title: Circulating Cell-Free RNA in Blood as a Host Response Biomarker for the Detection of Tuberculosis [validation_data]

first: Iwijn

last: De Vlaminck

summary: Tuberculosis (TB) remains a leading cause of death from an infectious disease worldwide. This is partly due to a lack of tools to effectively screen and triage individuals with potential TB. Whole blood RNA signatures have been extensively studied as potential biomarkers for TB, but they have failed to meet the World Health Organization's (WHOs) optimal target product profiles (TPPs) for a non-sputum triage or diagnostic test. In this study, we investigated the utility of plasma cell-free RNA (cfRNA) as a host response biomarker for TB. We used RNA profiling by sequencing to analyze plasma samples from individuals with a cough lasting at least two weeks, who were seen at outpatient TB clinics in Uganda, Vietnam, and the Philippines. We split the samples into a discovery set for model training and testing, and reserved a validation set from a separate cohort to validate the model performance. We trained 15 machine learning classification models and developed a 6-gene signature that has a high performance in differentiating TB positive and negative individuals (Area Under the Curve, AUC = 0.95, 0.92, 0.95 for the test, training and validation sets respectively). This 6-gene signature exceeds the optimal WHO TPPs for a TB triage test (sensitivity: 97.1% [95% CI: 80.9-100%], specificity: 85.2% [95% CI: 72.4-100%]) and was robust to differences in geographic location, sample collection, and HIV status. Analysis of matched whole blood samples from the validation cohort highlighted the differences in origin of plasma and whole blood RNA. Overall, our results demonstrate the utility of plasma cfRNA for the detection of TB and suggest the potential for a point-of-care, gene expression-based assay to aid in early detection of TB.

overall_design:
In the study presented here 251 patients who presented with a cough >= 2 weeks were identified in three separate cohorts, across three different countries (Uganda, Vietnam, Philippines). Patients were identified as TB positive or negative at clinics (tb status indicated in the “tb” column of the metadata). We extracted the cell-free RNA from the plasma of patients and conducted RNA sequencing. We split these samples into discovery (cohort 1 and 2) and validation (cohort 3). And then further split the discovery into 70% for machine learning model training and 30% for model testing. The validation cohort was evaluated independently. The data shown here are the gene transcript counts from the validation data samples.
Please note that the records have been updated with raw sequencing data on Mar 27, 2024.

first: Adrienne

last: Chang

first: Conor

middle: J

last: Loy

first: Daniel

last: Eweis-LaBolle

first: Joan

last: Lenz

first: Amy

last: Steadman

first: Alfred

last: Andama

first: Nguyen

last: Viet Nhung

first: Charles

last: Yu

first: William

last: Worodria

first: Claudia

middle: M

last: Denkinger

first: Payam

last: Nahid

first: Adithya

last: Cattamanchi


title: Transcriptomic profiling of cardiac resident macrophages upon high-fat diet or with aging

status: Public on Jan 24 2024

city: Shanghai

first: Congfeng

last: Xu

address: NO. 600 Yishan road

country: China

institute: Shanghai Jiaotong University School of Medicine Affiliated Sixth People's Hospital

type: Expression profiling by high throughput sequencing

summary: Cardiac resident macrophages (CRM) are essential for cardiac homeostais and physiological/pathological responses. CRM of high-fat diet fed mice or aged mice were sorted by FACS and subjected to SMART-seq.

overall_design: CRM were sorted from the hearts of control, aged or high-fat diet fed mice ( n = 3 ea.) . More than 5000 cells from each sample (murine heart) were used to extract total RNA and SMART-seq assay.

first: Simeng

last: Zhu

first: Guofang

last: Xia


title: Cbfβ regulates Wnt/β-catenin, Hippo/Yap, and TGFβ signaling pathways in articular cartilage homeostasis and protects from ACLT surgery-induced osteoarthritis

status: Public on Jan 19 2024

city: New Orleans

first: Yi-Ping

last: Li

state: LA

address: 1441 Canal St, Room 318

department: Pathology and Laboratory Medicine

country: USA

institute: Tulane University School of Medicine

summary:
As the most common degenerative joint disease, osteoarthritis (OA) contributes significantly to pain and disability during aging. Several genes of interest involved in articular cartilage damage in OA have been identified. However, the direct causes of OA are poorly understood. Evaluating the public human RNA-seq dataset showed that Cbfβ, (subunit of a heterodimeric Cbfβ/Runx1,Runx2, or Runx3 complex) expression is decreased in the cartilage of patients with OA. Here, we found that the chondrocyte-specific deletion of Cbfβ in tamoxifen-induced Cbfβf/fCol2α1-CreERT mice caused a spontaneous OA phenotype, worn articular cartilage, increased inflammation, and osteophytes. RNA-sequencing analysis showed that Cbfβ deficiency in articular cartilage resulted in reduced cartilage regeneration, increased canonical Wnt signaling and inflammatory response, and decreased Hippo/YAP signaling and TGF-β signaling. Immunostaining and western blot validated these RNA-seq analysis results. ACLT surgery-induced OA decreased Cbfβ and Yap expression and increased active β-catenin expression in articular cartilage, while local AAV-mediated Cbfβ overexpression promoted Yap expression and diminished active β-catenin expression in OA lesions. Remarkably, AAV-mediated Cbfβ overexpression in knee joints of mice with OA showed the significant protective effect of Cbfβ on articular cartilage in the ACLT OA mouse model. Overall, this study, using loss-of-function and gain-of-function approaches, uncovered that low expression of Cbfβ may be the cause of OA. Moreover, Local admission of Cbfβ may rescue and protect OA through decreasing Wnt/β-catenin signaling, and increasing Hippo/Yap signaling and TGFβ/Smad2/3 signaling in OA articular cartilage, indicating that local Cbfβ overexpression could be an effective strategy for treatment of OA.
Using unbiased genome-wide RNA-seq data from Cbfβf/f;Col2α1-Cre hip joint articular cartilage and Cbfβf/f;Aggrecan-cre knee joint articular cartilage and their controls, we examined Cbfβ-mediated transcriptional targets for articular cartilage regeneration in OA.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for mice WT, Cbfβf/f;Col2α1-Cre hip joint articular cartilage and Cbfβf/f;Aggrecan-cre articular cartilage

first: Wei

last: Chen

first: Siyu

first: Abigail

last: McVicar


title: Evidence of small interfering RNA in the fungus Fusarium fujikuroi

status: Public on Jan 21 2024

city: Sevilla

first: Javier

last: Avalos

address: Av. Reina Mercedes sn

department: Genetica

country: Spain

institute: Universidad de Sevilla

type: Non-coding RNA profiling by high throughput sequencing

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

sample_organism:
Fusarium fujikuroi
Fusarium oxysporum f. sp. lycopersici

overall_design: Refer to individual Series


title: Evidence of small interfering RNA in the fungus Fusarium fujikuroi (miRNA-Seq)

summary: RNA interference (RNAi) mechanisms play key regulatory roles in many biological systems, and components of the RNAi pathway are conserved in a wide range of eukaryotic genomes, including those of filamentous fungi. The biotechnological fungus Fusarium fujikuroi, widely used in secondary metabolism studies, contains the complete set of genes expected for RNAi pathways, including dcl1 and dcl2 dicer genes. We have analyzed by means of a high-throughput sequencing technology the content of sRNAs in F. fujikuroi grown in the dark or after one hour of illumination. For comparative purposes, the study was extended to the phytopathogenesis model Fusarium oxysporum, grown under the same conditions. Total RNA samples from each species and growth condition were used to construct RNA libraries, which were subjected to massive sequencing. sRNA preparations included a size cut-off below 150 nt, which covered sRNAs and their precursors. The size distributions and 5' nucleotide preferences of the sRNA reads showed a higher proportion of 5' uracil in the RNA samples of the expected sizes in both species, more noticeable in F. fujikuroi, indicating the occurrence of genuine sRNAs. Consistently, the number of sRNAs mapped at CDS loci was significantly higher in F. fujikuroi compared to F. oxysporum. F. fujikuroi carries at least one transcriptionally expressed copy of a Ty1/copia-like retrotransposable element, in which sRNAs were found in both sense and antisense DNA strands, whereas in F. oxysporum Skippy-like elements are expressed and show siRNA formation. The finding of sRNA in these mobile elements is an indication of an active siRNA-based RNAi pathway. The dcl2 deletion mutants did not show phenotypic alterations or changes in their global transcriptome, while no dcl1 deletion mutants could be obtained.

platform_organism:
Fusarium fujikuroi
Fusarium oxysporum f. sp. lycopersici

overall_design: This sRNA analysis was carried by selecting transcripts with a size <150 nt in total RNA samples used in a previously published experiment [Ruger-Herreros et al. 2019. BMC Genomics. 20: 67. doi:10.1186/s12864-019-5430-x; GEO Series accession number GSE107342], in which the influence of light and CarS protein in F. fujikuroi and F. oxysporum transcriptomes was analyzed. They were sequenced on Illumina's Hiseq platform in mode 50 bp single read. Raw reads for all samples were trimmed, filtered, and quality controlled with AfterQC.

last: Pardo-Medina

first: Tim

middle: A

last: Dahlmann

first: Minou

last: Nowrousian

first: M

middle: C

last: Limón


title: 3D genome structures of single differentiating mouse ES cells reveal a unique reorganisation of chromatin in the formative state

city: Cambridge

first: David

last: Lando

address: Department Biochemistry , Sanger Bulding, Tennis Court Road

department: Biochemistry

country: United Kingdom

institute: University of Cambridge

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Pluripotency is the ability of an embryonic stem (ES) cell to differentiate into all the different cell lineages of a mature organism. Naïve and primed pluripotent ES cells in vitro correspond to the pre- and post-implantation populations in the embryo, and they are developmentally linked to each other via progression through a ‘formative’ transition state where naïve ES cells mature in response to inductive cues prior to becoming specified towards certain cell fates. Studying the formative state is thus crucial for understanding the mechanisms of multi-lineage decision-making. Here we show that in the formative state there is a profound reorganisation of 3D genome structure, where genes whose expression levels are changing become located in multi-way chromatin hubs and highly intermingled regions of the genome. Our results suggest that the reorganisation of 3D genome structure is driven by changes in DNA methylation and Polycomb-mediated heterochromatin formation, which is accompanied by a global increase in transcriptional bursting, leading to a reconfiguration of enhancer-promoter interactions – consistent with changes in the epigenome priming promoters for lineage specific gene expression.

overall_design: ChIP-seq, single-cell RNA-seq and single-cell and population Hi-C in haploid and diploid mouse embryonic stem cells

first: Ernest

middle: D

last: Laue


title: Single-cell and spatial transcriptomics characterisation of the immunological landscape in the healthy and PSC human liver

status: Public on Jan 18 2024

city: Toronto

first: Diana

last: Nakib

state: ON

address: 101 College Street

country: Canada

institute: University of Toronto

summary: Primary sclerosing cholangitis (PSC) is a rare liver disease characterized by chronic inflammation and irreparable damage to the bile ducts. Due to a limited understanding of the underlying pathogenesis of disease, there remains a paucity of treatment options. As such, we sequenced healthy and diseased livers to compare the activity, interactions, and localization of immune and non-immune cells. This revealed that outside PSC scar regions, hepatocytes are transitioning to bile duct cells, whereas within the scars, there is an accumulation of immune cells. Of these cells, macrophages that typically contribute to tissue repair were enriched in immunoregulatory genes and were less responsive to stimulation. These cells are likely involved in maintaining hepatic inflammation and could be targeted in novel therapeutic development.

overall_design: Spatial transcriptomics on OCT-embedded tissue

first: Tallulah

last: Andrews

first: Gary

last: Bader

first: Ian

last: McGilvray

first: Sonya

last: MacParland


title: Fetal growth delay caused by loss of non-canonical imprinting is resolved late in pregnancy and culminates in offspring overgrowth

status: Public on Jan 26 2024

city: Clayton

first: Patrick

middle: S

last: Western

state: VIC

address: 27-31 Wright Street

department: Centre for Reproductive Health

country: Australia

institute: Hudson Institute of Medical Research

summary: Germline epigenetic programming, including genomic imprinting, substantially influences offspring development. Polycomb Repressive Complex 2 (PRC2) plays an important role in Histone 3 Lysine 27 trimethylation (H3K27me3)-dependent imprinting, loss of which leads to growth and developmental changes in mouse offspring. In this study, we show that offspring from mouse oocytes lacking the PRC2 protein Embryonic Ectoderm Development (EED) were initially developmentally delayed, characterised by low blastocyst cell counts and substantial growth delay in mid-gestation embryos. This initial developmental delay was resolved as offspring underwent accelerated fetal development and growth in late gestation resulting in offspring that were similar stage and weight to controls at birth. The accelerated development and growth in offspring from Eed-null oocytes was associated with remodelling of the placenta, which involved an increase in fetal and maternal tissue size, conspicuous expansion of the glycogen enriched cell population and delayed parturition. Despite placental remodelling and accelerated offspring fetal growth and development, placental efficiency and fetal blood glucose levels were low, and the fetal blood metabolome was unchanged. Moreover, while expression of the H3K27me3-imprinted gene and amino acid transporter Slc38a4 was increased, fetal blood levels of individual amino acids were similar to controls, indicating that placental amino acid transport was not enhanced. Genome-wide analyses identified extensive transcriptional dysregulation and DNA methylation changes in affected placentas, including a range of imprinted and non-imprinted genes. Together, while deletion of Eed in growing oocytes resulted in fetal growth and developmental delay and placental hyperplasia, our data indicate a remarkable capacity for offspring fetal growth to be normalised despite inefficient placental function and the loss of H3K27me3-dependent genomic imprinting.

overall_design: Examination of the impact of EED deletion in oocytes on placental transcription and offspring development

first: Ruby

last: Oberin

first: Sigrid

last: Petautschnig

first: Ellen

middle: G

last: Jarred

first: Zhipeng

last: Qu

first: Tesha

last: Tsai

first: Neil

last: Youngson

first: Gabrielle

last: Pulsoni

first: Thi

middle: T

last: Truong

first: Dilini

last: Fernando

first: Heidi

last: Bildsoe

first: Rheannon

middle: O

last: Blucher

first: Marteen

last: van den Buuse

middle: K

last: Gardner

first: Natalie

last: Sims

middle: L

last: Adelson


title: Effect of interfering Itgb6 gene expression on renal proximal tubular epithelial cells during hypoxia/reoxygenation injury

city: Guangzhou

first: Ruilin

last: Zheng

address: Zhongshan Second Road

institute: The First Affiliated Hospital, Sun Yat-sen University

summary: Integrin αvβ6 holds promise as a therapeutic target for organ fibrosis, yet targeted therapies are hampered by inflammatory-related side effects. Here, we revealed that upregulated αvβ6 in proximal tubule cells (PTCs) is remarkably positively correlated with renal inflammation both in CKD patients and in murine model of ischemia-reperfusion injury (IRI)-induced renal fibrosis. Knockout of αvβ6 significantly reduced the inflammatory response in IRI kidneys, especially the infiltration of pro-inflammatory macrophages. To delve deeper into the mechanism by which αvβ6 modulates inflammatory response, an unbiased RNA-sequencing of si-NC and si-Itgb6-transfected hypoxic TKPTS cells was performed.

overall_design: TKPT cells were exposed to a hypoxic incubator (1% O2, 5% CO2, 96% N2, 37°C) for 24 h, and then the cells were placed back into a normal oxygen incubator (5% CO2, 37°C) for 12 h. When TKPT cells were cultured in 6-well culture plates to a density of 60%, RNA transfection reagent was used as a carrier to transfect TKPT cells with Itgb6 siRNA. An unbiased RNA-sequencing of si-NC and si-Itgb6-transfected hypoxic TKPTS cells was performed.

first: Changjian

last: Zhu

first: Zhou

last: Liang

first: Yi

last: Zhou


title: Onocgenic cell marking and single-cell transcriptomics reveal cell type and time-resolved actions of von Hippel Lindau inactivation in the kidney

status: Public on Jan 16 2024

city: Oxford

first: David

middle: Robert

last: Mole

address: Roosevelt Drive, Headington

department: Nuffield Department of Medicine

institute: University of Oxford

summary: Defining the first events in oncogenesis and the cellular responses they entrain, even in advance of morphological abnormality, is a fundamental challenge in understanding cancer initiation. As a paradigm to address this we studied the tumor suppressor von Hippel Lindau (VHL), whose loss ultimately drives clear cell renal cancer. Vhl inactivation was directly coupled to activation of a tdTomato reporter within a single allele, allowing accurate visualization of affected cells in their native context and retrieval from the kidney for single-cell RNA sequencing. This strategy uncovered cell-type specific responses to Vhl inactivation, defined a novel proximal tubular class with oncogenic potential, and revealed longer term adaptive changes in the renal epithelium and the interstitium. Oncogenic cell marking also revealed markedly heterogeneous cellular effects including time-limited proliferation and elimination of specific cell types. Striking heterogeneity in these responses suggests that very early events define the tissue specificity of VHL tumor suppressor action.

overall_design: Vhl(wt/flox) (Control) and Vhl(KO/flox) (KO) mice carrying the renal tubular epithelium specific Pax8-CreERT2 were dosed with 5x 2 mg tamoxifen and harvested either at an early (1-3 weeks) or late (4-12 months) timepoints after dosing. Cre-mediated recombination marked Vhl recombination with the expression of tdTomato. tdTomato-positve and tdTomato-negative cells were retrieved by FACS and subjected to scRNA-seq using the 10X Chromium NextGEM platform.

first: Samvid

last: Kurlekar

first: Ran

last: Li


title: The phosphate starvation response regulator PHR2 antagonizes arbuscule maintenance in Medicago

city: Changsha

first: Peng

last: Wang

address: Lushan Road

institute: Hunan Normal university

summary: • Phosphate starvation response (PHR) transcription factors play essential roles in regulating phosphate uptake in plants through binding to the P1BS cis-element in the promoter of phosphate starvation response genes. Recently, PHRs were also shown to positively regulate arbuscular mycorrhizal colonization in rice and lotus by controlling the expression of a large set of symbiotic genes. However, their role in arbuscule development has remained unclear. In Medicago we previously showed that arbuscule degradation is controlled by two SPX proteins that are highly expressed in arbuscule-containing cells. Since SPX proteins bind to PHRs and repress their activity in a phosphate-dependent manner, we investigated whether arbuscule maintenance is also regulated by PHR.  • Here, we show that MtPHR2 is a major regulator of the phosphate starvation response in Medicago. Knock-out of mtphr2 showed reduced phosphate starvation response, symbiotic gene expression and AM colonization levels. However, the arbuscules that formed showed less degradation, suggesting a negative role for PHR2 in arbuscule maintenance. This was supported by the observation that overexpression of MtPHR2 led to enhanced degradation of arbuscules. Although many arbuscule-induced genes contain P1BS elements in their promoters, we found that the P1BS cis-elements in the promoter of the symbiotic phosphate transporter MtPT4 are not required for arbuscule-containing cell specific expression. • These results indicate that MtSPX1 and MtSPX3 control arbuscule maintenance independent from PHR2. While MtPHR2 potentiates symbiotic gene expression and colonization, its activity in arbuscule-containing cells needs to be tightly controlled to maintain a successful symbiosis in Medicago.

overall_design: To investigate gene differential expression between wild type R108 and Mtphr2 mutant under mycorrizal condition.


title: Decoding the Significance of Amino Acid-dependent Signal Recognition Particle 9 Nuclear Translocation: Implications for Pancreatic Cancer Prognosis and Functionality [RIP-seq]

status: Public on Jan 12 2024

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

country: Japan

institute: NGS core facility, Research Institute for Microbial Diseases, Osaka University

summary: Signal recognition particles (SRPs) are important components in regulating intracellular protein transport and secretion, and tumors with high expression of Signal recognition particle 9 (SRP9) have tended to have poorer overall survival, although no reports have described the relationship between its localization and prognosis in pancreatic cancer. We conducted immunohistochemical staining for SRP9 for excised specimens from pancreatic cancer surgery cases without preoperative chemotherapy or radiotherapy performed. We found that in some cases SRP9 was preferentially expressed in the nucleus of the cancerous regions, which was hardly detected in other cases, suggesting SRP9 was transported to nucleus in the former cases. Therefore, the prognosis of patients with SRP9 nuclear translocation was compared by dividing them into two groups: those with a nuclear translocation rate of >50% and ≤50%. Patients with a nuclear translocation rate of >50% had significantly better recurrence-free survival compared with nuclear translocation rate of ≤50% group (p = 0.037). Subsequent in vitro experiments also showed that its nuclear translocation rate is reduced under amino acid deficient conditions. Further, to investigate the mechanism of SRP9 nuclear translocation, we studied in vitro by introducing the genes of SRP9 splicing variants (v1 and v2) and their deletion mutants lacking C-terminal regions into MiaPaCa pancreatic cancer cells. As a result, it was confirmed that both splicing variants showed nuclear translocation regardless of C-terminal deletions. Taking advantage of the fact that SRP9 is an RNA-binding protein, we next decided to sequence RNAs that bind to nuclear translocated v1 and v2 and evaluate their function in nuclear translocation. RNA Immunoprecipitation Sequencing of nuclear translocated v1 and v2 showed that pathways involved in cancer progression and protein translation were downregulated. The further study of nuclear translocation of SRP9 may open an avenue to optimization of precise evaluation and therapeutic control of pancreatic cancer.

overall_design: Taking advantage of the RNA-binding nature of SRP9, we transfected MiaPaCa cell lines with SRP9 v1 and v2 with nuclear translocation signals, immunoprecipitated them, and performed RNA immunoprecipitation (RIP) sequencing on them to investigate their functions in the nucleus.

first: Hiromichi

last: Sato

first: Sikun

last: Meng

first: Kazuki

last: Sasaki

first: Shogo

last: Kobayashi

first: Kansuke

last: Kido

first: Yoshiko

last: Tsuji

first: Yasuko

last: Arao

last: Saito

first: Yoshifumi

last: Iwagami

first: Daisaku

last: Yamada

first: Yoshito

last: Tomimaru

first: Takehiro

last: Noda

first: Hidenori

last: Takahashi

first: Shizuka

last: Uchida

first: Ken

last: Ofusa

first: Taroh

last: Satoh

first: Yuichiro

last: Doki

first: Hidetoshi

last: Eguchi

first: Tomoaki

last: Hara

first: Hideshi

last: Ishii


title: RNA sequencing of tumors from mice fed diets excluding methionine/tryptophan/niacin (Part 1)

first: Daisuke

last: Motooka

summary: In the past, animal experiments with rats, by depleting various amino acids in the diet, have shown that a deficiency of methionine inhibits tumor growth at the individual level and that methionine is important in the development and progression of cancer. This methionine is one of the essential amino acids and is the initiation codon that initiates protein translation from mRNA. Methionine is known to be converted to SAM, which is required for nucleic acid synthesis in cancer. Tumors are underdeveloped in an environment deficient in methionine, which is known as the "Hoffman effect". In fact, past reports have shown that in the absence of methionine, tumor growth is suppressed, and methionine has recently been shown to be essential in cancer stem cells or "tumor initiating cells". In addition, the focus on methionine in cancer is said to influence tumor-cell metabolism, histone patterns, and T cell immunity in the cancer microenvironment.  In cancer immunity, not only methionine but also its upstream pathways are said to be involved. Tryptophan/niacin, which is located upstream of the methionine cycle, is metabolized to form nicotinamide, and MNAM is formed from the SAM and nicotinamide via nicotinamide N-methyltransferase (NNMT). NNMT and MNAM have been reported to participate in the mechanism of inhibition of the apoptosis signal-regulated kinase 1-p38 MAPK pathway, resulting in increased colon cancer cell resistance to 5-FU. NNMTs promote nicotinamide adenine dinucleotide depletion and epigenetic reprogramming, which have been implicated in the development of metabolic plasticity, circumvention of the major tumor suppressive process of cellular senescence, acquisition of stem cell properties, and resistance to therapy and poor clinical outcomes. Also, MNAM synthesized in this methionine cycle is said to suppress T cells and promote cancer.  Hence, we implanted the colon cancer cell line HT-29 cultured in normal medium with all nutrients into NOD SCID mice and kept them for 2 weeks on a diet without the aforementioned methionine/tryptophan/niacin. Afterwards, tumors were removed and RNA-sequencing was performed, and a marked increase in the expression of RN7SL1, a non-coding RNA, was observed.

overall_design: All experiments in the present study were approved by the Osaka University Animal Experiments Committee, the formal ethics committee for institutional animal study at Osaka University, Japan, under the approval No. 30-011-053, and the experiments were conducted in accordance with the Osaka University Regulations on Animal Experiments. Five-week-old wild-type NOD SCID mice, derived from a mouse strain with a naturally occurring mutant, exhibiting an autosomal recessive inheritance pattern, and showing severe immunodeficiency due to the absence of T and B cells, which results in minimal rejection of xenogeneic cells and tissues, were procured from Japan SLC, Inc. All mice were female. Mice were randomly divided into 5 mice per cage. Given that not only methionine but also its upstream pathways are involved in cancer immunity. Tryptophan/niacin, which is located upstream of the methionine cycle, is metabolized to form nicotinamide, which is related to the methionine pathway, and 1-methyl-nicotinamide (MNAM) is formed from S-adenosylmethionine (SAM) synthesized from methionine, via nicotinamide N-methyltransferase, and thus MNAM synthesized in this methionine cycle is said to suppress T cells and promote cancer, we therefore subcutaneously implanted HT-29 cultured in regular medium containing all nutrients into the backs of 2 x 10⁵ NOD SCID mice, and the mice were then divided into four groups: those kept on normal food, those kept on food excluding methionine only, and those kept on food excluding niacin/tryptophan, and those kept on mice without methionine/tryptophan/niacin. And, they were reared for 2 weeks. Special diets for the mice were custom-ordered with as much appearance and taste as possible. Rearing conditions were such that the floor was changed daily to minimize the intake of excretory-derived amino acids. Room temperature was maintained around 23°C and lights were turned on in 12-hour cycles. Although our initial plan was to euthanize the mice if the tumor size exceeded 150 mm3 or if the mouse's body weight increased by more than 20% within a week, we in practice successfully completed the intended three-week experimental period without encountering any breaches of the restrictions pertaining to tumor size or body weight. At the conclusion of the animal experiment, we adhered to the following ethical guidelines: For mice weighing around 20g, the concentration of carbon dioxide used for scarification was 40%, with a flow rate of 0.5L/min for an inhalation time of 10 minutes; For smaller mice weighing 16g or less, the concentration of carbon dioxide was reduced within the range of 30% to 40%, with a lower flow rate between 0.2L/min to 0.5L/min for a shorter inhalation time between 5 to 10 minutes, to minimize the stress and pain to the mice during the procedure. Tumors were then removed and evaluated for RNA expression and type by RNA sequence. After confirming the cessation of mouse respiratory movements, the tumor was promptly excised within 30 minutes in a well-maintained environment with laboratory air conditioning and a clean experimental table in the laboratory. Subsequently, the tumor was immersed in RNA LATER reagent (Sigma-Aldrich), and RNA extraction was performed using RNAeasy kit (Qiagen), followed by RNA sequencing analysis.

first: Hiromichi

last: Sato

first: Sikun

last: Meng

first: Kazuki

last: Sasaki

last: Kobayashi


title: Rescue of Impaired Blood-Brain Barrier in a Tuberous Sclerosis Complex Patient Derived Neurovascular Unit

city: Nashville

first: Matthew

last: Cottam

state: TN

address: 2213 Garland Avenue South 9465

institute: Vanderbilt University

summary: Tuberous sclerosis complex (TSC) is a multi-system genetic disease that causes benign tumors in the brain and other vital organs. The most debilitating symptoms result from involvement of the central nervous system and lead to a multitude of severe symptoms including seizures, intellectual disability, autism, and behavioral problems. TSC is caused by heterozygous mutations of either theTSC1orTSC2gene. Dysregulation of mTOR kinase with its multifaceted downstream signaling alterations is central to disease pathogenesis. Although the neurological sequelae of the disease are well established, little is known about how these mutations might affect cellular components and the function of the blood-brain barrier (BBB). We generated disease-specific cell models of the BBB by leveraging human induced pluripotent stem cell and microfluidic cell culture technologies. Using these microphysiological systems, we demonstrate that the BBB generated fromTSC2heterozygous mutant cells shows increased permeability which can be rescued with treatment with rapamycin, an mTOR kinase inhibitor, but also by wild type astrocytes.

overall_design:
To investigate how TSC mutations affect cellular components and functions of the BBB, pluripotent stem cells from healthy human tissue and from patients with heterozygous TSC mutations were grown using microfluidic cell culture with continual perfusion.
Gene expression profiling via bulk RNA-seq was performed on control and TSC-mutant cells.

first: Jacquelyn

last: Brown

first: Shannon

last: Faley

first: Monika

last: Judge

first: Patricia

last: Ward

first: Rebecca

last: Ihrie

first: Robert

last: Carson

first: Laura

last: Armstrong

first: Mustafa

last: Sahin

first: John

middle: P

last: Wikswo

first: Kevin

last: Ess

first: Diana

last: Neely


title: RNA sequencing of tumors from mice fed diets excluding methionine/tryptophan/niacin (Part 2)

last: Motooka

overall_design: All experiments in the present study were approved by the Osaka University Animal Experiments Committee, the formal ethics committee for institutional animal study at Osaka University, Japan, under the approval No. 30-011-053, and the experiments were conducted in accordance with the Osaka University Regulations on Animal Experiments. Five-week-old wild-type NOD SCID mice, derived from a mouse strain with a naturally occurring mutant, exhibiting an autosomal recessive inheritance pattern, and showing severe immunodeficiency due to the absence of T and B cells, which results in minimal rejection of xenogeneic cells and tissues, were procured from Japan SLC, Inc. All mice were female. Mice were randomly divided into 5 mice per cage. Given that not only methionine but also its upstream pathways are involved in cancer immunity. Tryptophan/niacin, which is located upstream of the methionine cycle, is metabolized to form nicotinamide, which is related to the methionine pathway, and 1-methyl-nicotinamide (MNAM) is formed from S-adenosylmethionine (SAM) synthesized from methionine, via nicotinamide N-methyltransferase, and thus MNAM synthesized in this methionine cycle is said to suppress T cells and promote cancer, we therefore subcutaneously implanted HT-29 cultured in regular medium containing all nutrients into the backs of 2 x 10⁵ NOD SCID mice, and the mice were then divided into four groups: those kept on normal food, those kept on food excluding methionine only, and those kept on food excluding niacin/tryptophan, and those kept on mice without methionine/tryptophan/niacin. And, they were reared for 2 weeks (except for 08_T_zen (GSM7873764) and 09_T_3ketsu (GSM7873765), described in each Sample section). Special diets for the mice were custom-ordered with as much appearance and taste as possible. Rearing conditions were such that the floor was changed daily to minimize the intake of excretory-derived amino acids. Room temperature was maintained around 23°C and lights were turned on in 12-hour cycles. Although our initial plan was to euthanize the mice if the tumor size exceeded 150 mm3 or if the mouse's body weight increased by more than 20% within a week, we in practice successfully completed the intended three-week experimental period without encountering any breaches of the restrictions pertaining to tumor size or body weight. At the conclusion of the animal experiment, we adhered to the following ethical guidelines: For mice weighing around 20g, the concentration of carbon dioxide used for scarification was 40%, with a flow rate of 0.5L/min for an inhalation time of 10 minutes; For smaller mice weighing 16g or less, the concentration of carbon dioxide was reduced within the range of 30% to 40%, with a lower flow rate between 0.2L/min to 0.5L/min for a shorter inhalation time between 5 to 10 minutes, to minimize the stress and pain to the mice during the procedure. Tumors were then removed and evaluated for RNA expression and type by RNA sequence. After confirming the cessation of mouse respiratory movements, the tumor was promptly excised within 30 minutes in a well-maintained environment with laboratory air conditioning and a clean experimental table in the laboratory. Subsequently, the tumor was immersed in RNA LATER reagent (Sigma-Aldrich), and RNA extraction was performed using RNAeasy kit (Qiagen), followed by RNA sequencing analysis.

first: Shogo

first: Kansuke

last: Kido

first: Yoshiko

last: Tsuji

first: Yasuko

last: Arao

last: Saito

first: Yoshifumi

last: Iwagami

first: Daisaku

last: Yamada

first: Yoshito

last: Tomimaru

first: Takehiro

last: Noda

first: Hidenori

last: Takahashi

first: Shizuka

last: Uchida

first: Ken

last: Ofusa

first: Taroh

last: Satoh

first: Yuichiro

last: Doki

first: Hidetoshi

last: Eguchi

first: Tomoaki

last: Hara

first: Hideshi

last: Ishii


title: Single-nucleus RNA-seq of aortas from Ldlr-/- and Ldlr-/-Trem2-/- atherosclerotic mice

city: Würzburg

first: Alexander

middle: M.

last: Leipold

address: Josef-Schneider-Str. 2

country: Germany

institute: Helmholtz Institute for RNA-based Infection Research

summary: Single-nucleus RNA-seq of aortas was performed in Ldlr-/- and Ldlr-/-Trem2-/- mice fed a high fat diet for 10 weeks

overall_design: Ldlr-/- (n=3) and Ldlr-/-Trem2-/- (n=3) female mice were fed a high fat diet for 10 weeks to induce the formation of atherosclerotic plaques. Aortas were excised, cryoconserved and nuclei extracted for single-nucleus RNA-sequencing

first: Clement

last: Cochain

first: Tobias

last: Krammer

first: Antoine-Emmanuel

last: Saliba


title: Mapping BRD1 and HBO1 genomic binding in BRCA1 mutant cells

status: Public on Jan 11 2024

city: Boston

first: Sarah

middle: J.

last: Hill

state: MA

address: 450 Brookline Ave., Dana Building, Room 1410C

department: Medical Oncology

institute: Dana-Farber Cancer Institute

summary: The BRCA1 tumor suppressor has many functions in DNA damage repair, DNA replication, and beyond. Loss of any of these functions can lead to breast and ovarian carcinogenesis and also therapeutic sensitivity. In this study, we have discovered a novel role for BRCA1 in controlling replication initiation by regulating the bromodomain-protein BRD1 and the histone acetyltransferase HBO1. Specifically, we show that BRCA1 regulates localization of the BRD1-HBO1 complex to replication origins where the complex normally acetylates histone H3 on lysine 14. We performed chromatin-immunoprecipitation seqeuncing (ChIP-seq) for BRD1 and HBO1 in a BRCA1 mutant line (UWB1.289) and demonstrate that BRD1 and HBO1 co-occupy ORC2 binding sites and replication origins.

overall_design: UWB1.289 cells were harvested, chromatin prepared, and immunoprecipitation carried out using the Cell Signaling SimpleChIP Enzymatic Chromatin IP Kit following the manufacturer's protocol (Catalog #9003). 5ug chromatin were used per IP. Antibodies for ChIP included HBO1 (Cell Signaling Cat. #58418) and BRD1 (Abcm Cat. #ab181060). For the HBO1 and BRD1 ChIP, Active Motif's spike in controls were used according to the manufacturer's protocol (Cat. #61686 and 53083).


title: Targeting low levels of MIF expression as a potential therapeutic strategy for ALS

city: Beer Sheva

first: Adrian

last: Israelson

address: Ben Gurion Blv. 1

country: Israel

institute: Ben Gurion University of the Negev

summary: Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by motor neurons (MNs) loss. We previously discovered that macrophage migration inhibitory factor (MIF), whose levels are extremely low in spinal MNs, inhibits mutant SOD1 misfolding and toxicity. In this study, we show that a single peripheral injection of adeno-associated virus (AAV) delivering MIF into adult SOD1G37R mice, significantly improved their motor function, delayed disease progression and extended survival. Moreover, MIF treatment reduced neuroinflammation and misfolded SOD1 accumulation, rescued MNs and corrected dysregulated pathways as observed by proteomics and transcriptomics. Furthermore, we revealed low MIF levels in human induced pluripotent stem cell derived MNs from familial ALS patients with different genetic mutations, as well as in post-mortem tissues of sporadic ALS patients. Our findings indicate that peripheral MIF administration may provide a potential therapeutic mechanism for modulating misfolded SOD1 in vivo and disease outcome in ALS patients.

overall_design: RNA from the lumbar spinal cord of non-transgenic and SOD1-G37R transgenic mice not treated or treated with AAV-PHP.eB-MIF using single tail-vein injection after the disease onset.

first: Leenor

last: Alfahel

first: Shir

last: Zaccai


title: Assessing chromatin accessibility in maize using ATAC-seq

status: Public on Jan 10 2024

city: Taipei

first: Pao-Yang

last: Chen

address: 128 Sec. 2, Academia Rd, Nankang

department: Institute of Plant and Microbial Biology

country: Taiwan

institute: Academia Sinica

summary: The depiction of maize chromatin architecture using Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) provides great opportunities to investigate cis-regulatory elements, which is crucial for crop improvement. We demonstrate a streamlined ATAC-seq protocol for maize in which nuclei purification can be achieved without cell sorting, and using only a standard bench-top centrifuge. Our protocol, coupled with the bioinformatic analysis, provides a precise and efficient assessment of the maize chromatin landscape.

overall_design: We perform the Maize seedling ATAC-Seq using only 2500 and 5000 nuclei, as well as the naked gDNA as control, in a total of three samples with two biological replicates, providing a new way to study the role of chromatin accessibility.

first: Jo-Wei

last: Hsieh

first: Pei-Yu

last: Lin

first: Chi-Ting

last: Wang

first: Yi-Jing

last: Lee

first: Pearl

last: Chang

first: Rita

last: Lu

first: Chung-Ju

middle: r


title: RNA seq for Rat's heart infarction

city: Chongqing

first: Xue

last: Fangchao

address: XINQIAO

institute: AMU

summary: This project performed BulkRNA sequencing on the myocardial tissue of rats one month after myocardial infarction and attempted to reveal the transcriptome differences in the myocardial infarction area of rats among different groups.

overall_design: The experimental animals are SD rats, and the experimental groups include Normal, MI, SNAP, DTMS, and S-DTMS.

first: Shanlan

last: Zhao

first: Fangchao

last: Xue


title: Protein translation rate determines neocortical neuron fate

city: Berlin

first: Andrew

last: Newman

address: Charitéplatz 1

department: Institute for Cell and Neurobiology

institute: Charité Universitätsmedizin Berlin

summary: The mammalian neocortex comprises an enormous diversity regarding cell types, morphology, and connectivity. In this work, we discover a post-transcriptional mechanism of gene expression regulation, protein translation, as a determinant of cortical neuron identity. We find specific upregulation of protein synthesis in the progenitors of later-born neurons and show that translation rates and concomitantly protein half-lives are inherent features of cortical neuron subtypes. In a small molecule screening, we identify Ire1a as a regulator of Satb2 expression and neuronal polarity. In the developing brain, Ire1a regulates global translation rates, coordinates ribosome traffic, and the expression of eIF4A1. Furthermore, we demonstrate that the Satb2 mRNA translation requires eIF4A1 helicase activity towards its 5’-untranslated region. Altogether, we show that cortical neuron diversity is generated by mechanisms operating beyond gene transcription, with Ire1a-safeguarded proteostasis serving as an essential regulator of brain development.

overall_design:
Total RNAseq from P0 Cortices from wild-type and IRE1aEmx1Cre mutant mice as well as RNAseq from purified Polysomes from wild type and IRE1aEmx1Cre mice.
3 batches:
Batch 1: Total RNAseq from cortex
Batch 2: Total RNAseq from Purified Polysomes
Batch 3: Total RNAseq from Purified Light and Heavy Polysomes

Analysis scripts can be seen at:
https://github.com/qoldt/IRE1aKO_Polysome_RNAseq

first: Ekaterina

last: Borisova

first: Marta

last: Couce Iglesias

first: Rike

last: Dannenberg

first: Theres

last: Schaub

first: Bo

last: Qin

first: Alexandra

last: Rusanova

first: Marisa

last: Brockmann

first: Janina

last: Koch

first: Marieatou

last: Daniels

first: Paul

last: Turko

first: Olaf

last: Jahn

first: David

middle: R

last: Kaplan

last: Rosário

first: Takao

last: Iwawaki

first: Christian

last: Spahn

last: Rosenmund

last: Meierhofer

first: Matthew

last: Kraushar

first: Victor

last: Tarabykin

first: Mateusz

last: Ambrozkiewicz


title: Taurine‑mediated gene transcription and cell membrane permeability reinforced co‑production of bioethanol and Monascus azaphilone pigments for a newly isolated Monascus purpureus

status: Public on Jan 09 2024

city: Changzhou

first: Xia

last: Yi

address: Gehu Middle Road #21

institute: Changzhou University

summary:
To uncover the molecular mechanism of the enhanced ethanol and pigments fermentability after adding taurine, we carried out transcriptome assays for the isolated fungus
We then performed gene expression profiling analysis using data obtained from RNA-seq of 2 different cells at one time points.

overall_design: Comparative gene expression profiling analysis of Denovo RNA-seq data between the control groups and the taurine-treated groups for the wild M. purpureus cells.


title: Optimizing Cell Therapy by Sorting Cells with High Extracellular Vesicle Secretion

status: Public on Jan 07 2024

city: Los Angeles

first: Dino

last: Di Carlo

address: 420 Westwood plaza

institute: University of California, Los Angeles

summary: Selection and delivery of sub-populations of cells that secrete more extracellular vesicles leads to improved therapeutic efficacy.

overall_design: We sorted single cells based on their extracellular secretion phenotype and sequenced to generate unique genetic profiles of high extracellular vesicle secretors.

first: Doyeon

last: Koo

first: Xiao

last: Cheng

first: Shreya

last: Udani

first: Dashuai

last: Zhu

first: Junlang

last: Li

first: Brian

first: Natalie

last: Tsubamoto

first: Shiqi

last: Hu

first: Jina

last: Ko

first: Ke


title: Synergistic immunochemotherapy exerts efficiently anti-hepatocellular carcinoma effect to induce SAMD4B-APOA2 axis for inhibition of tumor immune evasion

status: Public on Jan 08 2024

city: Urumqi

first: Zhang

middle: Qing

last: hua

state: Xinjiang

address: Liudao Bay Street

institute: The Second Affiliated Hospital of Xinjiang Medical University

summary: The purpose of this study is to speculate TCC (Thalidomide+carmofur+ Cantharidin), may be a promising treatment strategy for patients with advanced HCC.However, there is few research on this novel clinical combination therapy, especially the mechanism of its anti-hepatocellular effect. Therefore, it is necessary to further explore the specific mechanism of triple drug combination in HCC treatment.Here, we prospectively collected the clinical data of 92 patients with unresectable HCC, observed the effective efficacy of TCC in clinical practice, and confirmed in 6 PDX models in mice.Later on, we collected the resected tumors conducted single cell sequencing (scRNA-seq).  We found that SAMD4B, especially its expression level, could affect the immune microenvironment. In order to understand the details of its immune regulation mechanism, such as which immune cells PD1 (i.e. PD-L1 receptor regulated by SAMD4B) comes from, we selected 4 patients from low- and high-SAMD4B groups for scRNA-seq.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: 10x Single-cell Transcriptome sequencing were used to obtain the characteristics of  6 PDX samples and 4 HCC samples.

first: Feng

last: Qi

first: Jian

last: Zhang

first: Jia

first: Donghe

last: Gao

first: Zhuoran

first: Xiuyan

last: Kong

first: Zhijie

last: Yu

first: Biwei

last: Yang

first: Jinglin

last: Xia


title: CBX4 in Lung Adenocarcinoma: A Dual-Edged Sword in Transcriptional Regulation and Tumor Progression [RNA-seq]

status: Public on Jan 05 2024

city: Tianjin

first: Chenghao

last: Xuan

address: 22 Qixiangtai Road

institute: Tianjin Medical University

summary: Lung adenocarcinoma (LUAD) remains a leading cause of cancer-related mortality worldwide. Understanding the dysregulated epigenetics governing LUAD progression is pivotal for identifying therapeutic targets. CBX4, a chromobox protein, is reported to be upregulated in LUAD. However, its regulatory role in LUAD progression warrants in-depth investigation. This study highlights the dual impact of CBX4 on LUAD proliferation and metastasis. Through a series of rigorous in vitro and in vivo experiments, we elucidate that CBX4 functions in promoting LUAD proliferation via upregulating PHGDH expression while concurrently suppressing LUAD metastasis by inhibiting ZEB2 transcription. Further investigation into the underlying mechanism reveals CBX4’s intricate interactions with distinct epigenetic factors. The inhibition of ZEB2 transcription involves CBX4-mediated recruitment of canonical PRC1 (cPRC1), establishing H2K119ub on the ZEB2 promoter. Simultaneously, CBX4 facilitates PHGDH transcription through interaction with GCN5, inducing heightened histone acetylation on the PHGDH promoter. These findings underscore CBX4's pivotal role as a regulator of LUAD progression, emphasizing its diverse transcriptional regulatory functions contingent upon interactions with specific epigenetic partners. Understanding the nuanced interplay between CBX4 and epigenetic factors sheds light on potential therapeutic avenues in LUAD.

overall_design: Examination of gene expression changes in CBX4 knockdown and control A549 cells.

first: Ran

last: Zhao


title: CBX4 in Lung Adenocarcinoma: A Dual-Edged Sword in Transcriptional Regulation and Tumor Progression

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

overall_design: Refer to individual Series


title: CBX4 in Lung Adenocarcinoma: A Dual-Edged Sword in Transcriptional Regulation and Tumor Progression [ChIP-Seq]

overall_design:
Chromatin immunoprecipitation sequencing (ChIP-seq) was performed to investigate the distribution of H2AK119ub in A549 cells.

***************************************************************
Raw sequence data access for Input provided at: China Genomic Sequence Archive (GSA) at https://ngdc.cncb.ac.cn/gsa/browse/CRA001102/CRR033172
***************************************************************

first: Xuanyuan


title: Large-scale, genome-wide iPSC chromatin co-accessibility analysis

status: Public on Jan 04 2024

city: La Jolla

first: Kelly

middle: Ann

last: Frazer

state: CA

address: 9500 Gilman Drive

department: Pediatrics

institute: UC San Diego

summary: The goal of this study is to characterize interactions between accessible chromatin sites within a diverse collection of human iPSCs. Here, we generated 150 bulk ATAC-seq libraries for iPSCs from the iPSCORE collection. We identified sites of open chromatin and calculated the pairwise correlation between sites to characterize functional coordination of epigenetic modifications.

overall_design:
Chromatin accessibility maps of iPSCs.

**Submitter states that the raw data will be available in dbGaP (phs000924)**

first: Frazer

last: Kelly A

first: Arthur

last: Timothy D

first: D'Antonio

last: Matteo

first: Nguyen

last: Jennifer


title: Transcriptome analysis reveals the key role of overdominant expression of photosynthetic and respiration-related genes in the formation of tobacco(Nicotiana tabacum L.) biomass heterosis

status: Public on Jan 01 2024

city: Guiyang

first: Renxiang

last: Liu

state: Guizhou

address: Huaxi

institute: Guizhou University

summary: Heterosis, a phenomenon where hybrids exhibit superior performance in various traits such as yield, quality, and resistance compared to their parents, has long been a focus of agricultural research. Although tobacco biomass significantly affects tobacco yield and economic profitability, the underlying reasons for the dominance of tobacco biomass hybrids and their formation remain unclear. To address this, we selected 5 tobacco varieties (lines) with large differences in tobacco biomass as the parents and formulated F1 hybrids to analyze the expression of tobacco biomass heterosis. Our findings demonstrate that tobacco leaf biomass exhibits a clear heterosis advantage. In particular, the hybrid Va116×GDH94 showed strong heterosis, with 76.69% of differentially expressed genes (DEGs) displaying Overdominant expression pattern. Notably, these Overdominant DEGs were significantly enriched in biological processes such as photosynthesis and respiration. During photosynthesis, transgressive up-regulation of Lhc improved light-harvesting efficiency, while the up-regulation of Psa and Psb in the photosystem enhanced the defense of chloroplasts against strong light. Additionally, the up-regulation of rbcl promoted the increase of photosynthetic products in the hybrids. During respiration, the down-regulation of MDH, ACO, and OGDH suppressed the tricarboxylic acid cycle (TCA cycle) and weakened respiratory consumption in the hybrids. Furthermore, we observed that the net photosynthetic rate and intercellular CO2 concentration of the hybrids were significantly higher than those of the parents. Taken together, our results suggest that the overdominant expression effect of DEGs plays a crucial role in the formation of tobacco biomass hybrid advantage. The overdominant expression effect of photosynthesis and respiration-related genes enhanced the photosynthetic capacity of the hybrids, reduced respiration consumption, and promoted the increase of tobacco biomass, thus demonstrating the obvious hybrid advantage.

overall_design: We subsequently selected the hybrid combination Va116 × GDH94, with the highest heterosis value 45 days after transplantation, for transcriptome sequencing analysis.

first: Anbin

last: Lu

first: Shuaibo

last: Zeng

first: Kai

last: Pi


title: Programmable RNA N6-methyladenosine editing with CRISPR/dCas13a in plants

city: Shenzhen

first: Chuanlin

last: Shi

address: Dapeng

institute: Chinese Academy of Agricultural Sciences

summary: N6-methyladenonsine (m6A) is the most prevalent modification on mRNA and plays critical roles in mRNA processing and metabolism. However, how the m6A modification on individual gene of interest regulates gene function and the links to phenotypic outcome in plants are mostly unknown. Here, we described the construction and characterization of programmable m6A editing tools by fusing the m6A writer, core catalytical domain of MTA and MTB complex, and eraser, ALKBH5, to catalytically dead Cas13a (dCas13a), respectively, for targeting methylation and demethylation of specific mRNA. We demonstrated that our m6A editors could efficiently and specifically add and remove the m6A modification on specific RNA transcript in both Nicotiana benthamiana and Arabidopsis. Moreover, targeting SHORT-ROOT (SHR) transcript with methylation editor could significantly increase its m6A levels with limited off-target effects and enhance its expression, giving rise to induced plant growth with enlarged leaf size and root, increased plant height, plant biomass and total grain weight in Arabidopsis. Collectively, these findings suggest that our programmable m6A editing tools can be applied to study m6A modification of specific genes in plants, and might also have great potential applications for crop improvement in future

overall_design: The MeRIP-seq is based on the protocols previously described. Total RNA from Arabidopsis inflorescences was isolated by TRIzol reagent, and 100 μg total RNA was polyA-selected to get ~2 μg intact mRNA using Dynabeads mRNA Purification Kit (Invitrogen). All the isolated mRNA was chemically fragmented using NEBNext Magnesium RNA Fragmentation Module (New England Biolabs). We aliquoted ~300 ng fragmented mRNA used as input for MeRIP-seq. The remaining fragmented mRNA was added into 500 μl IP buffer, incubated with 5 μg anti-m6A antibody (Synaptic Systems) and 5 μl of RNasin Plus RNase Inhibitor (Promega) and tumbled at 4 °C for 2 h. Then, the reaction mixture was incubated with protein A/G magnetic beads (MedChemExpress) and tumbled at 4°C for 1 h. Then, the reaction mixture was washed twice with IP buffer, twice with low-salt IP buffer, and twice with high-salt IP buffer at 4 °C for 10 min for each. After extensive washing, the m6A-enriched RNA was purified by TRIzol reagent with glycol blue coprecipitant (Invitrogen). All the input and IP RNA were used for RNA library construction using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer’s instructions. All the RNA libraries were sequenced (Paired-end 150nt) on an Illumina NovaSeq 6000 platform at Novogene, China.

last: shi

first: Qili

last: Fei

first: Lianguang

last: Shang


title: CCl4 treatment led to differential lncRNA expression in murine hepatocytes

city: Nanjing

first: Yuhui

last: Zang

address: 163 Xianlin Road

institute: Nanjing University

summary: Five-week old male C57BL/6J mice (20 ± 2 g) were intraperitoneally injected with of 20% CCl4 solution in sterile mineral oil at a dose of 2.5 ml CCl4 per kilogram body weight twice per week for eight weeks. Afterwards, the primary hepatocytes were isolated by pronase/collagenase perfusion digestion followed by subsequent density gradient centrifugation. The isolated primary hepatocytes were counted with a hemocytometer to determine the number and percentage of viable cells using the trypan blue method. The isolated cells with the viability above 90% were used for the experiments.

overall_design: Total RNA was extracted from the isolated hepatocytes with TRIzol reagent.

first: Zhiqian

last: Kang

first: Junfeng

last: Zhang


title: Jun is a key Regulator of Prostate Cancer Progression and Immune Microenvironment Dynamics

city: Vienna

first: Torben

last: Redmer

address: Veterinärplatz 1

country: Austria

institute: University of Veterinary Medicine Vienna

summary: Background: Prostate cancer remains one of the most diagnosed malignancies among men worldwide and is responsible for significant morbidity and mortality. Despite advances in diagnostics and therapeutics, effective management and understanding of its molecular biology continue to pose challenges. The disease develops through malignant transformation of the prostate epithelium in a stepwise, mutation-driven process. Although AP1 transcription factors have been implicated as presumed oncogenic drivers, the molecular programs contributing to prostate cancer progression are still not fully understood. Methods: To investigate the role of the AP1 transcription factor Jun in prostate cancer, we deleted Jun in a Pten deficient PCa mouse model.  We performed RNAseq analysis of sorted epithelial cells and immune-phenotyped the tumors of different genotypes by immunohistochemistry methods. We compared the results from the in vivo model to publicly available human expression datasets and prostate cancer tissue microarrays.  Results Decreased JUN expression levels corresponded with advanced stages of prostate cancer, effectively distinguishing high and low-risk human prostate tumors. Surprisingly, Jun expression was significantly increased in a Pten-deficient prostate cancer mouse model, recapitulating early stages of PCa development. Immune phenotyping of Pten-deficient prostates revealed high accumulation of tumor-infiltrating leukocytes, particularly innate immune cells, neutrophils and macrophages as well as high levels of Stat3 activation (pStat3Tyr705) and Il1-β production. Remarkably, Jun depletion prevented immune cell attraction accompanied by significantly reduced levels of pStat3Tyr705 and Il1-β production and accelerated growth of Jun/Pten-deficient (JunPEΔ/Δ;PtenPEΔ/Δ) tumors. Comparative whole transcriptome profiling of prostate epithelial cells revealed a SenMayo senescence associated gene signature, upregulation of pro-inflammatory processes and cytokines such as Il1b, Ccl3 and Ccl8 that are pivotal for immune cell attraction in Pten-deficient prostates. Strikingly, depletion of Jun reversed both senescence and senescence-associated immune infiltration and, as a consequence, further accelerated tumor expansion.

overall_design: EPCAM expressing cells of murine prostates of wildtype (wt) mice and mice with probasin (Pb) promoter-controlled Cre-mediated homozygous deletion of Jun (exon 1) and Pten (exons 4-5) and combined deletion of Jun and Pten were enriched by MACS sorting, collected and subjected to RNA extraction. Libraries yield from 200 ng of high-quality RNA were subjected to 75bp single-end sequencing.

first: Martin

last: Raigel

first: Sabine

last: Lagger

first: Lukas

last: Kenner


title: Decreased sperm production in aged testes and functional sperm defects in aged epididymides lead to decreased fertility in male mice.

city: Suita

first: Daisuke

last: Motooka

address: 3-1, Yamadaoka

institute: NGS core facility, Research Institute for Microbial Diseases, Osaka University

summary: To analyze mRNA expressions in aged mouse testes and cauda epididymides at genome wide level, we performed mRNA-Seq on these bulk samples at 2, 18, and 24 months.

overall_design: Bulk testes and cauda epididymides in wild-type C57BL/6J mice at 2, 18, and 24 months (five biological replicates for each organ) were placed in TRIzol, homogenized, and obtained the total RNAs.

first: Tsutomu

last: Endo

last: Okuzaki

first: Masahito

last: Ikawa


title: Transcriptomic analysis of mammary tumor biospy from MMTV-ErbB2 transgenic mice [Docetaxlel]

status: Public on Jan 02 2024

city: Berkeley

first: Jian-Hua

last: Mao

address: One Cyclotron Road

institute: Lawrence Berkeley National Laboratory

summary: The tyrosine kinase ErbB2 positive breast tumors have more aggressive tumor growth, poorer clinical outcome, and more resistance to radiotherapy, chemotherapy and hormone therapy. To identify the biomarkers for predicting the responses to chemotherapy, we treated genetically diverse F1 backcross (F1Bx) mice between C57BL/6J and FVB/N MMTV-Erbb2 transgenic mice with mammary tumor by docetaxel. Biopsies were taken in sterile conditions and processed for RNA and histological analysis before treatment.  This will help us to have a better understanding of the heterogeneous response to chemotherapy and permit us to design better and more individualized (personalized) treatment strategies for human ErbB2 positive breast cancer.

overall_design: 50 MMTV-ErbB2 mammary tumor biopsy before treatment were analyzed by Affymetrix microarrays.

first: Jesus

last: Perez-Losada


title: Expression of transcriptional factors in age-associated B cells

status: Public on Dec 31 2023

city: Shanghai

first: dai

last: dai

state: Shanghai

address: 上海市黄浦区山东中路仁济医院西院区

institute: Shanghai Jiao Tong University School of Medicine

summary: To gain insights into the nature of human age-associated B cells (ABCs), we sorted peripheral B cells from a patient with new-onset SLE and performed droplet-based scRNA-seq. Seven distinct clusters were revealed by unsupervised clustering with a two-dimensional uniform manifold approximation and projection (UMAP). These clusters were assigned to known peripheral B cell subsets including transitional B cells, naïve B cells, activated naïve B cells, ABCs, memory B cells, plasmablasts, and plasma cells by comparing differentially expressed genes with established landmark genes. ABCs preferentially expressed genes encoding key surface markers CD19, CD86, FCRLA, FCRL3/5, FCGR2B, MS4A1 and ITGAX. We found 43 differentially expressed TFs: 27 upregulated and 16 downregulated.

overall_design: The peripheral B cells from a patient with newly-onset SLE were sorted and performed droplet-based scRNA-seq. Then unsupervised clustering with a two-dimensional uniform manifold approximation and projection (UMAP) was used to identify distinct clusters of peripheral B cells.

first: Dai

last: Dai


title: Exonic knock-out/in gene editing in hematopoietic stem and progenitor cells fully rescues RAG1 immunodeficiency [OFFTARGET]

city: Segrate

first: Ivan

last: Merelli

state: Milano

address: via F.lli Cervi 93

department: Istituto di Tecnologie Biomediche

country: Italy

institute: Consiglio Nazionale delle Ricerche

summary: Recombination Activating Genes (RAG) are tightly regulated during lymphoid differentiation and their mutations cause a spectrum of severe immunological disorders. Haematopoietic stem/progenitor cell (HSPC) transplantation is the treatment of choice but limited by donor availability and toxicity. To overcome these issues, we developed and validated gene editing (GE) strategies targeting a corrective sequence into the human RAG1 gene by homology-directed repair (HDR). Off-target and RNA-Seq analyses were performed on edited human and patient-derived HSPCs to assesse the genome integrity and the trascriptomic profile. Whereas integration into intron 1 achieved suboptimal levels of correction, in-frame insertion into exon 2 drove faithful recapitulation of physiologic hRAG1 expression and activity.

overall_design: Off-target validation was performed by deep sequencing analysis on three different mPB HD-derived HSPCs edited with gRNA#13 RNP  and untreated cells were used as negative controls

first: Maria

last: Castiello

first: Chiara

last: Brandas

first: Simona

last: Porcellini

first: Gabriele

last: Pelosi

first: Anna

last: Villa


title: The transcription factor ZEB2 drives formation of age-associated B cells

address: 上海市黄浦区山东中路仁济医院西院区

summary: Age-associated B cells (ABCs) accumulate during infection, aging and autoimmunity, contributing to lupus pathogenesis. Here, we screened for transcription factors driving ABC formation and found Zeb2 was required for human and mouse ABC differentiation in-vitro. ABCs were reduced in ZEB2 haploinsufficient individuals and in mice lacking Zeb2 in B cells. In mice with TLR7-driven lupus, Zeb2 was essential for ABC formation and autoimmune pathology. Zeb2 binds to the +20kb intronic enhancer of Mef2b, repressing Mef2b-mediated germinal center B cell differentiation and promoting ABC formation. Zeb2 also targets genes important for ABC specification and function including Itgax. Zeb2-driven ABC differentiation required Jak-Stat signaling, and treatment with the Jak1/3 inhibitor tofacitinib reduced ABC accumulation in mice and autoimmune patients. Zeb2 thus emerges as a driver of B-cell autoimmunity.

overall_design: 1. Examination of in vitro derived B cells in the presence of R848, CD40, anti-IgM or for R848, CD40, anti-IgM, IFNg, IL-21 3 days. 2. Examination of in vitro derived ABCs from Zeb2 deficient mice. 3. Examination of in vitro derived ABCs with Cas9-mediated ZEB2 or T-bet ablation. 4. Examination of in vitro derived ABCs with ZEB2 overexpression. 5. Examination of in vitro derived ABCs treated with tofacitinib.


title: Exonic knock-out/in gene editing in hematopoietic stem and progenitor cells fully rescues RAG1 immunodeficiency [GUIDE2]

overall_design: Guide-Seq assay was performed on K562 cells edited by sgRNAs targeting the RAG1 exon  (gRNA#11 and #13), cells electroporated in absence of gRNA were used as negative control


title: Exonic knock-out/in gene editing in hematopoietic stem and progenitor cells fully rescues RAG1 immunodeficiency


title: Exonic knock-out/in gene editing in hematopoietic stem and progenitor cells fully rescues RAG1 immunodeficiency [RNA-Seq]

overall_design: Whole transcriptomic analysis was performed on a pool of HSPCs derived from 2 mPB HDs and 2 RAG1 patients. We compared HSPCs edited by gRNA#9 and AAV6.SA-co.hRAG1-BGHpA with untreated cells before and after the editing procedure.


title: The regulatory role of ZEB2 in ABC formation

address: 上海市黄浦区山东中路仁济医院西院区

summary: To elucidate the direct targets of ZEB2 in ABCs, we performed high-throughput sequencing of regulome by ATAC-seq, CUT & Tag, and CUT & RUN, leading to the identification the accessible sites with ZEB2 binding. Among the genes differentially expressed by Zeb2 deficiency, we found 33 candidate target genes of ZEB2, with 22 repressed and 11 activated by ZEB2. The critical transcription factor Mef2b, essential for GC development, was repressed by ZEB2. This direct regulation was mapped to a conserved region about 20kb downstream of Mef2b's exon I TSS, enriched with enhancer-associated features in both human and mouse.

overall_design: 1.Examination of chromatin accessibility of CD11c+T-bet+ and CD11c−T-bet− B cells sorted by flow cytometry from bm12-induced T-bet reporter mice. 2. Examination of chromatin accessibility of splenic B cells from Zeb2 deficient mice cultured in the presence of mouse ABC-skewing cocktail. 3. Examination of endogenous ZEB2 binding in the  in vitro derived B cells. 3. Examination of endogenous ZEB2 binding in the ex vivo sorted B cells.


title: Micro-C on K562 cell line

first: Kun

last: Sun

state: Guangdong

address: Rm A1921, Guangming International Innovation Center,

institute: Shenzhen Bay Laboratory

summary: The three-dimensional (3D) organization of genome is fundamental to cell biology. To explore 3D genome, emerging high-throughput approaches have produced billions of sequencing reads, which is challenging and time-consuming to analyze. Here we present Microcket, a package for mapping and extracting interacting pairs from 3D genomics data, including Hi-C, Micro-C, and derivant protocols. Microcket utilizes a unique read-stitch strategy that takes advantage of the long read cycles in modern DNA sequencers; benchmark evaluations reveal that Microcket runs much faster than the current tools along with improved mapping efficiency, and thus shows high potential in accelerating and enhancing the biological investigations into 3D genome. Microcket is freely available at https://github.com/hellosunking/Microcket.

overall_design: Micro-C experiments are performed on K562 cell lines with 10 biological replicates.

first: Yu

last: Zhao


title: Pleiotropic roles of LAMMER kinase, Lkh1 in stress responses and virulence of Cryptococcus neoformans

city: Jeongeup-si

first: Sunhak

last: Kwon

address: 29, Geumgu-gil

country: South Korea

institute: Korea Atomic Energy Research Institute

summary: To investigate the pleiotropic roles of Lkh1 in stress response and virulence, we constructed lkh1∆ mutant strains. In this study, we found that Tor1 was an upstream regulator of Lkh1 in C. neoformans. We then performed gene expression profiling analysis to elucidate signaling circuitry downstream of CnLkh1 in the TOR1-Lkh1 pathway using data obtained from RNA seq of 2 different strains (WT of lkh1∆ mutant) with or without rapamycin treatment.

overall_design: To investigate downstream genes regulated by TOR1-Lkh1 pathway, total RNA was isolated from wild-type (H99) and lkh1∆ mutant. Strains were grown in 30ml in a liquid YPD medium for 16hr at 30℃. Then, overnight culture was inoculated into 100 ml fresh YPD medium and adjusted to OD600=0.2. The cells were further incubated until OD600 reached approximately 0.6. 50 ml of cells were harvested for the zero-time sample and rapamycin (3 ng/ml) were treated to the remaining cells. After rapamycin treatment, cells were further incubated at 30℃ for 60 mins.

first: SunHak

first: Kwang

middle: W

last: Jung


title: ISRIB can temporarily maintain the cell fate of human trophoblast stem cell without activating WNT and inhibiting TGFβ

city: Beijing

first: Yue

last: Wang

address: 1 Beichen West Road, Chaoyang District, Beijing 100101, P.R.China

institute: Institute of zoology, Chinese Academy of Sciences

summary:
To investigate the role of ISRIB in human trophoblast stem cell(hTSC) maintenance without activation of WNT & TGFβ.
We performed gene expression profiling analysis using data obtained from RNA-seq of hTSCs cultured in the medium contained 0.5 µM ISRIB and 10 µM Y27652, after removing CHIR99021, A83-01, SB431542.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for hTSCs in the chemically deficient medium or in its original medium.

first: Shuwei

last: Xia

first: Hao

last: Wu


title: Exonic knock-out/in gene editing in hematopoietic stem and progenitor cells fully rescues RAG1 immunodeficiency [GUIDE1]

overall_design: Guide-Seq assay was performed on K562 cells edited by sgRNAs targeting the RAG1 intron 1 (gRNA#7 and #9), unedited cells were used as negative control

middle: C


title: suppression of Pyruvate carboxylase in M-wnt tumors

status: Public on Dec 29 2023

city: chapel hill

first: michael

middle: francis

last: coleman

state: North Carolina

address: 235 dauer drive

institute: UNC

summary: Metabolic reprogramming an immune evasion are established hallmarks of the tumor microenvironment (TME). Growing evidence supports tumor metabolic dysregulation as an important mediator of tumor immune evasion. High TME levels of lactate potently suppress antitumor immunity. Pyruvate carboxylase (PC), responsible for the anaplerotic conversion of pyruvate to oxaloacetate, is essential for lung metastasis in breast cancer. Conversely, PC may be dispensable in some cells in the TME, with loss of PC associated with immunosuppression. Here we test whether PC suppression alters tumor metabolism and immunosuppression.    Using multiple animal models of breast cancer, we identify a dimorphic role for PC expression in mammary cancer cells. PC supports metastatic colonization of the lungs; however, depletion of PC promotes primary tumor growth and suppresses histological and transcriptomic markers of antitumor immunity. We demonstrate that PC is potently suppressed by hypoxia, and that PC suppression is common in solid tumors, particularly those with higher levels of hypoxia. Using metabolomics, high resolution respirometry, and extracellular flux analysis, we show that PC-depleted cells produce more lactate and undergo less oxidative phosphorylation than scramble controls. Finally, we identify lactate metabolism as a targetable dependency of PC-depleted cells, which is sufficient to restore T cell populations to the TME of PC-depleted tumors.  Taken together these data demonstrate that elevated lactate following PC suppression by hypoxia may be a key mechanism through which primary tumors limit antitumor immunity. Thus, these data highlight PC directed tumor metabolism is a nexus of tumor progression and antitumor immunity.

overall_design: 10 M-Wnt tumors transduced with Doxycycline inducible PC targeting shRNA, from mice provided 150µg/ml doxycycline in drinking water (shPC, n=5), or not provided doxycycline (ctrl, n=5)

first: Michael

middle: F

last: Coleman

first: Stephen

middle: D

last: Hursting


title: DNA-PKcs Suppresses Illegitimate Chromosome Rearrangements

status: Public on Dec 30 2023

city: Stanford

first: Jinglong

state: California

address: 269 Campus Drive

department: Radiation Oncology

institute: Stanford Medicine

summary: Two DNA repair pathways, non-homologous end joining (NHEJ) and alternative end joining (A-EJ), are involved in V(D)J recombination and chromosome translocation. Previous studies reported distinct repair mechanisms for chromosome translocation, with NHEJ predominantly involved in human and A-EJ in mice. NHEJ depends on DNA-PKcs, a critical partner in synapsis formation and downstream component activation. While DNA-PKcs inhibition promotes chromosome translocations harboring microhomologies in mice, its synonymous effect in human is not known. We find partial DNA-PKcs inhibition in human cell lines leads to increased genome-wide translocations composed mostly of direct joints, indicating the continued involvement of dampened NHEJ in these processes. In contrast, complete DNA-PKcs inhibition and genetic inhibition DNA-PKcs kinase domain substantially increased microhomology-mediated end joining (MMEJ), thus bridging the two different translocation mechanisms between human and mice. Similar to a previous study on Ku70 deletion, DNA-PKcs deletion in G1/G0-phase mouse pro-B cell lines, impair the recombination of RAG1/2-mediated DNA double-strand breaks (DSBs). This DNA-PKcs-deficient repair mechanism exhibited reduced V(D)J recombination efficiency, increased end resection, decreased polymerase-mediated insertions, loss of recombination fidelity and generated relatively higher rates of chromosome translocation as a consequence of dysregulated coding and signal end joining.  Our study underscores DNA-PKcs in suppressing illegitimate chromosome rearrangement in both species.

overall_design: We induced DNA double-strand breaks (DSBs) using CRISPR-Cas9 technology in various cell lines, including K562-derived cell lines (K562-Bcl2 and K562-Bcl2-PKcs-/-) and the HCT116-derived cell lines (HCT116 DNA-PKcs+/-, HCT116 DNA-PKcs-/- and HCT116 DNA-PKcs KD/-). These cell lines were subjected to different treatment conditions. To capture translocation events, we utilized a DSB site either RAG1L located on chr11, as a bait, which allowed us to detect its ligation with the remaining DSB site(s) using high-throughput genome translocation sequencing (HTGTS). In parallel, we also captured the V-J recombination and chromosome translocations in murine DNA-PKcs catalytically dead v-Abl Pro-B cells using JK1CE and JK1SE as baits, respectively.

first: Richard

last: Frock


title: suppression of Pyruvate carboxylase

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

overall_design: Refer to individual Series


title: Gene expression signatures of megakaryocytes post-IR

status: Public on Dec 27 2023

city: Chongqing

first: Weinian

last: Liao

address: Gaotanyan street 30

institute: Army Medical University

summary: Platelets are anucleated blood cells that are produced by their progenitor cell, the megakaryocyte (MK). Platelets are centrally positioned in hemostasis and thrombosis and, according to the recent findings, play key roles in innate immunity, inflammation, atherogenesis, and cancer metastasis. The quantitative and qualitative properties of platelets are crucial determinants of their hemostatic function. While the regulatory mechanisms of platelet number and size have been studied separately, critical questions remain regarding the interplay of platelet number and size in hemostasis. In this study, using a mouse model of irradiation (IR)-induced thrombocytopenia, we show that mature MKs (mMKs) are resistant to IR and maintain the nadir platelet levels post IR. To identify the phenotype switching and function variation in BM MKs post IR, we performed microarray expression profiling of MKs from the bone marrow of mice at 1- or 3-days post IR and control mice (Ctrl).

overall_design: Radiation induced gene expression in MK was measured at 1 and 3 days after exposure to doses of 5 Gy g-rays.  Three independent experiments were performed at each time using different donors for each experiment.

first: Yiding


title: Ribonucleotides embedded in genomic DNA of Aicardi-Goutières syndrome (AGS)-orthologous mutants of Saccharomyces cerevisiae using ribose-seq protocol

status: Public on Dec 25 2023

city: Atlanta

first: Francesca

last: Storici

state: GA

address: 950 State Street Nw, Krone Engineering Biosystems Building

department: Biological Sciences

institute: Georgia Institute of Technology

summary: Ribonucleoside monophosphates (rNMPs) are abundantly found in DNA of cells, with over a million counts in the mammalian genome and thousands in budding yeast DNA. The embedded rNMPs alter DNA properties, increasing DNA fragility and mutability. Mutations in any of the three subunits of ribonuclease (RNase) H2, a key enzyme for rNMP removal, are associated with the Aicardi-Goutières syndrome (AGS), a severe neurological disorder. Here, we engineered two AGS orthologous mutations in Saccharomyces cerevisiae: rnh201-G42S and rnh203-K46W. Using the ribose-seq technique and the Ribose-Map bioinformatics tool, we studied the rNMP incorporation frequency, distribution, composition, and patterns in these yeast mutants. We found significantly more abundant rNMPs in the nuclear genome of rnh201-G42S than in wild-type and rnh203-K46W-mutant cells. Moreover, we observed distinct sequence contexts and genomic sites with abundant rNMP incorporation (hotspots) in rnh201-G42S and rnh203-K46W cells, anticipating similar rNMP patterns in human DNA carrying the orthologous AGS mutations.

overall_design: Strain BY4742, a derivative of S288C that was used in the S. cerevisiae gene disruption project, and have MATα mating type. Standard genetic and molecular biology methods were used for growth, gene disruption, isolation of mutants, selection, genome engineering, colony PCR, and sequence analysis of genomic DNA. The ribose-seq libraries derived from the wild-type RNase H2 or null-rnh201 genotype from S. cerevisiae cells containing the AGS mutations, affect RNase H2 activity on single rNMPs in DNA but allow cleavage at long RNA/DNA hybrids. We engineered two AGS orthologous mutations in Saccharomyces cerevisiae: rnh201-G42S and rnh203-K46W.  We constructed three or more ribose-seq libraries for each genotype using two or three different sets of restriction enzymes (RE1: DraI, EcoRV, and SspI; RE2: AluI, DraI, EcoRV, and SspI; and RE3:  HaeIII and RsaI). This strategy allowed us (i) to verify that the conclusions taken from our analyses of ribose-seq data are not influenced by a particular set of restriction enzymes used to fragment the DNA extracted from the different yeast species and genotypes, and (ii) to further confirm reproducibility of the results. After ribose-seq, the fastq files were trimmed and aligned to sacCer2 reference genome using Ribosemap toolkit to make the respective bam files available. Coordinate module of Ribose-Map and custom scripts were used to filter experimental artifacts and get the coordinates of rNMPs and converted to bedGraph format.

first: Deepali

last: Kundnani

first: Taehwan

last: Yang

first: Alli

last: Gombolay

first: Chance

last: Meers

first: Kuntal

last: Mukherjee

first: Gary

last: Newnam


title: Functional difference between TIF fusion protein and single protein

status: Public on Dec 22 2023

first: Guanxing

last: Zhai

address: 2901 Caolang Road, Shanghai

institute: Shanghai Public Health Clinical Center, Fudan University

summary: TIF is a fusion protein of TFF2-IFNα2-Fc. The same dose of TIF/IFNα2/TFF2 was used to stimulate THP-1 cells respectively in the presence of LPS. After extracting RNA, RNAseq technology was used to screen the expression of significantly differential genes induced by TIF/IFNα2/TFF2.

overall_design:
Compare whether there are differences in LPS-induced genes between TIF fusion protein and single protein IFNα2/TFF2, as well as changes over time.

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Submitter states that missing raw files are due to file loss.
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first: Weihui

last: Fu


title: The Rax homeoprotein in Müller glial cells is required for homeostasis maintenance of the postnatal mouse retina

status: Public on Dec 20 2023

first: Daisuke

last: Motooka

summary: To address the function of Rax in differentiated Müller glial cells, we generated Rax tamoxifen-induced conditional knock-out (Rax iCKO) mice, where Rax can be depleted in mTFP-labeled Müller glial cells upon tamoxifen treatment, by crossing Raxflox/flox mice with Rlbp1-CreERT2 mice and found histological characteristic of reactive gliosis and an enhanced gliosis of Müller glial cells in the Rax iCKO retina under normal and stress conditions, respectively. RNA sequencing using isolated Müller glial cells from the Rax iCKO retina by fluorescence-activated cell sorting demonstrated that reduced expression of suppressor of cytokine signaling-3 (Socs3), whose depletion leads to reactive gliosis in Müller glial cells. Reporter gene assays showed that Rax directly transactivates Socs3 and we observed decreased expression of Socs3 in Müller glial cells of the Rax iCKO retina by immunostaining. Taken together, these results suggest that the Rax homeoprotein suppresses reactive gliosis in Müller glial cells by transactivating Socs3 in vivo. Our results may shed light on the transcriptional regulatory mechanisms underlying Müller glial cell homeostasis.

overall_design: We generated Rax tamoxifen-induced conditional knock-out (Rax iCKO) mice, where Rax can be depleted in mTFP-labeled Müller glial cells upon tamoxifen treatment, by crossing Raxflox/flox mice with Rlbp1-CreERT2 mice.We then performed RNA sequencing using isolated Müller glial cells from the Rax iCKO retina by fluorescence-activated cell sorting demonstrated.

first: Takuya

last: Yoshimoto

first: Taro

last: Chaya

first: Leah

last: Varner

first: Makoto

last: Ando

first: Toshinori

last: Tujii

first: Kazuhiro

last: Kimura

first: Takahisa

last: Furukawa


title: Multi-omics in nasal epithelium reveals three axes of dysregulation for asthma risk in the African Diaspora populations [DNA methylation]

status: Public on Dec 23 2023

city: Baltimore

first: Rasika

middle: M

last: Mathias

state: MD

address: 5501 Bayview Cir

department: Allergy and Clinical Immunology

institute: Johns Hopkins University

type: Methylation profiling by genome tiling array

summary:
Background: Asthma, a complex chronic lung disease affecting the airways, has striking disparities across ancestral groups, but the molecular underpinning of these differences is poorly understood and minimally studied. A major goal of the Consortium on Asthma among African-ancestry Populations in the Americas (CAAPA) is to understand multi-omics signatures of asthma risk in the nasal epithelium focusing on populations of African ancestry.
Methods: DNA methylation (DNAm) quantification was performed using Illumina’s Infinium MethylationEPIC array® using genomic DNA from nasal airway epithelial cells collected across the 4 US recruitment sites (Baltimore, Chicago, Denver, and Washington DC) for 331 subjects (N=149 asthma cases, N= 182 never asthmatic controls). We performed association analysis to identify eQTMs (CpG-gene associations) for DEGs limiting to CpGs ≤5kb from the transcription start site or within enhancer regions identified through promoter-capture HiC in bronchial epithelial cells. CpGs from significant eQTMs (p<0.05) were tested for differential methylation by asthma (DMCs) to assess the relative contribution of expression and methylation in asthma risk. All models were fully adjusted for ancestry, sampling site, and appropriate latent factors.
Findings: Multi-omic analysis identified FKBP5 as a key contributor to asthma risk, where the association between nasal epithelium gene expression is likely regulated by methylation and is associated with increased use of inhaled corticosteroids. FKBP5 is a co-chaperone of glucocorticoid receptor signaling and known to be involved in drug response in asthma.
Interpretation: Our analyses reveal genes and networks in asthma that are differentially expressed in nasal epithelium of current asthma cases of African ancestry in CAAPA. Importantly, this work reveals molecular dysregulation on three axes – increased Th2 inflammation, decreased capacity for wound healing, and impaired drug response – that may play a critical role in asthma within the African Diaspora.

overall_design: DNA methylation data was generated from nasal epithelium samples in newly recruited subjects representing 4 locations across the African Diaspora in the United States: Baltimore, Washington DC, Chicago and Denver. We examined differential methylation by asthma (DMCs) to assess the relative contribution of expression and methylation in asthma risk.

first: Kathleen

last: Barnes


title: suppression of Pyruvate carboxylase in E0771 tumors [in vivo]

first: michael

last: coleman

overall_design: 11 E0771 tumors transduced with scramble control or PC targeting shRNA (scram n=4, shPC25 n=3, shPC28 n=4)

first: Michael

last: Coleman

first: Stephen

last: Hursting


title: Transcriptomic study reveals changes of lncRNAs in PBMCs from HIV-1 patients before and after ART

city: Wuhan

first: yingying

last: zhou

address: NO.169.Donghu Road, Wuhan

institute: Zhongnan Hospital of Wuhan university

summary: Background: Many studies have shown that long noncoding RNAs (lncRNAs) derived from the host and human immunodeficiency virus (HIV) itself play important roles in virus-host interactions and viral pathogenesis. It is unclear whether lncRNAs were involved in ART therapy of HIV/AIDS patients. Methods: Six HIV/acquired immunodeficiency syndrome (AIDS) subjects pre-HAART and post-HAART with effective control of plasma viremia (<20 HIV RNA copies/ml) and 6 healthy subjects were recruited in this study to perform RNA sequencing. The expression profiles of lncRNAs and mRNAs were obtained, to exploration of the potential roles of lncRNAs, trans- and cis-acting analysis was used. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to sort out functional categories of genes. Results: We identified a total of 974 lncRNAs whose expression levels were restored to normal after ART therapy. The results of the cis-acting analysis showed that only six lncRNAs have cis-regulated target genes, among which the target gene RP11-290F5.1, interferon regulatory factors 2 (IRF2), could promote HIV replication. We also identified lncRNA CTB-119C2.1, which regulates most mRNAs with differential expression between pre- and post-HAART, and the differences were significant. We selected lncRNA CTB-119C2.1 for qRT–PCR verification, and the results were consistent with those of RNA-seq. RAB3A and GADD45A, two of the lncRNA CTB-119C2.1-associated genes, have been shown to be associated with HIV infection. KEGG analysis of lncRNA CTB-119C2.1-associated genes revealed that most of the genes are involved in the p53 signaling pathway or pathways related to cell circulation and DNA replication. Conclusion: In this study, we used RNA-seq to systematically compare the expression profiles of lncRNAs in HIV subjects between untreated and treated time points. We successfully identified some lncRNAs with differential expression during certain periods (no HIV infection, HIV infection before treatment, and after treatment). Their expression is associated with HIV viral loads, and some of their regulating genes were found to be involved in HIV pathogenesis through bioinformatic analysis. These findings could help to reveal the underlying molecular mechanism of the progression of HIV infection.

overall_design: In order to reveal whether lncrna is involved in ART therapy for HIV/AIDS patients,six HIV/acquired immunodeficiency syndrome (AIDS) subjects pre-HAART and post-HAART with effective control of plasma viremia (<20 HIV RNA copies/ml) and 6 healthy subjects were recruited in this study to perform RNA sequencing. The expression profiles of lncRNAs and mRNAs were obtained, to exploration of the potential roles of lncRNAs, trans- and cis-acting analysis was used. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to sort out functional categories of genes.

first: Yingying

last: Zhou

first: Yuqing

last: Huang

first: Xiaoping

last: Chen

first: Tielong

first: Wenjia

last: Hu

first: Wei

last: Hou

first: Qi

last: Zhang

first: Yong

last: Xiong


title: An inflamed tumor cell subpopulation promotes chemotherapy resistance in triple negative breast cancer

status: Public on Dec 18 2023

city: San Francisco

first: Sourav

last: Bandyopadhyay

address: 1450 3rd Street

department: Helen Diller Family Comprehensive Cancer Center

institute: University of California, San Francisco


title: IGHMBP2 deletion suppresses translation and activates the integrated stress response [Ribo-Seq]

status: Public on Dec 19 2023

first: Jesslyn

last: Park

address: 513 Parnassus Ave

department: Cell and Tissue Biology

summary: IGHMBP2 is a non-essential, superfamily 1 DNA/RNA helicase with incompletely understood biological function. Mutagenesis in IGHMBP2 is found in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing IGHMBP2, we generated full and partial IGHMBP2 deletion K562 cell lines. We performed Ribo-seq and RNA-seq and identified diverse gene expression changes due to IGHMBP2 deletion, including ATF4 upregulation.

overall_design: We performed Ribo-seq and RNA-seq using two replicates per samples including K562 CRISPRi parental cells and K562 CRISPRi monoclonal cell lines with partial "HET" (two separate clones) versus full deletion "KO" (two separate clones) of IGHMBP2. We performed differential expression analysis among "HET" and "KO" genotypes relative to parental cell lines as the control.

last: Floor


title: Uncovering systemic immunological modification by type 2 diabetes mellitus in periodontitis patients via single-cell RNA analysis

city: Yangsan

first: Hansong

last: Lee

address: 49 Busandaehak-ro

institute: Pusan National University

summary: Type 2 diabetes mellitus (DM) is a complex metabolic disorder and leads to various complications including periodontitis (PD). Though bidirectional relationship of DM and PD has been reported, however, their immunological relationship remain poorly understood. This study aims to compare the immune response in patients with PD only and those with both PD and DM (PDDM), in order to expand our knowledge of the complicated connections between PD and DM. We collected peripheral blood mononuclear cells (PBMCs) from three distinct groups: 11 healthy control subjects, 10 patients with PD without DM, and 6 patients with PDDM. PBMCs were analyzed using single-cell RNA sequencing.

overall_design: PBMCs from subjects were isolated and libraries for scRNA-seq were generated using the chromium controller according to the 10× chromium Next GEM Single Cell 3’ v3.1 protocol. Cells are sequenced on Illumina HiSeq platform.

first: Jiyoung

last: Joo

first: Jae-Min

last: Song

first: Hyun-Joo

last: Kim

first: Yunhak

first: Hae-ryoun


title: An inflamed tumor cell subpopulation promotes chemotherapy resistance in triple negative breast cancer [RNA-Seq]

summary: Individual cancers are composed of heterogeneous tumor cells with distinct phenotypes and genotypes, with triple negative breast cancers (TNBC) demonstrating the most heterogeneity among breast cancer types. Variability in transcriptional phenotypes could meaningfully limit the efficacy of monotherapies and fuel drug resistance, although to an unknown extent. To determine if transcriptional differences between tumor cells lead to differential drug responses we performed single cell RNA-seq on cell line and PDX models of breast cancer revealing cell subpopulations in states associated with resistance to standard-of-care therapies. We found that TNBC models contained a subpopulation in an inflamed cellular state, often also present in human breast cancer samples. Inflamed cells display evidence of heightened cGAS/STING signaling which we demonstrate is sufficient to cause tumor cell resistance to chemotherapy. Accordingly, inflamed cells were enriched in human tumors taken after neoadjuvant chemotherapy and associated with early recurrence, highlighting the potential for diverse tumor cell states to promote drug resistance.

overall_design: For bulk RNA seq total RNA was isolated from FACS sorted cells using the RNAeasy Mini kit (Qiagen). RNA was quantified using a Qubit RNA BR (Broad-Range) Assay kit while RNA integrity was determined by the Agilent 2100 Bioanalyzer system. A complementary DNA library was prepared and Illumina RNAseq performed by Novogene (https://www.novogene.com/us-en/).

first: Jacobo

last: Jacobo

first: H

middle: J

last: Donnella

first: S


title: IGHMBP2 deletion suppresses translation and activates the integrated stress response [RNA-Seq]

first: Stephen


title: suppression of Pyruvate carboxylase in E0771 cells [in vitro]

first: michael

last: coleman

overall_design: 9 replicates of E0771 cell culture using E0771 cells transduced with scramble control or PC targeting shRNA (scram n=3, shPC25 n=3, shPC28 n=3)

first: Michael

last: Coleman

last: Hursting


title: The intricate cellular ecosystem of human peripheral veins as revealed by single-cell transcriptomic analysis

city: Miami

first: Anthony

last: Griswold

state: FL

address: 1501 NW 10th Ave, BRB 318

institute: University of Miami

summary: We applied single-cell RNA sequencing to 4 non-diseased human veins (3 basilic, 1 cephalic) obtained from organ donors, followed by bioinformatic and histological analyses.

overall_design: Veins were collected in cold cell culture media and enzymatically digested to generate single-cell suspensions. Samples were run using the Chromium Single Cell 3′ Library & Gel Bead Kit v3 (10X Genomics) with a target capture of 10,000 cells or as indicated otherwise. Libraries were sequenced on the Illumina NovaSeq 6000. FASTQ files were generated with Cell Ranger's mkfastq pipeline (version 6.0.2). The Cell Ranger's count pipeline (version 6.0.2) was used to generate raw gene-barcode matrices from alignment to the 10X Genomics pre-built Cell Ranger human reference package (version 2020-A), from GRCh38 Ensembl build 98/GENCODE v32 gene annotations.

first: Laisel

last: Martinez

first: Roberto

middle: I

last: Vazquez-Padron


title: An inflamed tumor cell subpopulation promotes chemotherapy resistance in triple negative breast cancer [scRNA-Seq]

first: Sourav

overall_design: Single-cell RNA-seq libraries were generated for five breast cancer samples including four breast cancer cell lines (MCF7, SKBR3, MDA-MB-231, HCC38) and one patient-derived PDX sample (HCI-002) using the 10x Genomics Chromium platform and sequenced on the Illumina HighSeq 2500