title: Hypothalamic thyroid hormone deficiency underlies reversible anorexia in a mammalian hibernator

status: Public on Apr 03 2024

city: New Haven

first: Viktor

last: Feketa

state: Connecticut

address: 333 Cedar St SHM BE58

department: Cellular & Molecular Physiology

institute: Yale University School of Medicine

summary: Mammalian hibernators survive prolonged periods of cold and resource scarcity by temporarily modulating normal physiological functions, but the mechanisms underlying these adaptations are poorly understood. The hibernation cycle of thirteen-lined ground squirrels lasts for 5–7 months and comprises weeks of hypometabolic, hypothermic torpor interspersed with 24–48-hour periods of an active-like interbout arousal (IBA) state. We show that ground squirrels, who endure the entire hibernation season without food, have negligible hunger drive during IBAs. We further demonstrate that squirrels exhibit reversible inhibition of the hypothalamic feeding center, such that hypothalamic arcuate nucleus neurons have reduced sensitivity to the orexigenic and anorexigenic effects of ghrelin and leptin, respectively. However, hypothalamic infusion of thyroid hormone during an IBA is sufficient to rescue hibernation anorexia. Our results reveal that thyroid hormone deficiency underlies hibernation anorexia and demonstrate the functional flexibility of the hypothalamic feeding center.

overall_design: Single cell gene expression in the arcuate nucleus of hypothalamus and median eminence was compared between active and hibernating thirteen-lined ground squirrels by single cell RNA sequencing on the 10X Genomics platform

first: Sarah

last: Mohr

first: Rafael

last: Dai Pra

first: Maryann

last: Platt

middle: V

first: Marya

last: Shanabrough

first: Luis

last: Varela

first: Ashley

last: Kristant

first: Haoran

last: Cao

first: Dana

last: Merriman

first: Tamas

middle: L

last: Horvath

first: Sviatoslav

last: Bagriantsev

first: Elena

last: Gracheva


title: Primitive macrophages induce sarcomeric maturation and functional enhancement of contractile human cardiac microtissues via efferocytic pathways

status: Public on Apr 02 2024

city: Toronto

first: Slava

last: Epelman

state: Ontario

address: 101 College St.

country: Canada

institute: University Health Network

type: Expression profiling by high throughput sequencing

summary: The first macrophages that seed the developing heart originate from the yolk sac during fetal life. While murine studies reveal important homeostatic and reparative functions in adults, we know little about their roles in the earliest stages of human heart development due to a lack of accessible tissue. Generation of bioengineered human cardiac microtissues from pluripotent stem cells models these first steps in cardiac tissue development, however macrophages have not been included in these studies. To bridge these gaps, we differentiated human embryonic stem cells (hESCs) into primitive LYVE1+ macrophages (hESC-macrophages; akin to yolk sac macrophages) that stably engrafted within cardiac microtissues composed of hESC-cardiomyocytes and fibroblasts to study reciprocal interactions. Engraftment induced a tissue resident macrophage gene program resembling human fetal cardiac macrophages, enriched in efferocytic pathways. Functionally, hESC-macrophages induced production and maturation of cardiomyocyte sarcomeric proteins, and enhanced contractile force, relaxation kinetics, and electrical properties. Mechanistically, the primary effect of hESC-macrophages was during early microtissue formation, where they engaged in phosphatidylserine dependent ingestion of apoptotic cardiomyocyte cargo, which reinforced core resident macrophage identity, reduced microtissue stress and drove hESC-cardiomyocytes to become more similar to human ventricular cardiomyocytes found in early development, both transcriptionally and metabolically. Inhibiting efferocytosis of hESC-cardiomyocytes by hESC-macrophages led to increased cell stress, impaired sarcomeric protein maturation and reduced cardiac microtissue function (contraction and relaxation). Taken together, macrophage-engineered human cardiac microtissues represent a considerably improved model for human heart development, and reveal a major beneficial, yet previously unappreciated role for human primitive macrophages in enhancing cardiac tissue function.

overall_design: We generated immuno-engineered human cardiac microtissues to investigate hESC-macrophage education and their subsequent role in cardiomyocyte function. We performed gene expression profiling (bulk RNA-seq) of sorted hESC-macrophages (day 14; N=3 per group) and hESC-cardiomyocytes (day 3; N=4 per group).

first: Homaira

last: Hamidzada


title: Single cell RNA sequencing reveals heterogeneity and dynamics of mouse early inguinal white adipose tissue (iWAT) during  development

status: Public on Apr 01 2024

city: Shanghai

first: Yeung

last: Sivannn

address: Dongan Road

country: China

institute: Fudan University

summary: In this study, we comprehensively compared the changes in adipose tissue from 1 week (7 d) to 4 weeks (28 d) postnatally. To analyze the different cell groups and how their amount and function vary along with developmental time, we collected iWAT from 7-, 14-, 18-, 21-, and 28-day-old mice and enzymatically disassociated the tissues into single cells and performed single-cell RNA sequencing.

overall_design: To profile cells in an unbiased manner from iWAT considering the apparent alterations in phenotype and gene expression in mouse iWAT during early life, we collected iWAT from 7-, 14-, 18-, 21-, and 28-day-old mice and enzymatically disassociated the tissues into single cells. Stromal vascular cell fractions (SVFs) were separated from mature lipid-containing adipocytes by centrifugation and were subjected to single-cell RNA-seq.

first: Chenyang

last: Zhang


title: Chromosome structure in Drosophila is determined by boundary pairing not loop extrusion

status: Public on Apr 04 2024

city: Princeton

first: Wenfan

last: Ke

state: New Jersey

address: 119 Lewis Thomas Laboratory

department: Department of Molecular Biology

country: USA

institute: Princeton University

summary: Two different models have been proposed to explain how the endpoints of chromatin looped domains (“TADs”) in eukaryotic chromosomes are determined.  In the first, a cohesin complex extrudes a loop until it encounters a boundary element roadblock, generating a stem-loop (and an unanchored loop).  In this model, boundaries are functionally autonomous: they have an intrinsic ability to halt the movement of incoming cohesin complexes that is independent of the properties of neighboring boundaries.  In the second, loops are generated by boundary:boundary pairing.  In this model, boundaries are functionally non-autonomous, and their ability to form a loop depends upon how well they match with their neighbors.  Moreover, unlike the loop-extrusion model, pairing interactions can generate both stem-loops and circle-loops.  We have used a combination of MicroC to analyze how TADs are organized and experimental manipulations of the even skipped TAD boundary, homie, to test the predictions of the “loop-extrusion” and the “boundary-pairing” models.  Our findings are incompatible with the loop-extrusion model and instead suggest that the endpoints of TADs in flies are determined by a mechanism in which boundary elements physically pair with their partners, either head-to-head, or head-to-tail, with varying degrees of specificity.  Although our experiments do not address how partners find each other, the mechanism is unlikely involve loop extrusion.

overall_design: microC for Drosophila st14 whole embryo samples, 2-3 independent replicates for each genotype

first: Xinyang

last: Bing

first: Miki

last: Fujioka

first: Amina

last: Kurbidaeva

last: Levitt

first: Mike

last: Levine

first: Paul

last: Schedl

first: James

last: Jaynes


title: Effect of anti-tumor efficacy of depletion of Nrf2 in effector T cells

city: Yangsan

first: YUNA

last: JO

address: 49, Busandaehak-ro

country: South Korea

institute: Pusan National University

summary: To investigate the function of Nrf2-deficient CD8 T cells in the regulation of anti-tumor response, we established adoptive transfer models. We then performed gene expression profiling analysis using data obtained from RNA-seq of Nrf2-deficient and WT CD8 T cells in the tumor.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Nrf2-deficient and WT CD8 T cells in the tumor

first: Yuna

last: Jo

first: Changwan

last: Hong


title: Regulation of IkB kinase family crosstalk by an N4BP1-caspase-8 axis [RNA-seq NGS3799]

city: South San Francisco

first: Rohit

last: Reja

state: CA

address: 1 DNA Way

institute: Genentech

summary: The cell death protease caspase-8 plays an essential role in controlling inflammation, as severe immunodeficiency results from its loss. We previously found that caspase-8 promotes inflammatory responses by cleaving NEDD4-binding protein 1 (N4BP1), a suppressor of cytokine production, but the underlying mechanisms remained unclear. Here we find that N4BP1 curtails the duration, rather than initial induction, of proinflammatory signaling through a mechanism involving noncanonical IKK (ncIKK)-mediated inhibition of the canonical IkB kinase (IKK) complex, a crosstalk event among the IKK family facilitated by N4BP1. Accordingly, co-deletion of the ncIKKs or their adaptor protein TANK largely phenocopied deletion of N4BP1, augmenting cytokine responses by macrophages upon engagement of TRIF-independent toll-like receptors (TLR) 1/2, TLR7, or TLR9. Like N4BP1, TANK was largely prevented from inhibiting the TRIF-dependent TLR4 response due to caspase-8. Biochemically, N4BP1 binds both the canonical and noncanonical IKK complexes, in a manner promoted by linear and/or K63-linked polyubiquitin chain binding by N4BP1 and independent of its RNAse activity. Consistent with this, a knock-in mutant of N4BP1 with diminished ubiquitin chain-binding capacity led to increased proinflammatory cytokine responses. These findings thereby unveil a mechanism of late-phase inflammatory gene control, whereby N4BP1 prevents persistent IKK activity through ncIKK-mediated inhibition. This molecular crosstalk among caspase-8, N4BP1, and the IKKs and ncIKKs may have implications for our understanding of genetic immune diseases caused by mutations in caspase-8 or TBK1 and suggest a novel ‘guarding’ mechanism against pathogens that attempt to subvert the ncIKKs.

overall_design: Time course of N4bp1 knockout or wildtype mouse Bone Marrow Derived Macrophages (BMDMs) treated with R837 and/or Actinomycin D

first: R


title: Regulation of IkB kinase family crosstalk by an N4BP1-caspase-8 axis [scRNA-seq]

overall_design: Single cell transcriptome on wildtype and N4bp1 mouse bone marrow derived macrophages  (BMDMs) that were co-cultured with GFP expressing BMDMs, with and without TLR7 treatment


title: tumor-infiltrating CD8+ T cells from tumor-bearing mice transferred Nrf2-deficient CD8+ T cells.

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Regulation of IkB kinase family crosstalk by an N4BP1-caspase-8 axis [NGS4102 RNA-seq]

overall_design: Untreated and 8h treatment of R837 for the following genotypes: TBK1 wildtype (TBK1-WT ∆control), TBK1 wildtype with IKKe knockout (TBK1-WT ∆IKKe), TBK1 conditional knockout (TBK1-cKD ∆control), and TBK1 conditional knockout with IKKe knockout (TBK1-cKD ∆IKKe)


title: Regulation of IkB kinase family crosstalk by an N4BP1-caspase-8 axis [RNA-seq NGS3611]

overall_design: Time course of N4bp1 knockout or wildtype mouse Bone Marrow Derived Macrophages (BMDMs) treated with Pam3csk4 or R837


title: Regulation of IkB kinase family crosstalk by an N4BP1-caspase-8 axis


title: Regulation of IkB kinase family crosstalk by an N4BP1-caspase-8 axis [ChIP-seq]

overall_design: ChIP-seq of RNA Polymerase II of WT and N4bp1 KO of mouse Bone Marrow Derived Macrophages (BMDMs), untreated and 2,4,6 hrs post-treatment with R837


title: Stem-loop and circle-loop TADs generated by directional pairing of boundary elements have distinct physical and regulatory properties

summary: The chromosomes in multicellular eukaryotes are organized into a series of topologically independent loops called TADs. In flies, TADs are formed by physical interactions between neighboring boundaries. Fly boundaries exhibit distinct partner preferences, and pairing interactions between boundaries are typically orientation dependent. Pairing can be head-to-tail or head-to-head. The former generates a stem-loop TAD, while the latter gives a circle-loop TAD. The TAD that encompasses the Drosophila even skipped (eve) gene is formed by the head-to-tail pairing of the nhomie and homie boundaries. To explore the relationship between loop topology and the physical and regulatory landscape, we flanked the nhomie boundary region with two attP sites. The attP sites were then used to generate four boundary replacements: λ DNA, nhomie forward (WT orientation), nhomie reverse (opposite of WT), and homie forward (same as WT homie). The nhomie forward replacement restores the WT physical and regulatory landscape: In MicroC experiments, the eve TAD is a volcano triangle topped by a plume, and the eve gene and its regulatory elements are sequestered from interactions with neighbors. The λ DNA replacement lacks boundary function: the endpoint of the “new” eve TAD on the nhomie side is ill-defined, and eve stripe enhancers activate a nearby gene, eIF3j. While nhomie reverse and homie forward restore the eve TAD, the topology is a circle-loop, and this changes the local physical and regulatory landscape. In MicroC experiments, the eve TAD interacts with its neighbors, and the plume at the top of the eve volcano triangle is replaced by a cloud of contacts with the next-door TADs. Consistent with the loss of isolation afforded by the stem-loop topology, the eve enhancers weakly activate genes in the neighboring TADs. Conversely, eve function is partially disrupted.


title: Comparing axon regeneration in male and female mice after peripheral nerve injury

city: Seoul

first: Eun-Mi

last: Hur

address: 1, Gwanak-ro, Gwanak-gu

department: College of Veterinary Medicine

institute: Seoul National University

summary: In the mammalian peripheral nervous system, axon regeneration occurs spontaneously after injury. We compared the transcriptome profile of male and female dorsal root ganglia to examine if injury responses after sciatic nerve injury is sex-dependent.

overall_design: mRNA profile of male and female mice after sciatic nerve injury (SNI) were generated by deep sequencing, in triplicate, using a NextSeq 500 (Illumina) system.

first: Eun-Hae

last: Jang

first: Yun-Hee

last: Bae

first: Eun-Mo

last: Yang

first: Yunho

last: Gim

first: Hyun-Jun

last: Suh

first: Subin

last: Kim


title: Epigenetic maintenance of neural stem cell quiescence in the adult mouse hippocampus via Setd1a [RNA-Seq]

status: Public on Mar 31 2024

city: philadelphia

first: Ting

last: Zhao

address: 415 curie blvd

institute: University of Pennsylvannia

summary: Quiescence, a hallmark of adult neural stem cells (NSCs), is required for maintaining the NSC pool to support life-long continuous neurogenesis in the adult dentate gyrus (DG). Whether epigenetic mechanisms can maintain NSC quiescence over long term in the adult brain is not well-understood. Here we showed that adult mice with haploinsufficiency of Setd1a, a schizophrenia risk gene encoding a histone H3K4 methyltransferase, exhibited substantially enlarged DG with increased number of dentate granule cells. Deletion of Setd1a specifically in quiescent NSCs in the adult DG promoted their activation and neurogenesis, which was countered by inhibition of histone demethylase LSD1. Mechanistically, RNA sequencing and CUT & RUN analyses of cultured quiescent adult NSCs revealed Setd1a deletion-induced transcriptional changes and Setd1a targets, among which down-regulation of Bhlhe40 promoted quiescent NSC activation in the adult DG. Together, our study revealed a Setd1a-dependent epigenetic mechanism that sustains NSC quiescence in the adult DG.

overall_design: Comparative gene expression profiling time-course analysis of RNA-seq data for wild-type (WT) and Setd1a knockout (KO) mouse quiescent neural progenitor cells (NPCs) cutured in vitro

first: Yan

first: Bowen

last: Yan

first: Suming

last: Huang

first: Guo-li

last: Ming

first: Hongjun

last: Song


title: Chromatin accessibility analysis of tumor-infiltrating CD8+ T cells from tumor-bearing mice transferred Nrf2-deficient CD8+ T cells. [ATACseq]

first: YUNA

summary: To identify unique chromatin regions predicting the anti-tumor efficacy of Nrf2-deleted smart CD8+ T cell therapy, we performed an assay for transposase-accessible chromatin sequencing (ATAC-seq) using tumor-infiltrating CD8+ T cells from tumor-bearing mice transferred Nrf2-deficient CD8+ T cells. From the analysis of ATAC-seq data, we identified quantitatively distinct open regions of chromatin that distinguish Nrf2-deficient CD8+ T cells or WT CD8+ T cells.

overall_design: Comparative Chromatin accessibility analysis of ATAC-seq data for Nrf2-deficient and WT CD8+ T cells in the solid tumor

first: Yuna


title: Epigenetic maintenance of neural stem cell quiescence in the adult mouse hippocampus via Setd1a [Cut & Run]

overall_design: Cleavage Under Targets & Release Using Nuclease (CUT&RUN) for Setd1a in mouse quiescent neural progenitors (NPCs) cultured in vitro


title: Epigenetic maintenance of neural stem cell quiescence in the adult mouse hippocampus via Setd1a


title: CRISPR  pooled screen and QuantSeq 3' mRNA sequencing to identify  microRNAs (miRNAs) important for Breast Cancer 3D growth

city: Brighton

first: Leandro

last: Castellano

address: Falmer, JMS Building

country: United Kingdom

institute: University of Sussex


title: CRISPR Screening Uncovers a Long-Range Enhancer for ONECUT1 in Pancreatic Differentiation and Links a Diabetes Risk Variant [H3K27ac_ChIP-seq]

status: Public on Mar 29 2024

city: New York

first: Samuel

middle: Joseph

last: Kaplan

state: New York

address: 430 E 67th St

department: Developmental Biology Program

institute: Sloan Kettering Institute

summary: Functional enhancer annotation is a valuable first step for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants for investigation. However, unbiased enhancer discovery in physiologically relevant contexts remains a major challenge. To discover regulatory elements pertinent to diabetes, we conducted a CRISPR interference (CRISPRi) screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers uncovered, we focused on a long-range enhancer ~664 kb from the ONECUT1 promoter, as coding mutations in ONECUT1 cause pancreatic hypoplasia and neonatal diabetes. Homozygous enhancer deletion in hESCs was associated with a near-complete loss of ONECUT1 gene expression and compromised pancreatic differentiation. We then identified a type 2 diabetes (T2D) associated variant (rs528350911) in the enhancer which disrupts a GATA motif. Introduction of the risk variant into hESCs revealed substantially reduced binding of key pancreatic transcription factors (GATA4, GATA6 and FOXA2) on the edited allele, accompanied by a subtle reduction of ONECUT1 transcription, supporting a causal role for this risk variant in metabolic disease. This work expands our knowledge about transcriptional regulation in pancreatic development through the characterization of a long-range enhancer and highlights the utility of enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for H3K27ac in WT and ONECUT1e-664kb deletion hPSC cells (heterozygous and homozygous) differentiated to the pancreatic lineage.

first: Danwei

last: Huangfu

first: Hyein

middle: S

last: Cho

middle: J


title: CRISPR Screening Uncovers a Long-Range Enhancer for ONECUT1 in Pancreatic Differentiation and Links a Diabetes Risk Variant [RNA-seq]

overall_design: RNA-sequencing (RNA-seq) in WT and ONECUT1e-664kb deletion hPSC cells (heterozygous and homozygous) differentiated to the pancreatic lineage.


title: CRISPR  pooled screen and QuantSeq 3' mRNA sequencing to identify  microRNAs (miRNAs) important for Breast Cancer 3D growth [CRISPR screen]

summary: Breast cancer (BC) is a heterogeneous malignancy with distinct molecular subtypes and clinical characteristics. Tumor-initiating cells (TICs) constitute a small subset of cancer cells responsible for tumor initiation and progression. MicroRNAs (miRNAs), small non-coding RNA molecules, play a pivotal role in regulating gene expression and have been implicated in cancer development and progression. In this study, we conducted genome-wide CRISPR-Cas9 screens within 3D tumorspheres of two BC cell lines, MCF7 (ER+) and HCC1395 (TNBC). Our objective was to identify miRNAs that govern TIC viability, employing CRISPR/Cas9 pooled screens that targeted a total of 19,052 genes and 1,864 miRNAs. By suppressing relevant miRNAs following QuantSeq 3' mRNA sequencing we identify the genes regulated by those miRNAs

overall_design: We conducted genome-wide CRISPR screens in 2D and 3D cultures of two BC cell lines, MCF7 (ER+) and HCC1395 (TNBC) to identify miRNAs and their targets important for breast cancer tumorigenesis.

first: Tom

last: Stiff


title: CRISPR Screening Uncovers a Long-Range Enhancer for ONECUT1 in Pancreatic Differentiation and Links a Diabetes Risk Variant [long_read_seq]

overall_design: Long-read sequencing with adaptive sampling of ONECUT1 locus and controls regions of genomic DNA extracted from H1 iCas9 hPSCs with rs528350911 (clone N11).


title: Hematopoietic stem cell membrane-coated vesicles for targeted drug delivery to the bone marrow and elimination of leukemia cells

status: Public on Mar 28 2024

city: Hangzhou

first: Li

last: jinxin

address: 1369 West Wenyi Road

institute: Zhejiang University

summary: In order to study the mechanism of apoptosis of leukemia cells induced by HSC-lipo, we conducted transcriptome sequencing of leukemia cells treated with HSC-lipo.

overall_design: Leukemia cells were incubated with free chemotherapeutic drugs, Ara-C@Lipo and Ara-C@HSPC-Lipo, respectively. After 24h, cells were collected and RNA was extracted for transcriptome sequencing.

first: Jinxin

last: Li

first: Honghui

last: Wu

first: Zebin

last: Yu

first: Qiwei

last: Wang


title: Application of Translation Complex Profile sequencing (TCP-seq) to track the course of translational reprogramming in the exponentially growing culture of budding yeast (Saccharomyces cerevisiae, BY4741) subjected to glucose starvation for 10 minutes.

city: Melbourne

first: Attila

last: Horvath

state: Victoria

address: 305 Grattan St

country: Australia

institute: Peter MacCallum Cancer Centre

summary:
Work summary: Full-transcriptome methods have brought versatile power to protein biosynthesis research, but remain difficult to apply for the quantification of absolute protein synthesis rates. Here we propose and, using modified translation complex profiling, confirm co-localisation of ribosomes on messenger(m)RNA resulting from the ribosomal diffusional dynamics. We demonstrate that the stochastically co-localised ribosomes are linked with the translation initiation rate and provide a robust variable to model and quantify specific absolute protein output from mRNA. The new type of signal originating from the stochastic co-localisation of ribosomes on mRNA is evidenced by long disome-derived (potentially but rarely, multisome-derived) footprints outside the regions of non-random elongation stalls. Using the stochastic co-localisation component of the translation complex profiling data, we propose a new approach to assess absolute translation rates across mRNA, based on the calculation of Stochastic Translation Efficiency (STE) measure. STE employs a machine learning model trained with multiple TCP-seq-sourced variables. The variables, among others, include stochastic disome signal derived from the total disome footprint data and normalised to the ribosome (RS) footprint abundance across the respective open reading frames (ORFs). The variables also include SSU footprint occurrences over the respective start codons normalised to the RS signal averaged and normalised by the ORF length, to better distinguish between the cases of actively initiated or densely populated but slowly initiated mRNAs. Importantly, STE does not employ any variables resulting from normalisation of the signals of different nature, such as any normalisation of the footprint data by relative RNA abundance measured by RNA-seq. We propose STE as a more robust, sometimes more convenient, alternative to the classical TE (translation efficiency). The main distinguishing features of the STE are the inherent single-molecule-event nature of the main measure component (the long stochastic disome footprint signal), similar type of data used for the numerator(s) and denominator(s) of the calculated variables, resistance to the abundance changes and variations in the differential localisation of RNA and insensitivity to the library preparation techniques. We further hypothesise that similar high-throughput data-derived measures based on parameters dependent on random individual molecular co-incidences can be utilised to provide robust assessment of many molecular processes in dynamic biological systems.
Methods summary: Wild-type (WT) yeast cell line BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0; Dharmacon) (Non Starved, NS) was used for the glucose starvation (10 minutes; Starved, S10) studies. 2-3 replicates were used for each type of footprinting library and the NS and S10 biological points. The cells were subjected to snap-chilling followed by rapid formaldehyde fixation. The fixed material was separated into the ribosome-associated and non-associated fractions using ultracentrifugation. The ribosome-associated fraction was RNase-digested and separated into small ribosomal subunit (SSU), monosomal (ribosomes, RS), and disomal (disomes, DS) fractions of complexes using ultracentrifugation. The resultant translational complexes were de-crosslinked, and their RNA isolated to construct TCP-seq libraries, which were deep sequenced.
Results summary: Preliminary theoretical investigations revealed monotonous dependence of the stochastic DS frequency on translation initiation rate in a broad range of initiation and elongation rates and variances, well-covering all biologically meaningful combination of parameters. Biomimetic in silico modelling utilising elongation and initiation rate distributions extrapolated from in vivo single-molecule measurements available in the published data confirmed the link of stochastic DS signal with the initiation frequency. The in silico-modelled stochastic DS frequency has shown resistance to local alterations of the elongation rate over ORFs and monotonous dependence on the initiation rate, even in the extreme cases of elongation rate discontinuity. DS footprint fractions derived from the TCP-seq material prominently demonstrated the presence of longer footprints, and revealed a complex pattern of footprint lengths. The TCP-seq DS footprint pattern allowed us to propose a generalised case of ribosomal co-localisation, whereby DS can form as a result of non-random co-localisations of serially occurring ribosomes, as well as co-localisations resulting from the spatial proximity and the geometry and spatial arrangement of the respective source poly(ribo)somes. Bioinformatically segregating all of the DS signal into its stochastic and non-random components, and combining the derived components with the other TCP-seq-sourced variables and known ORF lengths, we further implemented a machine learning approach that results in Stochastic Translation Efficiency (STE) measure with high correlation (Pearson’s r ~0.7 for train and ~0.6 for test data, respectively) to the absolute protein synthesis rates as directly measured in the published data using metabolic labelling and mass spectrometry. Applying STE to the NS and S10 cell stress scenarios, we identified mRNAs from 5,238 genes with distinct translational behaviour. Multiple classes of mRNAs demonstrated acute (~50) and moderate (~50) translational up-regulation in the starved cells, while others were moderately (~300) and severely (~280) down-regulated. We dissected TCP-seq variable contribution strength into the STE, revealing strong input and role of the scanning process and stochastic co-localisation of ribosomes in the definition of translation rate. We found that the stressed cells retained high median elongation rate capacity, while losing their ability to translate proteins with the maximum rate, thus highlighting that the safety translation shutdown is permissive of translation and is largely aimed at capping the highest-producing mRNAs. Using STE, we demonstrated that translational control in rapid stress response is vastly differential, with mRNAs showing highly specific magnitude and direction of the response.
Conclusions: The stochastic disome signal, together with the other measurements sourced from TCP-seq and calculated as Stochastic Translation Efficiency (STE) measure, provide a link to the absolute translation initiation and protein biosynthesis rates. Using STE, it is possible to rank mRNAs by the absolute protein output and thus, characterise the ‘power’ of translation control elements across transcripts in a single setting or between different conditions. STE does not use bias-inducing normalisation to the RNA abundance or signals of different types and relies on self-normalised signal pairs. STE thus evades imprecisions arising from the different accessibility across mRNAs to translation machinery (such as in nucleus, phase-separated foci or condensates, etc.) and coincidental transcription- and stability-driven mRNA abundance alterations, and provides a robust measure suitable for investigating protein biosynthesis during rapidly changing transcriptome background, as occurs in cell stress response and reprogramming. With STE, we demonstrate that the prototypical example of translational control during yeast response to glucose starvation is more complex than previously thought and includes translational acceleration as well as re-adjustment of the RNA metabolism to suit new translational demands. More details can be uncovered in the future, as STE application is permissive to finer dynamics dissection and elucidation of very rapid cell responses at the level of RNA translation.

overall_design: budding yeast TCP-seq libraries from naive and starved cells

first: Yoshika

last: Janapala

first: Ross

last: Hannan

first: Eduardo

last: Eyras

first: Thomas

last: Preiss

first: Nikolay

last: Shirokikh


title: Atopic dermatitis complicated by recurrent eczema herpeticum is characterized by multiple, concurrent epidermal inflammatory endotypes

status: Public on Mar 26 2024

city: Denver

first: Nathan

last: Jackson

state: CO

address: 1400 Jackson St.

department: Center for Genes, Environment, and Health

institute: National Jewish Health

summary: A subgroup of atopic dermatitis (AD) patients suffers from recurrent, disseminated herpes simplex virus (HSV) skin infections, eczema herpeticum (EH). To determine transcriptional mechanisms of the skin and immune system pathobiology that underlies ADEH disease development, we performed RNA-sequencing analysis of non-lesional skin (epidermis, dermis) from AD patients with (ADEH+, n=15) and without (ADEH-, n=13) history of EH, and healthy controls (HC, n=15). We also performed RNA-sequencing on participants’ plasmacytoid dendritic cells (pDCs) infected in vitro with HSV-1. ADEH+ patients exhibited dysregulated gene expression, limited in dermis (differentially expressed genes [DEGs]=14) and more widespread in epidermis (DEGs=129). ADEH+-upregulated epidermal DEGs were enriched in type 2 cytokine (T2) (IL4R, CCL22, CRLF2, IL7R), interferon (CXCL10, ICAM1, IFI44, IRF7), and IL-36γ (IL36G) inflammatory gene pathways. All ADEH+ participants exhibited T2 and interferon endotypes and 87% were IL36G-high. In contrast, these endotypes were more variably expressed among ADEH- participants. ADEH+ skin also dysregulated epidermal differentiation complex (EDC) gene expression within LCE, S100, and SPRR families, which are involved in skin barrier function and antimicrobial activities. pDC transcriptional responses to HSV-1 infection were unaltered by ADEH status. Pathobiology underlying ADEH+ risk is associated with a unique, multi-faceted epidermal inflammation that accompanies dysregulation of EDC genes.  These findings will help direct future studies that define how these inflammatory patterns may drive risk of eczema herpeticum in AD.

overall_design: Normal human embryonic keratinocytes (NHEK) from a single donor were purchased from ThermoFisher Scientific and maintained in EpiLife medium containing 0.06 mM CaCl2 and S7 supplement in 5% CO2 at 37°C. For NHEK differentiation, in triplicate, cells were cultured in EpiLife medium containing 1.3 mM CaCl2 for 3 days, then treated with 200 ng/ml of IL-36-gamma  (R&D systems) for 2 days. After treatment, the cells were harvested for RNA extraction using a Qiagen RNeasy Kit according to the manufacturer’s instructions. Whole transcriptome libraries were constructed using the KAPA mRNA HyperPrep library kit (Roche Sequencing and Life Science, Kapa Biosystems, Wilmington, MA) from 250ng of total input RNA according to the manufacturers protocol. Barcoded libraries were pooled and sequenced using 150bp paired-end reads on the Illumina HiSeq 2500 system (Illumina, San Diego, CA). Raw sequencing reads were trimmed using skewer (v0.2.2) with the following parameters: end-quality=15, mean-quality=25, min=30. Read alignment to the human reference genome GRCh38 was performed using HISAT2 (v2.1.0) with default parameters. Gene quantification was performed using htseq-count (v0.9.1) and GRCh38 Ensemble v84 gene transcript model with the following parameters: stranded=reverse, a=20, mode=intersection-nonempty.

middle: D

last: Dyjack

first: Elena

last: Goleva

first: Lianghua

last: Bin

first: Michael

middle: T

last: Montgomery

first: Cydney

last: Rios

first: Jamie

last: Everman

first: Patricia

last: Taylor

first: Caroline

last: Bronchick

first: Brittany

middle: N

last: Richers

first: Donald

middle: M

last: Leung

first: Max

middle: A

last: Seibold


title: CRISPR  pooled screen and QuantSeq 3' mRNA sequencing to identify  microRNAs (miRNAs) important for Breast Cancer 3D growth [RNA-seq]

first: Leandro

last: Castellano

overall_design: We inhibited the top 6 miRNAs  (using anti-miRNA inhibitors) detected by the CRISPR screen and clinically relevant following QuantSeq 3' mRNA sequencing

first: Tom

last: Stiff


title: An innate immunity against Xanthomonas oryzae activated by ATM-mediated host DSB damage response in rice

city: Yangzhou

first: Mingnan

last: Qu

state: Jiangsu

address: Pazhou Dadao Rd, 17-19

institute: Yangzhou Univerisity

summary: We examined the transcriptional data in rice leaves of TOP6A3 overexpressed line (TOP6A3-OE1) with WT (K3) plants under normal growth.

overall_design: RNA-seq

first: Zhan

last: Xu

first: Peipei

first: Jiehua

last: Qiu

first: Min

last: Yang

first: Yanjun

last: Kou

first: Jemaa

last: Essemine

first: Xu

last: Wang

first: Yong

last: Xiang

first: Weixiong

last: Chen


title: The influence of OGA knockdown on naive hESC and its transition to primed state

first: Qianyu

last: Liu

address: YuhangTang Road

summary: In order to study the effect of OGA knockdown on naive hESC and its transition to the primed state, we knocked down OGA in PXGL-naive hESC and transformed it into the primed state，as well as collected RNA samples during and after transformation to performe  RNA-seq.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for PXGL-naïve hESC， its KD derivatives and their transformed cells.

first: Wen

last: Yi


title: Depletion-assisted multiplexing cell-free RNA sequencing reveals distinct human and microbial signatures in plasma versus extracellular vesicle

status: Public on Mar 27 2024

city: Beijing

first: Zhi

middle: John

last: Lu

address: Shuangqing Road

department: School of Life Sciences

institute: Tsinghua University

summary: Cell-free RNAs in biofluids provide opportunities to monitor cancer in a non-invasive manner. Although extracellular microRNAs are extensively characterized, fragmented cell-free long RNAs are not well investigated. Here, we developed Detector-seq (depletion-assisted multiplexing cell-free total RNA sequencing) to enable the deciphering of the cell-free transcriptome. After demonstrating the superior performance of detecting fragmented cell-free long RNAs, we applied Detector-seq to compare cell-free RNAs in human plasma and its extracellular vesicle (EV). Distinct human and microbial RNA signatures were revealed. Structured circular RNA, tRNA, and Y RNA were enriched in plasma, while mRNA and srpRNA were enriched in EV. Meanwhile, cell-free RNAs derived from the virus were more enriched in plasma than in EV. We identified RNAs that showed a selective distribution between plasma and EV and uncovered their distinct functional pathways, that is RNA splicing, antimicrobial humoral response enriched in plasma and transcriptional activity, cell migration, and antigen receptor-mediated immune signals enriched in EV. Although distinctive cancer-relevant RNA signals were identified in plasma and EV, a comparable performance of distinguishing cancer patients from normal individuals could be achieved. Compared to human RNAs, microbe-derived RNA features enabled better classification between colorectal and lung cancer. And for these microbial RNAs, plasma RNAs outperformed EV RNAs for the discrimination of cancer types. Overall, our work provides insights into the unexplored difference of cell-free RNA signals between plasma and EV, thus offering practical guidance for proper selection (with/without EV enrichment) when launching an RNA-based liquid biopsy study. Furthermore, with the ability to capture understudied cell-free long RNA fragments, Detector-seq offers new possibilities for transcriptome-wide characterization of cell-free RNAs to facilitate the understanding of extracellular RNA biology and clinical advances of liquid biopsy.

overall_design: In this study, we sequenced 113 cell-free RNA samples from human plasma and its extracellular vesicle (EV). Our datasets included 23 plasma RNA and 19 EV RNA samples of colorectal cancer, 19 plasma RNA and 19 EV RNA samples of lung cancer, and 19 plasma RNA and 14 EV RNA samples of age- and gender-matched healthy donors.

first: Hongke

first: Qing

last: Zhan


title: Brain metastasis from EGFR-mutated non-small cell lung cancer: Secretion of IL11 from astrocytes up-regulates PDL1, increases apoptosis of T lymphocytes, and promotes immune escape.

status: Public on Mar 23 2024

city: Dalian

first: Mengyi

last: Tang

address: 467 Zhongshan Road

institute: the Second Affiliated Hospital of Dalian Medical University

summary: To investigatethe mechanism responsible for the astrocyte-induced upregulation of PDL1 in BM cells, we constructed the tumor cell-astrocyte co-culture system.

overall_design:
We then performed gene expression profiling analysis using data obtained from RNA-seq of cells co-cultured compaired with single cells.
Comparative gene expression profiling analysis of RNA-seq data for astrocytes co-cultured with PC9-BrM3 cells and single astrocytes or PC9-BrM3 cells co-cultured with astrocytes and single PC9-BrM3 cells.

first: Wenwen

first: Qi


title: Gene expression profile at single cell level of Wild type and Plagl1 knock out retina at P7

status: Public on Mar 24 2024

first: yacine

last: touahri

address: 2075 Bayview Ave

institute: Sunnybrook Research Institute

summary: Plagl1 controls Muller glial cells proliferation in the retina.  We used single cell RNA sequencing (scRNA-seq) to analyze the prolirative state of Muller glial cells and identify the signals that might be involved in this process

overall_design: Retinal cells were isolated from P7 wild type and Plagl1 KO retinas at P7. Dead cells removed by magnetic cell sorting and cells were analyzed using csRNAseq

first: Yacine

last: Touahri

first: Chao

first: Carol

last: Schuurmans


title: Loss of Lamin A leads to the nuclear translocation of AGO2 and compromised RNA interference

status: Public on Mar 21 2024

city: Gothenburg

first: Aishe

last: Sarshad

address: Medicinaregatan 9C

department: Dept.of Biochemistry & Cell Biology

country: Sweden

institute: University of Gothenburg

summary: In mammals, RNA interference (RNAi) was historically studied as a cytoplasmic event, however, in the last decade growing number of reports convincingly show nuclear localization of the AGO proteins.  Nevertheless, the mechanism and the extent of nuclear RNAi remains to be fully elucidated. We found that reduced Lamin A levels significantly induced nuclear influx of AGO2 in SHSY5Y neuroblastoma and A375 melanoma cancer cell lines, which normally have no nuclear AGO2. Lamin A KO manifested a more pronounced effect in SHSY5Y cells compared to A375 evident by changes in cell morphology, increased cell proliferation and oncogenic miRNA expression. Furthermore, in SHSY5Y cells, AGO fPAR-CLIP in Lamin A KO cells revealed significantly reduced activity of RNAi. Further exploration of the nuclear AGO interactome by mass spectrometry indicated that AGO2 is in complex with FAM120A, an RNA binding protein and known interactor of AGO2. By performing FAM120A fPAR-CLIP we discovered that FAM120A co-binds AGO targets and that this competition reduces the activity of RNAi. Therefore, loss of Lamin A triggers nuclear AGO2 translocation, RNAi impairment and selective upregulation of oncogenic miRNAs to facilitate cancer cell proliferation.

overall_design: We performed AGO fPAR-CLIP to assess AGO proteins occupancy on RNA transcripts in SHSY5Y and A375 WT and Lamin A KO cells from either the cytoplasmic or nuclear fractions. FAM120A  fPAR-CLIP was carried out in SHSY5Y and A375 Lamin A KO cells from either the cytoplasmic or nuclear fractions.

first: V

last: Lobo

first: I

last: Nowak

first: C

last: Fernandez

first: A

middle: I

last: Muler

first: J

middle: O

last: Westholm

first: H

middle: C

last: Huang

last: Fabrik

last: Huynh

first: Evgeniia

last: Shcherbinina

first: M

last: Kanik

last: Härtlova

first: D

last: Benhalevy

last: Angeletti


city: Tel Aviv

first: Daniel

address: Tel Aviv University, Green Bldg. Room 205

department: Shmunis School of Biomedicine and Cancer Research

country: Israel

institute: Tel-Aviv University


title: Single-cell Multi-omics Analysis of Human Testicular Germ Cell Tumor Reveals its Molecular Features and Microenvironment

city: Bei jing

first: Xiaojian

last: Lu

state: Bei jing

address: No.3, Datusi Road, Chaoyang District, Beijing

department: State Key Laboratory of Stem Cell and Reproductive Biology

institute: Institute of Zoology，Chinese Academy of Sciences

summary: Seminoma is the most common malignant solid tumor in 14 to 44 year-old men. However, its molecular features and tumor microenvironment (TME) is largely unexplored. Here, we performed a series of studies via genomics profiling (single cell multi-omics and spatial transcriptomics) and functional examination using seminoma samples and a seminoma cell line. We identified key gene expression programs shared between seminoma and primordial germ cells, and further characterized the functions of TFAP2C in promoting tumor invasion and migration. We also identified 15 immune cell subtypes in TME, and found that subtypes with exhaustion features were located closer to the tumor region through combined spatial transcriptome analysis. Furthermore, we identified key pathways and genes that may facilitate seminoma breaking through the seminiferous tubules. These findings advanced our knowledge of seminoma tumorigenesis and produced a multi-omics atlas of in situ human seminoma microenvironment, which could help discover potential therapy targets for seminoma.

overall_design: scRNA-Seq 4 samples

first: Xichen

last: Nie

first: Hao

last: Bo

first: Jingtao

last: Guo


title: Stem-cell states converge in multi-stage cutaneous squamous cell carcinoma development [scRNA-seq]

status: Public on Mar 22 2024

city: San Francisco

first: MARK

middle: ALAN

last: TAYLOR

department: Hellen Diller Family Comprehensive Cancer Center

institute: UCSF

summary: Stem cells play a critical role in cancer development by contributing to cell heterogeneity, lineage plasticity, and drug resistance. We created gene expression networks from hundreds of mouse normal and tumor samples, and integrated these with lineage tracing and single-cell RNA-seq, to identify convergence of cell states in pre-malignant tumor cells expressing markers of lineage plasticity and drug resistance. Two of these cell states representing multilineage plasticity or proliferation were inversely correlated, suggesting a mutually exclusive relationship. Treatment of carcinomas in vivo with chemotherapy repressed the proliferative state and activated multilineage plasticity, while inhibition of differentiation repressed plasticity and potentiated responses to cell-cycle inhibitors. Manipulation of this cell state transition point may provide a rich source of potential combinatorial targets for cancer therapy.  These scRNAseq data encompass normal skin, papilloma, and squamous cell cacinoma samples.

overall_design: To investigate cell state identities of individual cells in mouse squamous cell carcinomas as they grow from normal skin, to papilloma, to carcinoma, and are then treated with cisplatin.  These cancers were initiated with DMBA and promoted with TPA.  Several samples were lineage traced using Lgr6-EGFP-CreERT2 mice carrying the Rosa26-LSL-Tomato reporter (Lgr6eGFPtdTomato) lineage tracing system to track Lgr6+ cells (GRF+) and their progeny (tomato).

first: Eve

last: Kandyba

first: Di

last: Wu

first: Reyno

last: Delrosario


title: Gene-regulatory potential of 1α,25-dihydroxyvitamin D3

status: Public on Mar 20 2024

city: Olsztyn

first: Carsten

last: Carlberg

state: Warmia-Mazury

address: Tuwima 10

department: Food Technology

country: Poland

institute: Institute of Animal Reproduction and Food Research, Polish Academy of Sciences

summary: In this study, we analysed the in-vitro modulation of the transcriptome of human PBMCs isolated from 14 individuals after 24 hours of stimunation with 10 nM 1α,25-dihydroxyvitamin D3 (1,25D)

overall_design: PBMCs of 14 healthy donors were isolated within one hour after peripheral blood collection and treated for 24 h either with 1,25(OH)2D3 (10 nM) or solvent (0.1% EtOH).

first: Ranjini

middle: G

last: Dastidar


title: 7q11.23 CNV alters ribosomal biogenesis, mTOR and neuronal intrinsic excitability [ribosome footprinting]

city: Milano

first: Alessandro

last: Vitriolo

state: MI

address: Via Rita Levi Montalcini 1

department: Neurogenomics

country: Italy

institute: Fondazione Human Technopole

summary: Copy number variations (CNVs) at 7q11.23 cause Williams-Beuren (WBS) and 7q microduplication syndromes (7Dup). Both neurodevelopmental disorders (NDDs) feature mild to moderate intellectual disability, accompanied by symmetrically opposite neurocognitive and behavioral features. Although significant progress has been made in understanding the molecular mechanisms underlying 7q11.23-related pathophysiologies, the interplay between different layers of gene expression and the propagation of opposite symmetry arising from CNV remains elusive.  Here, we uncovered 7q11.23 dosage-dependent symmetrically opposite dynamics in neuronal differentiation and intrinsic excitability. By integrating transcriptomics, translatomics and proteomics of patient-derived and isogenic induced neurons, we found that genes related to neuronal transmission follow 7q11.23 dosage and are transcriptionally controlled, while translational factors and ribosomal genes are post-transcriptionally buffered. Consistently, we found a deregulated mTOR pathway, with phospho-S6 (pS6) being downregulated in WBS and upregulated in 7Dup. Surprisingly phospho-4E-BP (p4E-BP) was going in the opposite direction, reflecting the total 4E-BP levels, thus highlighting the different roles of pS6 and p4E-BP during neurogenesis. Therefore, our work emphasizes the importance of multilayer research when investigating complex NDDs, highlighting ribosomal biogenesis and paving the way for novel treatments by uncoupling the role of pS6 and p4E-BPs in NDDs.

overall_design: Using a 7q11.23 duplication lines, isogenic lines with 2 (CTL) and 1 (WBS) copies of the 7q11.23 were generated via CRISPR. All three lines were differentiated to iNeurons in triplicates, before ribosome footprinting.

first: Marija

last: Mihailovic

first: Pierre-Luc

last: Germain

first: Reinald

last: Shyti

first: Giuseppe

last: d’Agostino

last: Testa


title: 7q11.23 CNV alters ribosomal biogenesis, mTOR and neuronal intrinsic excitability

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

overall_design: Refer to individual Series


title: 7q11.23 CNV alters ribosomal biogenesis, mTOR and neuronal intrinsic excitability [RNA-seq]

overall_design: We differentiated isogenic and patient-derived lines to induced Neurons (iNeurons) using Ngn2 overexpression, and profiled their transcriptome. In addition, we tested the effect of REST inhibition in WBS isogenic lines.


title: Organic phosphate but not inorganic phosphate regulates Fgf23 expression through MAPK and TGF-ꞵ signaling

status: Public on Mar 18 2024

city: Rotterdam

first: Bram

middle: CJ

last: van der Eerden

address: Wytemaweg 80

department: Internal medicine

country: Netherlands

institute: Erasmus University MC

summary: One of the main regulators of phosphate homeostasis is fibroblast growth factor 23 (FGF23), secreted by osteocytes. The effects of organic versus inorganic dietary phosphate on this homeostasis is unclear. This study used MC3T3-E1 osteocyte-like cells to examine the transcriptomic responses to these phosphates. Most importantly, the expression and secretion of FGF23 was only increased in response to organic phosphate. Gene ontology terms related to a response to environmental change were only enriched in osteocytes treated with organic phosphate while osteocytes treated with inorganic phosphate were enriched for terms associated with regulation of cellular phosphate metabolism. Inhibition of MAPK signaling diminished the response of Fgf23 to organic phosphate, suggesting it activates FGF23. TGF-β signaling inhibition increased Fgf23 expression after the addition of organic phosphate, while the negative TGF-β regulator Skil decreased this response. In summary, the observed differential response of osteocytes to phosphate types may have consequences for phosphate homeostasis.

overall_design: RNA sequencing was performed on MC3T3-E1 (day 28) after 16 hour treament with 4 mM BGP or 4 mM NaHPO4

first: Danielle

last: Ratsma

last: Van der Eerden


title: Genome-wide Transcription Response of Staphylococcus epidermidis to Heat Shock and Medically-Relevant Glucose Levels

status: Public on Mar 19 2024

city: Stanford

first: Drew

last: Endy

address: 443 Via Ortega, Shriram Center, Room 248

department: Bioengineering

institute: Stanford University

summary: Skin serves as both barrier and interface between body and environment. Skin microbes are intermediaries evolved to respond, transduce, or act in response to changing environmental or physiological conditions. Here, we quantify genome-wide changes in gene expression levels for one abundant skin commensal, Staphylococcus epidermidis, in response to an internal physiological signal, glucose levels, and an external environmental signal, temperature. We find 85 of 2354 genes change up to ~34-fold in response to medically-relevant changes in glucose concentration (0 mM to 17 mM; adj P value ≤ 0.05). We observed carbon catabolite repression in response to a range of glucose spikes, as well as upregulation of genes involved in glucose utilization in response to persistent glucose. We observed 366 differentially expressed genes in response to a physiologically-relevant change in temperature (37°C to 45°C; adj P value ≤ 0.05) and an S. epidermidis heat-shock response that mostly resembles the heat-shock response of related staphylococcal species. DNA motif analysis also revealed CtsR and CIRCE operator sequences arranged in tandem upstream of dnaK and groESL operons. We further identified 38 glucose-responsive genes as candidate ON or OFF genes for use in controlling synthetic genetic systems. Such systems might be used to instrument the in-situ skin microbiome or help control microbes bioengineered to serve as embedded diagnostics, monitoring, or treatment platforms.

overall_design:
To investigate the Staphylococcus epidermidis heat-shock response, we shifted mid-exponential phase ATCC 12228 cells from 37°C to 45°C (Sample HS). We used RNA sequencing to analyze gene expression profiles and then compared the expression profiles of heat-shocked cells to those of unstressed cells. There were three biological replicates for this experimental condition.
To investigate the Staphylococcus epidermidis response to persistent glucose, we cultured ATCC 12228 cells overnight in medium supplemented with 0.2% glucose (Sample A) or 1% glucose (Sample C). There were two biological replicates for each experimental condition. We then performed gene expression profiling analysis using data from RNA-Seq.
To investigate the Staphylococcus epidermidis response to medically-relevant glucose concentrations, we subjected ATCC 12228 cells to a range of 20-minute glucose spikes. Each sample (G2, G5, G10, G17, G50) represents a different glucose concentration (2 mM, 5 mM, 10 mM, 17 mM, and 50 mM respectively). G0 (0 mM) served as the control condition. There were three biological replicates for each experimental condition. We then performed gene expression profiling analysis using data from RNA-Seq.
To investigate the Staphylococcus epidermidis response to a step down in glucose concentration from 10 mM to 2 mM, we subjected ATCC 12228 cells to a 10 mM glucose spike followed by a 2 mM glucose spike (sample SD). G10 (10 mM) served as the control condition. There were three biological replicates for this experimental condition. We then performed gene expression profiling analysis using data from RNA-Seq.

first: Kaisha

last: Benjamin

first: Aditi

last: Goyal

first: Ramesh

last: Nair


title: Human promoter directionality is determined by transcriptional initiation and the opposing activities of INTS11 and CDK9 (RBBP6_POINT-Seq)

city: Exeter

first: Steven

last: West

state: Devon

address: Stocker Rd

department: Living Systems Institute

institute: University of Exeter

summary: RNA polymerase II (RNAPII) transcription initiates bidirectionally at many human protein-coding genes. Sense transcription usually dominates and leads to messenger RNA production, whereas antisense transcription rapidly terminates. The basis for this directionality is not fully understood. Here, we show that sense transcriptional initiation is more efficient and focused than in the antisense direction.  After transcription begins, directionality is maintained by the opposing functions of Integrator (INTS11) and cyclin-dependent kinase 9 (CDK9).  INTS11 terminates antisense transcription, whereas sense transcription is protected from this attenuation by CDK9.  However, INTS11 terminates transcription in both directions upon CDK9 inhibition, and the engineered recruitment of CDK9 abrogates attenuation by INTS11. Therefore, transcriptional initiation and the asymmetric activities of CDK9 and INTS11 explain the attenuation of antisense transcription, the more extensive nature of sense transcription, and promoter directionality.

overall_design: Comparative gene expression and meta analysis of chromatin RNA by polymerase intact nascent transcript (POINT-Seq) for HCT116 RBBP6-dTAG degron (with or without KD)

first: Joshua

last: Eaton

first: Jessica

last: Board

first: Lee

last: Davidson

first: Chris

last: Estell


title: Human promoter directionality is determined by transcriptional initiation and the opposing activities of INTS11 and CDK9 (INTS11-dTAG_ChIP-seq)

overall_design: Chromatin immunoprecipitation DNA-seq (ChIP) for RNA Polymerase (Pol) II occupancy in HCT116 INTS11-dTAG degron cells (with or without KD)


title: Human promoter directionality is determined by transcriptional initiation and the opposing activities of INTS11 and CDK9 (TS11dTAG_NVP2_POINT-seq)

overall_design: Comparative gene expression and meta analysis of chromatin RNA by polymerase intact nascent transcript (POINT-Seq) for HCT116 INTS-dTAG degron (with or without dTAG and with or without NVP2)


title: Human promoter directionality is determined by transcriptional initiation and the opposing activities of INTS11 and CDK9 (INTS11dTAG_POINT-seq)

overall_design: Comparative gene expression and meta analysis of chromatin RNA by polymerase intact nascent transcript (POINT-Seq) for HCT116 INTS-dTAG degron (with or without dTAG)


title: Human promoter directionality is determined by transcriptional initiation and the opposing activities of INTS11 and CDK9 (INTS11dTAG_sPOINT-seq)

overall_design: Comparative gene expression and meta analysis of chromatin RNA by short-read polymerase intact nascent transcript (POINT-Seq) for HCT116 INTS11-dTAG degron (with or without dTAG and with or without TEXO treatment)


title: Human promoter directionality is determined by transcriptional initiation and the opposing activities of INTS11 and CDK9


title: A network-based framework to discover treatment-response-predicting biomarkers for complex diseases

status: Public on Mar 13 2024

city: Waltham

first: Susan

middle: Dina

last: Ghiassian

address: 500 Totten Pond Rd, 6th Floor

institute: Scipher Medicine

summary: Precision medicine's potential to transform complex autoimmune-disease treatment is often challenged by limited data availability and inadequate sample size when compared to the number of molecular features found in high-throughput multi-omics datasets. Addressing this issue, the novel framework PRoBeNet (Predictive Response Biomarkers using Network medicine) was developed. ProBeNet operates under the hypothesis that the therapeutic effect of a drug propagates through a protein–protein interaction network to reverse disease states. ProBeNet prioritizes biomarkers by considering (1) therapy-targeted proteins, (2) disease-specific molecular signatures, and (3) an underlying network of interactions among cellular components (the human interactome). With ProBeNet, biomarkers were discovered predicting patient responses to both an established autoimmune therapy (infliximab) and an investigational compound (a MAPK3/1 inhibitor). Predictive power of ProBeNet biomarkers was validated with retrospective gene-expression data from ulcerative-colitis and rheumatoid-arthritis patients and prospective data from ulcerative-colitis and Crohn’s disease patient-derived tissues. Machine-learning models using ProBeNet biomarkers significantly outperformed models using either all genes or randomly selected genes, especially when data were limited (fewer than 20 samples). These results illustrate the value of ProBeNet for reducing features and for constructing robust machine-learning models when limited data are available. ProBeNet may be used to develop companion and complementary diagnostic assays for complex autoimmune-disease therapies, which may help stratify suitable patient subgroups in clinical trials, approve new drugs, and improve patient outcomes.

overall_design:
To investigate the power of treatment-response-predicting biomarkers identified by the ProBeNet framework, we established ex vivo biopsy-tissue cultures from colon or ileum tissue sections of patients with either ulcerative colitis or Crohn’s disease.
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Authors state that raw data is not availabe due to "privacy concerns".

first: Ghiassian

last: Dina

first: Shanthamallu

last: Uday


title: O-GlcNAc transferase acts as a critical nutritional node for the control of liver homeostasis

status: Public on Mar 12 2024

city: Paris

first: Fadila

last: Benhamed

address: 24 rue du Faubourg St Jacques

country: France

institute: Institut Cochin

summary: O-GlcNAcylation is a reversible post-translational modification controlled by the activity of two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). In the liver, O-GlcNAcylation has emerged as an important regulatory mechanism underlying normal liver physiology and metabolic disease.To address whether OGT acts as a critical hepatic nutritional node, mice with a constitutive hepatocyte-specific deletion of OGT (OGTLKO) were generated.Analyses of 4-week-old OGTLKO mice revealed significant oxidative and endoplasmic reticulum stress, and DNA damage, together with inflammation and fibrosis, in the liver. Susceptibility to oxidative and endoplasmic reticulum stress- induced apoptosis was also elevated in OGTLKO hepatocytes. Although OGT expression was partially recovered in the liver of 8- week-old OGTLKO mice, hepatic injury and fibrosis were not rescued but rather worsened with time. Interestingly, weaning of OGTLKO mice on a ketogenic diet (low carbohydrate, high fat) fully prevented the hepatic alterations induced by OGT deletion, indicating that reduced carbohydrate intake protects an OGT-deficient liver. These findings pinpoint OGT as a key mediator of hepatocyte homeostasis and survival upon carbohydrate intake and validate OGTLKO mice as a valuable model for assessing therapeutical approaches of advanced liver fibrosis.

overall_design: We used liver from 5 mice OGTLWT or OGTLKO at 4 or 8 weeks of age

first: Paula

last: Ortega-Prieto

first: Lucia

last: Parlati

first: Marion

last: Régnier

first: Isadora

last: Cavalcante

first: Mélanie

last: Montabord

first: Rachel

last: Onifarasoaniaina

first: Maryline

last: Favier

first: Natasa

last: Pavlovic

first: Julie

last: Magusto

first: Michèle

last: Cauzac

first: Patrick

last: Pagesy

first: Jérémie

last: Gautheron

first: Chantal

last: Desdouets

first: Sandra

last: Guilmeau

first: Tarik

last: Issad

first: Catherine

last: Postic


title: Coat colour of brushtail possum

status: Public on Mar 10 2024

city: Dunedin

first: Donna

last: Bond

address: 270 Great King Street

department: Department of Anatomy

country: New Zealand

institute: University of Otago

summary: The fur of the common brushtail possum (Trichosurus vulpecula), a model marsupial, is lightweight, soft and has superior insulation properties. Two predominate fur colours exisit: grey or black. RNA-sequencing of skin from grey (n=3) and black (n=3) possums was performed to investigate gene expression changes between these colour morphs.

overall_design: Gene expression analysis of RNA-sequencing data of skin from grey and black fur common brushtail possums (Trichosurus vulpecula)

first: Andrew

last: Veale

first: Alana

last: Alexander

first: Timothy

last: Hore


title: Anti-tumor activity of a novelLAIR1antagonist in combination with anti-PD-1to treat collagen-rich solid tumors (mouse)

status: Public on Mar 04 2024

city: South San Francisco

first: Lee

middle: Benjamin

last: Rivera

address: 333 Oyster Point Blvd.

department: Biology

institute: NGM Bio

summary: We recently reported that resistance to PD-1-blockade in a refractory lung cancer-derived model involved increased collagen deposition and the collagen-binding inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), and thus we hypothesized that LAIR1 and collagen cooperated to suppress therapeutic response. Here, we report LAIR1 is associated with tumor stroma and is highly expressed by intratumoral myeloid cells in both human tumors and mouse models of cancer. Stroma-associated myeloid cells exhibit a suppressive phenotype and correlate with LAIR1 expression in human cancer. NGM438, a novel humanized LAIR1 antagonist monoclonal antibody, elicits myeloid inflammation and allogeneic T cell responses by binding to LAIR1 and blocking collagen engagement. Further, a mouse-reactive NGM438 surrogate antibody sensitized refractory KP mouse lung tumors to anti-PD-1 therapy and resulted in increased intratumoral CD8+ T cell content and inflammatory gene expression. These data place LAIR1 at the intersection of stroma and suppressive myeloid cells and support the notion that blockade of the LAIR1/collagen axis can potentially address resistance to checkpoint inhibitor therapy in the clinic.

overall_design: The murine lung cancer cell line 344SQ was derived from K-rasLA1/+p53R172H∆g/+ mice. 6-8 week old 129/Sv mice were obtained from Charles River Laboratories. For studying the tumor growth in syngeneic immunocompetent models, 344SQ cells (1x106) were implanted subcutaneously into the right flank of male and female mice. When tumors reached an average size of 50-100 mm3 anti-LAIR was dosed 10 mg/kg I.P. twice per week and/or anti-PD-1 (Bioxcell) was dosed 8 mg/kg once per week. Tumor growth was measured with digital calipers once a week, and tumor sizes were calculated using the formula: ½(length × width × width) at indicated time points. At end points, mice were sacrificed to examine the tumor weight and metastatic lung nodules. GEMM (KrasLSL-G12D/p53fl/fl) were generated as previously described (47,49). Mice were infected at 3-4 months of age with 2.5x107 titer pfu adenovirus-cre by intratracheal intubation. Treatment with antibodies were performed at 2 months post-infections when tumors were visualized by micro-CT imaging. All mice were immunocompetent and assessed for health daily by the department of veterinary medicine and surgery at The University of Texas MD Anderson Cancer Center. All animal procedures were reviewed and approved by The University of Texas MD Anderson Cancer Center Animal Care and Use Committee.

first: B

middle: L

last: Rodriguez

first: Jiawei

last: Huang

first: Laura

last: Gibson

first: Jared

last: Fradette

first: Hung-I

last: Chen

first: Kikuye

last: Koyano

first: Czrina

last: Cortez

first: Betty

last: Li

first: Carmence

last: Ho

first: Amir

last: Ashique

first: Vicky

last: Lin

first: Suzanne

last: Crawley

last: Roda

first: Peirong

first: Bin

last: Fan

first: Jeong

last: Kim

first: James

last: Sissons

first: Jonathan

last: Sitrin

first: Daniel

last: Kaplan

first: Don

last: Gibbons


title: Anti-tumor activity of a novelLAIR1antagonist in combination with anti-PD-1to treat collagen-rich solid tumors

first: Lee

last: Rivera

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Anti-tumor activity of a novelLAIR1antagonist in combination with anti-PD-1to treat collagen-rich solid tumors (GeoMx)

overall_design: Freshly cut sections were first confirmed to contain adequate RNA quality using RNAscope LS 2.5 Probe Hs-PPIB (Advanced Cell Diagnostic, 313908). Adjacent tissue sections were then subjected to a whole transcriptome assay using guidelines from the manufacturer (GeoMx Human Whole Transcriptome Atlas; manual reference MAN-10115-05). Briefly, slides were deparaffinized and rehydrated in DEPC-treated water before being subjected to antigen retrieval using Tris-EDTA pH 9. Antigen retrieval was performed for 15 minutes at 99oC in a steamer using prewarmed buffer, and slides were then washed in PBS at room temperature for 5 minutes. Slides were then transferred to a 1 ug/mL proteinase K in PBS solution prewarmed to 37oC and incubated for 15 minutes, then washed in PBS for 5 minutes, and finally postfixed in 10% neutral buffered formalin (NBF) for 5 minutes. Slides were then washed twice in NBF stop buffer, and once in PBS. Slides were wiped and 200 µuL hybridization solution was then applied. Hybridization was allowed to proceed overnight at 37oC for 16 hours. After hybridization, slides were subjected to 2 stringent washes at 25 minutes each at 37oC, then washed twice in 2X SSC buffer. Cells were then blocked with Buffer W at room temperature for 30 minutes in the dark, then subjected to staining with the morphology panel for 1 hour in the dark at room temperature. The morphology panel consisted of Syto-13 (ThermoFisher, S7575), anti-smooth muscle actin A488 clone 1A4, anti-pan cytokeratin A594 clone AE-1/AE-3, and anti-CD68 A647 clone KP1. Slides were then washed twice in 2X SSC and immediately transferred to the GeoMx instrument for stromal and tumor nest ROI selection. CD68+ cells were masked within these ROIs, and 8 um-wide contour-based TME masks spaced 2 µum away from the edge of the CD68 masks were then applied using ImageJ (Fig S2A). Probes from all masks were collected and subjected to library preparation and subsequent sequencing using an Illumina HiSeq 4000. FASTQ files were then processed into DCC files using GeoMx NGS pipeline implemented at NGM.

first: B

last: Rodriguez

first: Jiawei

last: Huang

first: Laura

last: Gibson

first: Jared

last: Fradette

first: Julie


title: EPA Transcriptomic Assessment Product (ETAP) for Perfluoro-3-Methoxypropanoic Acid

status: Public on Mar 05 2024

city: RTP

first: Logan

last: Everett

state: North Carolina

address: 109 T.W. Alexander Dr

department: Office of Research and Development

institute: U.S. Environmental Protection Agency

summary: The EPA Transcriptomic Assessment Product (ETAP) is a human health assessment that utilizes a standardized short-term in vivo study design and data analysis procedures to develop transcriptomic-based reference values for data poor chemicals. The reference value represents the daily dose of the chemical that would likely be without appreciable risk to human health over a lifetime. This dataset corresponds to the transcriptomic studies on perfluoro-3-methoxypropanoic acid (DTXSID70191136). More information about ETAP is available at: https://www.epa.gov/etap

overall_design: A 5-day repeated dose exposure study for perfluoro-3-methoxypropanoic acid was performed in both male and female Sprague Dawley rats separated into 9 dose groups plus vehicle control animals. The vehicle was deionized water and route of exposure was oral gavage (5 ml/kg). Tissues were removed and preserved 24 hours following the last dose.  Transcriptomic measurements were performed using targeted RNA sequencing in kidney, liver, adrenal gland, brain, heart, lung, ovary (females), spleen, testis (males), thyroid, thymus, and uterus (females). More details on the study design are available at: https://doi.org/10.23645/epacomptox.25352962

first: Scott

last: Auerbach

first: Amanda

last: Brennan

first: Warren

last: Casey

first: Dan

last: Chang

first: John

last: Cowden

first: Sarah

last: Davidson-Fritz

first: Jeffry

last: Dean

first: Mike

last: DeVito

first: Jermaine

last: Ford

first: Alison

last: Harrill

first: Susan

last: Hester

first: Michael

last: Hughes

first: Jason

last: Lambert

first: Lucina

last: Lizarraga

first: Roman

last: Mezencev

first: Denise

last: Macmillan

first: Grace

last: Patlewicz

first: Aero

last: Reyner

first: Kristina

last: Thayer

first: Russell

last: Thomas

first: Kelsey

last: Vitense

first: Leah

last: Wehmas


title: Mapping the tumor microenvironment response to bone mineral content

city: Ithaca

first: Iwijn

last: De Vlaminck

address: 237 Tower Rd

department: Meinig School of Biomedical Engineering

institute: Cornell University

summary: Decreased mineral density is a risk factor for skeletal pathologies including bone metastasis, the leading cause of mortality in patients with advanced breast cancer, but the underlying mechanisms are poorly understood. While reduced mineral density can drive pathological bone remodeling via direct effects on select cell types, indirect effects due to broad changes of the microenvironment may be similarly important. However, how bone mineral content affects microenvironmental heterogeneity remains to be elucidated. Here, we leverage decellularized bone matrices with varied mineral content in combination with single-cell RNA-sequencing to study how reduced bone mineral content affects microenvironmental complexity and tumor growth. We performed single-cell RNA sequencing on implanted decellularized bovine bone scaffolds in which the mineral was either maintained at physiological levels or removed to simulate scenarios of impaired bone mineralization as, for example, present during aging. Using this approach, we explored the heterogeneous stromal response to varied bone mineral content in both an immunocompromised and immunocompetent, syngeneic mouse model in the presence and absence of cancer cells.

overall_design: Single-cell RNA sequencing of metastatic breast cancer cell microenvironment on decellularized bovine bone scaffolds with varied mineral levels in immunocompromised and immunocompetent mouse models

first: Matthew

last: Whitman

first: Madhav

last: Mantri

first: Emmanuel

last: Spanos

first: Claudia

last: Fischbach


title: Archival chromatin sequencing in formalin-fixed mouse specimens

status: Public on Mar 01 2024

city: Canberra

first: Erin

middle: Elizabeth

last: Hahn

state: ACT

address: Clunies Ross

department: National Research Collections Australia

institute: CSIRO

summary: Formalin induces inter- and intra-molecular crosslinks within exposed cells. This cross-linking can be exploited to characterise chromatin state as in the FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) and MNase (micrococcal nuclease) assays. Here, we optimised the FAIRE and MNase assays for application upon heavily-fixed tissues as is typically found in historical formalin-preserved museum specimens. We demonstrate these assays in formalin-fixed mouse specimens and compare the chromatin signatures to specimen-matched fresh tissues. We found that heavy formalin fixation modulates rather than eliminates signatures of differential chromatin accessibility and that these chromatin profiles are reproducible, tissue-specific and sex-specific in vertebrate specimens.

overall_design: RNA-Seq of Mus musculus liver tissue in two genetic backgrounds (C57BL/6 and wild caught).

middle: E

first: Jiri

last: Stiller

first: Marina

last: Alexander

first: Alicia

last: Grealy

first: Jennifer

last: Taylor

first: Nicola

last: Jackson

first: Celine

middle: H

last: Frere

first: Clare

last: Holleley


title: Single-cell analyses of metastatic bone marrow in human neuroblastoma reveals microenvironmental remodeling and metastatic signature

status: Public on Mar 03 2024

city: Boston

first: Shenglin

last: Mei

state: MA

address: 10 Shattuck Street

institute: Harvard medical school (DMBI)

summary: To comprehensively elucidate the cellular composition of neuroblastoma bone metastatic and non-metastatic microenvironment.  Bone marrow tissue from neuroblastoma patients were obtained from surgical room.  We used 10× Geno- mics platform to profile single cell RNA sequencing, including 8 metastatic and 7 non-metastatic samples

overall_design:
Human bone marrow tissue were disaggregated and profiled using the Chromium Single Cell 3’ Library and Gel Bead Kit v2 (10X Genomics) .

***Please note that the records have been updated with raw data files (except the 'NB34_Bone' sample) on Aug 5, 2024****


title: The Human Milk Oligosaccharide 3'Sialyllactose Promotes Selective Modulation of Inflammation and Reduces Atherosclerosis Development in Mice

city: La Jolla

first: Nathanael

middle: Judge

last: Spann

address: 9500 Gilman Drive CMMW 219A

department: Cellular and Molecular Medicine

institute: UCSD

summary: Macrophages contribute to the induction and resolution of inflammation and play a central role in the chronic low-grade inflammation in cardiovascular diseases caused by atherosclerosis. Human milk oligosaccharides (HMOs) are complex unconjugated glycans unique to human milk that benefit infant health and act as innate immune modulators. Here, we identify the HMO 3’sialyllactose (3’SL) as a natural inhibitor of TLR4-induced low-grade inflammation in macrophages and endothelium. Transcriptome analysis in macrophages revealed that 3’SL attenuates a selected set of inflammatory gene expression and promotes activity of genomic regions occupied by LXR and SREBP. These acute anti-inflammatory effects of 3’SL were associated with reduced histone H3K27 acetylation at a subset of LPS-inducible enhancers distinguished by preferential enrichment for NFKB, ATF3, IRF2, BCL6, and other transcription factor recognition motifs. In a murine atherosclerosis model, both subcutaneous and oral administration of 3’SL significantly reduced atherosclerosis development and the associated inflammation. This study provides evidence that 3’SL attenuates inflammation by a transcriptional mechanism to reduce atherosclerosis development in the context of cardiovascular disease.

overall_design:
H3K27ac, ATAC-Seq, p300, p65, LXR and SREBP ChIP-seq in BMDMs (bone marow-derived macrophages), from C57BL/6J mice, stimulated with or without the human milk oligosaccharide (HMO) 3’sialyllactose (3’SL) at dose of 100µg/ml in presence of 10ng/ml LPS, or with GW3965 for duration as indicated. Data from BMDMs for both p65 ChIP-Seq and ATAC-Seq, under vehicle and LPS (KLA) treatment conditions, were taken from GSE79423 and GSE109965, respectively.

For p65 and ATAC-Seq re-analysis of data from GSE79423 and GSE109965 respectively: Relevant raw FASTQ files were mapped to mm10 genome with Bowtie2 with default parameters (Langmead and Salzberg, 2012). HOMER was used to convert aligned reads into “tag directories” for further analysis (Heinz et al., 2010). For p65 peaks were called with HOMER findPeaks for each tag directory with default peak finding parameters -style factor against the corresponding input directory. Peak files across various timepoints for vehicle and KLA treated timepoints were merged using HOMER mergePeaks. Merged peaks were annotated for H3K27ac and p300 signal as indicated. For ATAC-Seq, mm10 mapped, IDR defined peaks from GSE109965, for both vehicle and KLA treatment conditions, were used. Peaks were merged using HOMER mergePeaks, then merged peak file was used for further analysis as indicated in text.

Please note that the duplicated sample records of the re-analyzed samples are included for the convenient retrieval of the complete raw data from SRA.

******Please note that the records have been updated on Aug 1st, 2024*****

middle: J

first: Ariane

middle: R

last: Pessentheiner

first: Chloe

last: Autran

first: Joanna

last: Latasiewicz

first: Philip

last: Gordts

first: Lars

last: Bode

first: Christopher

middle: K

last: Glass


title: Archival chromatin sequencing in eastern water dragon museum specimens

summary: Formalin induces inter- and intra-molecular crosslinks within exposed cells. This cross-linking can be exploited to characterise chromatin state as in the MNase (micrococcal nuclease) assays. Our team aims to optimise these assays for application in museum preserved formalin-exposed specimens. To do so, we applied an optimised MNase assay to fresh-frozen and archival eastern water dragon specimens, as old as 1905.  We found that heavy formalin fixation modulates rather than eliminates signatures of differential chromatin accessibility and that these chromatin profiles are sex-specific and environmental condition dependent.

overall_design: MNase treatment of fresh-frozen and archival formalin-fixed eastern water dragon liver tissue.

middle: M


title: The HIF-1α/PLOD2 axis integrates extracellular matrix organization and cell metabolism leading to aberrant musculoskeletal repair.

status: Public on Mar 15 2024

city: Dallas

first: Sneha

last: Korlakunta

state: TX

address: 6000 Harry Hines Blvd

department: Surgery

institute: UT Southwestern Medical Center

summary: While hypoxic signaling has been shown to play a role in many cellular processes, its role in metabolism linked extracellular matrix (ECM) organization and downstream processes of cell fate after musculoskeletal injury remains to be determined. Heterotopic ossification (HO) is a debilitating condition where abnormal bone formation occurs within extra-skeletal tissues. Hypoxia and hypoxia-inducible factor 1α (HIF-1α) activation have been shown to promote HO. However, the underlying molecular mechanisms by which the HIF-1α pathway in mesenchymal progenitor cells (MPCs) contributes to pathologic bone formation remain to be elucidated. Here we used a proven mouse injury-induced HO model to investigate the role of HIF-1α on aberrant cell fate. Using single-cell RNA-sequencing (scRNA-Seq), we found that collagen ECM organization is the most highly up-regulated biological process in MPCs. Zeugopod mesenchymal cell-specific deletion of Hif1α (Hoxa11-CreERT2; Hif1afl/fl) significantly mitigated HO in vivo. ScRNA-Seq analysis of these Hoxa11-CreERT2; Hif1afl/fl mice identified the PLOD2/LOX pathway for collagen cross-linking as downstream of the HIF-1α regulation of HO. Importantly, our scRNA-seq data and mechanistic studies further uncovered that glucose metabolism in MPCs is most highly impacted by HIF-1α deletion.

overall_design:
To investigate the effects of Hoxa11(+) lineage specific deletion of Hif1a on burn/tenotomy injury-induced heterotopic ossification, cells from tenotomy inury site were isolated at 7 days post-injury for scRNA-seq.

Sample A1,B1: 10 week old male C57BL/6 mice were subjected to the burn/tenotomy procedure. Samples were collected 7 days post-injury

first: Heeseog

last: Kang

first: Chase

middle: A

last: Pagani

first: Robert

last: Tower

first: Benjamin

last: Levi


title: Archival chromatin sequencing in formalin-fixed mouse specimens [FAIRE and Mnase]

first: Erin

last: Hahn

overall_design: Mnase and FAIRE treatment of fresh-frozen and formalin-fixed Mus musculus liver tissue in two genetic backgrounds (C57BL/6 and wild caught).

first: Jiri

last: Stiller

first: Marina

last: Alexander

first: Alicia

last: Grealy

first: Jennifer

last: Taylor


title: PRDM3/16 Regulate Chromatin Accessibility Required for NKX2-1 Mediated Alveolar Epithelial Differentiations and Function [Cut&Run]

city: Cincinnati

first: Yan

last: Xu

state: OH

address: 3333 Burnet Ave

institute: Cincinnati Children's Hospital Medical Center

summary: Differential chromatin accessibility accompanies and mediates transcriptional control of diverse cell fates and their differentiation during embryogenesis. While the critical role of NKX2-1 and its transcriptional targets in lung morphogenesis and pulmonary epithelial cell differentiation is increasingly known, mechanisms by which chromatin accessibility alters the epigenetic landscape and how NKX2-1 interacts with other co-activators required for alveolar epithelial cell differentiation and function are not well understood. Here, we demonstrate that the paired domain zinc finger transcriptional regulators PRDM3 and PRDM16 regulate chromatin accessibility to mediate cell differentiation decisions during lung morphogenesis. Combined deletion of Prdm3 and Prdm16 in early lung endoderm caused perinatal lethality due to respiratory failure from loss of AT2 cell function. Prdm3/16 deletion led to the accumulation of partially differentiated AT1 cells and loss of AT2 cells. A combination of single cell RNA-seq, bulk ATAC-seq, and CUT&RUN demonstrated that PRDM3 and PRDM16 enhanced chromatin accessibility at NKX2-1 transcriptional targets in peripheral epithelial cells, with all three factors binding together at a multitude of cell-type specific cis-active DNA elements. Network analysis demonstrated that PRDM3/16 regulated genes critical for perinatal AT2 cell differentiation, surfactant homeostasis, and innate host defense. Lineage specific deletion of PRDM3/16 in AT2 cells led to lineage infidelity, with PRDM3/16 null cells acquiring a partial AT1 fate. Together, these data demonstrate that NKX2-1-dependent regulation of alveolar epithelial cell differentiations is mediated by epigenetic modulation via PRDM3/16.

overall_design:
Lungs were harvested from pooled fetal wild type C57Bl6 mice at E17.5 and the epithelial cells isolated using EpCAM+ microbeads.
We investigated binding regions via Cut&Run with PRDM16, PRDM3, and NKX2-1 antibodies in C57BL6 EpCAM+ sorted lung epithelial cells at E17.5 timepoint

first: Hua

last: He

first: Shelia

last: Bell

first: Ashley

last: Davis

first: Shuyang

last: Zhao

first: Anusha

last: Sridharan

first: Cheng-Lun

first: Minzhe

last: Guo

first: John

last: Snowball

first: William

last: Zacharias

first: Daniel

middle: T

last: Swarr

first: Mineo

last: Kurokawa

first: Susumu

last: Goyama

first: Jeffery

last: Whitsett


title: PRDM3/16 Regulate Chromatin Accessibility Required for NKX2-1 Mediated Alveolar Epithelial Differentiations and Function [ATAC-seq]

summary: Differential chromatin accessibility accompanies and mediates transcriptional control of diverse cell fates and their differentiation during embryogenesis. While the critical role of NKX2-1 and its transcritional targets in lung morphogenesis and pulmonary epithelial cell differentiation is increasingly known, mechanisms by which chromatin accessibility alters the epigenetic landscape to regulate NKX2-1 activites required for alveolar epithelial cell differentiation and function are not well understood. Herein, we demonstrate that the paird domain zinc finger transcriptional regulators PRDM3 and PRDM16 regulate chromatin accessibility required for NKX2-1 mediated AT1 and AT2 cell differentiation decisions during lung morphogenesis and for control of the induction of AT@ cell gene expression controlling alveolar function and ventilation at birth. PRDM3 and 16 were required for activation of AT2 cell-associated gene expression, enhancing chromatin accessibility at NKX2-1 transcriptional targets. NKX2-1, PRDM3, and PRDM16 bound together at cis-active DNA elements in AT2 and AT1 cell selective transcriptional target genes, including those critical for perinatal AT2 cell differentitaion, surfactant homeostasis, and innate host defense. Deletion of PRDM3/16 inhibited NKX2-1-dependent gene regulatory networks controlling surfactant lipid and protein production, resulting in respiratory failure at birth. NKX2-1-dependent regulation of alveolar epithelial cell differentiation is mediated by PRDM3/16.

overall_design:
Floxed alleles of Prdm16 and Prdm3 were knocked out using the sonic hedgehog CRE transgene. Lungs were harvested from fetal mice at E17.5 and EpCAM+ lung epithelial cells were isolated with microbeads.
We investigated differential changes in chromatin accessibility between control mice and those lacking functional Prdm16 and Prdm3 genes in EpCAM+ sorted lung epithelial cells at the E17.5 timepoint.


title: PRDM3/16 Regulate Chromatin Accessibility Required for NKX2-1 Mediated Alveolar Epithelial Differentiations and Function

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

overall_design: Refer to individual Series


title: Single-cell TCR sequencing analysis of the course of Mycobacterium avium complex pulmonary disease

city: Seoul

first: Su-Young

last: Kim

address: 81, Irwon-ro, Gangnam-gu

institute: Samsung Medical Center

summary: We performed scTCR-seq to investigate overall immune cell populations and distribution of MAC-PD patients, according to their clinical outcome.

overall_design: A total of twenty-one patients with newly diagnosed MAC-PD were included and divided into three categories based on clinical course. Group A included patients with spontaneous culture conversion, while group B patients experienced stable disease without antibiotics administration, and patients with progressive disease that required antibiotics administration were categorized as group C.

first: Byung

middle: W

last: Jhun


title: Loss of PPARα function promotes epigenetic dysregulation of lipid homeostasis driving ferroptosis and pyroptosis lipotoxicity in Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD) [MouseMethylation]

city: Wilrijk

first: Claudia

last: Theys

state: Antwerpen (nl)

department: FBD

country: Belgium

institute: Universiteit Antwerpen

type: Methylation profiling by genome tiling array

summary: Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD) is a growing epidemic with an estimated prevalence of 20‑30% in Europe and the most common cause of chronic liver disease worldwide. The onset and progression of MASLD are orchestrated by an interplay of the metabolic environment with genetic and epigenetic factors. Emerging evidence suggests altered DNA methylation pattern as a major determinant of MASLD pathogenesis coinciding with progressive DNA hypermethylation and gene silencing of the liver‑specific nuclear receptor PPARα, a key regulator of lipid metabolism. To investigate how PPARα loss of function contributes to epigenetic dysregulation in MASLD pathology, we studied DNA methylation changes in liver biopsies of WT and hepatocyte‑specific PPARα KO mice, following a 6‑week CDAHFD (choline-deficient, L-amino acid-defined, high-fat diet) or chow diet. Interestingly, genetic loss of PPARα function in hepatocyte‑specific KO mice could be phenocopied by a 6-week CDAHFD diet in WT mice which promotes epigenetic silencing of PPARα function via DNA hypermethylation, similar to MASLD pathology. Remarkably, genetic and lipid diet‑induced loss of PPARα function triggers compensatory activation of multiple lipid sensing transcription factors and epigenetic writer-eraser-reader proteins, which promotes the epigenetic transition from lipid metabolic stress towards ferroptosis and pyroptosis lipid hepatoxicity pathways associated with advanced MASLD. In conclusion, we show that PPARα function is essential to support lipid homeostasis and to suppress the epigenetic progression of ferroptosis‑pyroptosis lipid damage associated pathways towards MASLD fibrosis.

overall_design: Methylation profiles of liver biopsies of hepatocyte-specific PPARα KO C57BL/6J (PPARafl/fl AlbuminCreTg/+) mice and WT C57BL/6J (PPARafl/fl AlbuminCre+/+) mice were determined after a 6 week chow or CDAHFD

first: Anne

last: Scheepers

first: Joe

last: Ibrahim


title: Parallel tracking dosage compensation in the early mouse embryo

first: Chunyao

last: Wei

address: 185 Cambridge St.

department: Molecular Biology

institute: Massachusetts General Hospital

type: Methylation profiling by high throughput sequencing

summary: We develop So-Smart-seq to interrogate a comprehensive transcriptome inclusive of coding, noncoding, and repetitive element transcription. We reveal that, surprisingly, de novo silencing pertains only to a subset of  paternal X (Xp) genes. In contrast, a set of evolutionarily older genes and LINEs demonstrate constitutive Xp silencing, adopt epigenetically distinct signatures, and do not require Xist to initiate silencing. On maternal X (Xm), however, these elements are hyperactivated. Thus, a significant subset of Xp elements appears to be inherited in a “pre-inactive” state. We also find that Xm-hyperactivation is timed to Xp silencing on an individual gene basis, giving rise to heterogeneity in the parallel track of dosage compensation as well. These conclusions have significant implications for the mechanism and evolution of imprinting in early mammals.

sample_organism:
Mus musculus
Mus musculus castaneus

platform_organism:
Mus musculus
Mus musculus castaneus

overall_design: Examining the full transcriptome of single preimplantation embryos at various stages from WT and Xist mutant mouse.

first: Barry

last: Kesner

first: Jeannie

last: Lee


title: Loss of PPARα function promotes epigenetic dysregulation of lipid homeostasis driving ferroptosis and pyroptosis lipotoxicity in Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD) [RNA-seq]

summary: Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD) is a growing epidemic with an estimated prevalence of 20‑30% in Europe and the most common cause of chronic liver disease worldwide. The onset and progression of MASLD are orchestrated by an interplay of the metabolic environment with genetic and epigenetic factors. Emerging evidence suggests altered DNA methylation pattern as a major determinant of MASLD pathogenesis coinciding with progressive DNA hypermethylation and gene silencing of the liver‑specific nuclear receptor PPARα, a key regulator of lipid metabolism. To investigate how PPARα loss of function contributes to epigenetic dysregulation in MASLD pathology, we studied transcriptome profile changes that could induce changes in DNA methylation  in liver biopsies of WT and hepatocyte‑specific PPARα KO mice, following a 6‑week CDAHFD (choline-deficient, L-amino acid-defined, high-fat diet) or chow diet. Interestingly, genetic loss of PPARα function in hepatocyte‑specific KO mice could be phenocopied by a 6-week CDAHFD diet in WT mice which promotes epigenetic silencing of PPARα function via DNA hypermethylation, similar to MASLD pathology. Remarkably, genetic and lipid diet‑induced loss of PPARα function triggers compensatory transcription of multiple lipid sensing transcription factors and epigenetic writer-eraser-reader proteins, which promotes the epigenetic transition from lipid metabolic stress towards ferroptosis and pyroptosis lipid hepatoxicity pathways associated with advanced MASLD. In conclusion, we show that PPARα function is essential to support lipid homeostasis and to suppress the epigenetic progression of ferroptosis‑pyroptosis lipid damage associated pathways towards MASLD fibrosis.

overall_design: mRNA profiles of liver biopsies of hepatocyte-specific PPARα KO C57BL/6J (PPARafl/fl AlbuminCreTg/+) mice and WT C57BL/6J (PPARafl/fl AlbuminCre+/+) mice were determined after a 6 week chow or CDAHFD

first: Ligia

last: Mateiu


title: Single-cell RNA sequencing analysis of the course of Mycobacterium avium complex pulmonary disease

summary: We performed scRNA-seq to investigate overall immune cell populations and distribution of MAC-PD patients, according to their clinical outcome.


title: Anti-tumor activity of a novelLAIR1antagonist in combination with anti-PD-1to treat collagen-rich solid tumors (human)

first: Lee

last: Rivera

overall_design: Dendritic cells were derived from monocytes, which were purified from PBMCs using a human pan monocyte isolation kit (Miltenyi). DCs were generated by culturing monocytes in X-VIVO 15 media (Lonza) containing 50 ng/mL recombinant human GM-CSF (Peprotech) and 50 ng/mL recombinant human IL-4 (Peprotech) for 5-6 days. 96-well tissue culture plates (Corning) were coated with 5 µg/mL human collagen type 1 (Millipore Sigma) in PBS overnight at 4° C, then washed in PBS. 1 x 105 DCs were added to the plates with anti-LAIR1 or an isotype control antibody at the listed concentrations then incubated for 2 days at 37° C. Conditioned media was collected the next day and analyzed using Luminex (ProcartaPlex, ThermoFisher).

first: B

last: Rodriguez

first: Jiawei

last: Huang

first: Laura

last: Gibson

first: Jared

last: Fradette

first: Hung-I

last: Chen

first: Kikuye

last: Koyano

first: Czrina

last: Cortez

first: Betty

last: Li

first: Carmence

last: Ho

first: Amir

last: Ashique

first: Vicky

last: Lin

first: Suzanne

last: Crawley

first: Julie

last: Roda

first: Peirong

first: Bin

last: Fan

first: Jeong

first: James

last: Sissons

first: Jonathan

last: Sitrin

last: Kaplan

first: Don

last: Gibbons


title: transcriptome-wide collateral effects of Cas13d variants

status: Public on Feb 28 2024

city: Shanghai

first: Zilong

last: Geng

address: 800 Dongchuan Rd, Minhang District, Shanghai, China

institute: Shanghai Jiao Tong University

summary: The intrinsic collateral cleavage activity of Cas13d,which refers to the gRNA-independent degradation of bystander RNA, significantly limits its therapeutic potential. To extensively evaluate the collateral effects of hpCas13d genome-wide, we conducted RNA-seq analysis of the transcriptome in HEK293T cells after the transfections with dCas13d, wtCas13d, hpCas13d, and hfCas13d . Compared to dCas13d, wtCas13d with a PPIA gRNA (reported to induce the significant collateral cleavage) resulted in significant downregulations of thousands  genes. In sharp contrast, hpCas13d and hfCas13d showed much less off-target genes, respectively. These findings demonstrate that hpCas13d exhibits reduced collateral activity, rendering it suitable for in vivo applications.

overall_design: To assess the transcriptome-wide off-target effect, we sorted 1x10^6 GFP-positive HEK293T cells using the BD FACS Aria III, 48 hours after transfection with Cas13d variants. Total RNA was then extracted and RNA sequencing was performed.

first: Ping

last: Yang

first: Bing

last: Zhang


title: Effects of formalin-fixation on FAIRE-Seq and MNase-Seq signatures in yeast (RNA-Seq)

first: Erin

last: Hahn

summary: Formalin induces inter- and intra-molecular crosslinks within exposed cells. This cross-linking can be exploited to characterise chromatin state as in the FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) and MNase (micrococcal nuclease) assays. Our team aims to optimise these assays for application in museum preserved formalin-exposed specimens. To do so, we first sought to understand the effect of prolonged formalin fixation on the read alignment signatures resulting from FAIRE and MNase treatment. Here we cultured yeast (Saccharomyces cerevisiae) under normal and heat-shock conditions then fixed the cells with formalin for 15 minutes, 1 hour, 6 hours, and 24 hours. We found that heavy formalin fixation modulates rather than eliminates signatures of differential chromatin accessibility and enables semi-quantitative estimates of relative gene expression in this yeast model.

overall_design: FAIRE and MNase treatment of Saccharomyces cerevisiae (strain BJ5464 auxotroph ΔURA3) in a formalin-fixation time series

first: Jiri

last: Stiller

first: Marina

last: Alexander

first: Alicia

last: Grealy

first: Jennifer

last: Taylor

first: Nicola

last: Jackson

first: Celine

last: Frere

first: Clare

last: Holleley