptional signature with involvement of ECM remodeling. The present study provides mechanistic and functional insights about the activation and reprogramming of CAFs in the ovarian tumor microenvironment mediated by nonvesicular paracrine signaling. Moreover, it provides a translational based approach to reprogram normal fibroblasts from both uterine and ovarian origin into CAFs using tumor-derived conditioned media. Using these resources, further development of therapeutics that possess potentiality and specificity towards CAF/ECMmediated chemoresistance in OC are further warranted.

overall_design: Normal human uterine fibroblasts (HUFs) were reprogramed into cancer associated fibroblasts (CAFs) by using ovarian cancer conditioned media. Reprogrammed CAFs were functionally characterized and demonstrated an activated fibroblast status characteristic of primary CAFs. RNA-seq and gene expression analysis was performed in 3 replicates of each of the following: HUFs (negative control), primary ovarian CAFs (positive control), and reprogramed CAFs derived from the conditioned media of two different ovarian cancer cell lines (KURAMOCHI and SKOV3).

first: Hailey

last: Axemaker

first: Simona

last: Plesselova

first: Kristin

last: Calar

first: Megan

last: Jorgensen

first: Jared

last: Wollman


title: scRNA-Seq of tumour using different dissocation condition

city: Vestec by Prague

first: Radek

last: Šindelka

address: Průmyslová 595

country: Czech Republic

institute: Institute of Biotechnology, Czech Academy of Science, v.v.i.

summary: We performed single-cell RNA-Seq to study the effect of temperature and the transcriptional inhibitor ActD, on the representation of individual cellular populations within the tumour sample. We collected tumour tissue after 10 days of the growth, dissociated it and analysed it using scRNA-Seq. The ScRNA-Seq analysis contained 654 cells, which were divided into four main cellular types: Tumour cells, Macrophages, T cells and Natural killer (NK) cells.

overall_design: Balb/c mice were subcutaneously injected with 1x106 of 4T1 cells. After 10 days, tumours were collected, weighted and dissociated at different conditions. The final single-cell suspensions were divided into two technical replicates and single-cell libraries were prepared using ddSEQ single cell isolator.

first: Pavel

last: Abaffy

first: Jaroslav

last: Truksa


title: Small Nucleolar RNAs in the Dlk1-Gtl2 Imprinting Locus Maintains LT-HSC Self-renewal by Regulation of rRNA Modification and Translation [ncRNA-Seq]

city: Hangzhou

first: Jiang

last: Penglei

address: Yuhangtang Road 866

department: School of basic medicine science

institute: Zhejiang University

summary: Small nucleolar RNA (snoRNA) are non-coding RNAs, which participate in the chemical modification of ribosomal RNAs (rRNAs) and small nuclear RNAs. However, the roles of snoRNA in homeostasis of hematopoietic stem cells(HSCs) have not been studied. We isolated four hematopoietic stem and progenitor cells (HSPCs) (LT-HSCs, IT-HSCs, ST-HSCs, and MPPs) from the bone marrow (BM) of C57BL/6 mice and performed small RNA-seq. We found SnoRNAs belonging to SNORD113-114 cluster were specifically enriched in LT-HSCs, and their expression decreased dramatically with HSC differentiation. Here, we used a modified small RNA-seq method to examine snoRNA profiles of hematopoietic progenitor and mature cells in WT mice.

overall_design: Examination of snoRNA profiles of hematopoietic progenitor and mature cells from WT mice.

first: Hui

last: Wang

first: Zhaoru

last: Zhang

first: Yingli

last: Han

first: Penglei

last: Jiang

first: Pengxu

last: Qian