title: RNA-seq analysis of Mettl3fl/fl and LysM-cre, Mettl3fl/fl BMDMs (bone-marrow-derived macrophages) Transcriptomes

city: Hangzhou

first: Jia

last: Xiong

address: 866 Yuhangtang Road

institute: Zhejiang University

summary: To comprehensively learn about the mechanisms of METTL3-mediated pro-tumoral functions, we performed RNA-sequencing to identify differentially expressed genes in bone-marrow-derived macrophages (BMDMs) in Mettl3fl/fl (WT) and LysM-cre, Mettl3fl/fl (KO) mice after co-cultured with MC38 cell line.

overall_design: RNA-seq profiles of  BMDMs in 8 weeks Mettl3fl/fl and LysM-cre, Mettl3fl/fl mice were generated by deep sequencing, with biologic replicates.


title: Expression data from Stx2-injected (i.p.) mouse kidneys

status: Public on Jan 01 2022

city: Yonago

first: Fumiko

last: Obata

state: Tottori

address: 86 Nichicho

department: Bacteriology

country: Japan

institute: Tottori University

summary:
Shiga toxin type 2 (Stx2) from Escherichia coli is thought to be a main factor to casue renal dysfunction in Enterohemorrhagic E. coli (EHEC) infection. The renal dysfunction caused by the proximal tubular defects can be detected in the earlier EHEC infection. However, the precise information of gene expression from proximal tubular epithelial cells has yet to be clarified.
We performed microarray experiments using Stx2-injected mouse kidney and Stx2-treated human renal proximal tubular epithelial cells (RPTEC), and extracted common genes that were differentially expressed.

overall_design:
Stx2-injected mouse kidneys from various time points were used for RNA extraction and hybridization on Affymetrix microarrays.
time course

first: Ryo

last: Ozuru


title: m6A-seq analysis of Mettl3fl/fl (WT) and Mettl3fl/fl,LyzM-cre  (cKO) bone-marrow-derived macrophages (BMDMs) transcriptomes after co-culturing with MC38 cells

country: China

type: Expression profiling by high throughput sequencing

summary: To investigate the role of RNA methyltransferase METTL3-mediated m6A modification in macrophage after co-culturing with MC38 cells, we performed m6A-sequencing to map the m6A modification in bone-marrow-derived macrophages (BMDMs) from Mettl3fl/fl (WT) and Mettl3fl/fl,LyzM-cre  (cKO) mice

overall_design: 4 Samples total.

first: Qingqing

last: Wang


title: Single-cell transcriptomics of adult gd T-cell lineage commitment highlights its TCR-instructive nature II

city: Marseille

first: Delphine

last: Potier

address: 27 boulevard Lei Roure

country: France

institute: CRCM

summary: Single cell RNA-seq study of T lymphocyte differentiation WT and LAT deficient mice in order to deciphy gd T-cell lineage commitment.

overall_design: Single cell RNA-seq study of T lymphocyte differentiation. Two datasets were produced, corresponding to either double negative cells (DN, i.e. CD8– and CD4–) or total thymocytes (TOT) that were both sorted for live cells.

first: Sara

last: Scaramuzzino

first: Robin

last: Ordioni

first: Pierre

last: Grenot

first: Dominique

last: Payet-Bornet

first: Hervé

last: Luche

first: Bernard

last: Malissen


title: RNA-seq analysis of Mettl3fl/fl and LysM-cre, Mettl3fl/fl MDSC Transcriptomes

summary: To comprehensively learn about the mechanisms of METTL3-mediated pro-tumoral functions, we performed RNA-sequencing to identify differentially expressed genes in isolated MDSC in Mettl3fl/fl (WT) and LysM-cre, Mettl3fl/fl (KO) MC38-bearing mice

overall_design: RNA-seq profiles of  MDSCs in 8 weeks Mettl3fl/fl and LysM-cre, Mettl3fl/fl mice were generated by deep sequencing, with biologic replicates.


title: Next-generation sequencing analysis to compare expression pattern of relapse and baseline MDS samples

status: Public on Jan 02 2022

city: Houston

first: Ganiraju

last: Manyam

state: TX

address: 1515 Holcombe Blvd

country: USA

institute: UT MD Anderson Cancer Center

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

overall_design: Refer to individual Series


title: Single-cell transcriptomics of adult gd T-cell lineage commitment highlights its TCR-instructive nature


title: Analysis of alkylation damage formation and base excision repair at yeast transcription factor binding sites

status: Public on Dec 31 2021

city: Albuquerque

first: Peng

last: Mao

state: NM

address: 915 Camino de Salud. NE

institute: The University of New Mexico Comprehensive Cancer Center

summary: DNA base damage arises frequently in all living cells and is an important contributor to mutations and genome instability. The main repair pathway for base damage is base excision repair (BER). How the formation and repair of base lesions are modulated by DNA-binding proteins is poorly understood. Here we used a high-throughput damage mapping method, N-methylpurine-sequencing (NMP-seq), to characterize alkylation damage distribution and BER at yeast transcription factor (TF) binding sites upon the treatment with alkylating agent methyl methanesulfonate (MMS). We found that formation of alkylation damage was mainly suppressed at the binding sites of yeat TFs Abf1 and Reb1, but individual hotspots with elevated damage formation were also observed. Furthermore, our data indicates that repair of alkyhlation damage by BER was significantly inhibited both within the TF core motif and its adajcent DNA. The modulation of damage formation and BER was caused by the TF binding, because lesion formation and repair can be restored by depletion of Abf1 or Reb1 from the nucleus. Finally, we show that repair of UV damage by nucleotide excision repair (NER) was also inhibited at the binding sites of Abf1 and Reb1. A comparision between alkylyation and UV damage repair reveals that NER was inhibited in a broader DNA region relative to BER. Thus, our analyses indicate that TF binding significantly modulates alkylation damage formation and inhibits repair by the BER pathway. The interplay between base damage formation and BER may play an important role in affecting mutation frequency in gene regulatory regions.

overall_design: Yeast cells were exposed to MMS and then repaired in fresh media. Alkylation damage was mapped with NMP-seq to characterize initial damage formation immediately after MMS treatment (i.e., no repair) and remaining damage (i.e., unrepaired) at each repair time point. Analysis of initial damage and remaining damage at yeast TF binding sites reveals the impacts of TF binding on base damage formation and BER.

first: Mingrui

last: Duan

first: John

last: Wyrick


title: A multi-tissue study of immune gene expression profiling highlights the key role of the nasal epithelium in COVID-19 severity

city: Santiago de Compostela

first: Alberto

last: Gómez-Carballa

state: A coruña

address: Choupana sn

department: Pediatrics

country: Spain

institute: GENVIP research group

summary: COVID-19 symptoms range from mild to severe illness; the cause for this differential response to infection remains unknown. Unraveling the immune mechanisms acting at different levels of the colonization process might be key to understand these differences. We carried out a multi-tissue (nasal, buccal and blood) gene expression analysis of immune-related genes from patients affected by different COVID-19 severities, and healthy controls.

overall_design: Blood, nasal epithelium, and saliva samples were sampled from patients and controls at the same timepoints. Blood samples (2.5ml) were collected in PAXgene tubes (BD). RNA was isolated using PAXgene blood miRNA extraction kit (Qiagen) following manufacturer recommendations. Saliva and nasal epithelium samples were collected in Oragene CP-190 kit (DNA Genotek). Patients and healthy controls were self-reported as of South-European ancestry; and cases were classified at the time of sample collection by severity illness: severe (ICU admission), moderate (non-ICU but admitted to hospital) and mild (domiciliary lockdown patients with mild symptoms or asymptomatic). Sample numbers are distributed as follows: (i) whole blood from 41 patients and 13 controls, (ii) nasal epithelium from 38 patients and 11 controls, and (iii) saliva from 41 patients and 12 controls. The complete tissue sample set could be collected for 27 out of the 52 patients. Immunological gene expression patterns of different tissues were evaluated using a SPRINT nCounter system (NanoString Technologies) and the Human Immunology v2 Panel.

first: A


title: Circulating exosomal microRNAs as predictive biomarkers of neoadjuvant chemotherapy response in breast cancer

city: Little Rock

first: Allen

last: Gies

state: AR

address: 4301 W. Markham

department: Biochemistry and Molecular Biology

institute: University of Arkansas for Medical Sciences

type: Non-coding RNA profiling by high throughput sequencing

overall_design: Differential expression analysis was performed to identify the differentially expressed exosomal miRNAs prior to  and post the initial dose of NACT in patients with pCR and non-pCR, and to prioritized miRNAs target genes. The  Cancer Genome Atlas (TCGA) database and other genomic software were used to explore the expression patterns  and survivability of the candidate miRNAs, and their potential targets based on the expression levels and copy  number variation (CNV) data.

first: Valentina

middle: K

last: Todorova

first: Stephanie

middle: D

last: Byrum

middle: J

first: Nathan

middle: S

last: Reyna

first: Cade

last: Haynie

first: Hunter

last: Smith

first: Issam

last: Makhoul


title: ChIP-seq on Zfp961 GFP flox/flox mESCs to verify Zfp961 repression capacity to transcription of ERVKs

city: Guangzhou

first: Bo

last: Yang

state: Guangdong

address: Guangzhou, 510150, Guangdong Province, China.

institute: The Third Affiliated Hospital of Guangzhou Medical University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We performed ChIP-seq assay with anti-KAP1 antibodies on Zfp961-GFPflox/flox ESCs.We found KAP1 signal decreased at PBS-Lys sites in Zfp961 KO cells compared to WT, suggesting Zfp961 recruiting KAP1 to ERV-K regions. We performed H3K27ac ChIP on Zfp961 WT or KO mESCs. We found that H3k27ac modification at PBS-Lys sites was elevated upon Zfp961 deletion, indicating Zfp961 serving as a repressive transcription factor.

overall_design: Basically, Zfp961-GFP floxed mESCs cell lines treated with 4-OHT or etOH as control were used for anti-KAP1 ChIP and anti-H3K27ac ChIP.


title: SARS-CoV-2 infects and replicates in photoreceptor and retinal ganglion cells of human retinal organoids

city: Muenster

first: Hans

middle: R

last: Schöller

state: NRW

address: Roentgenstrasse 20

department: Cell and Developmental Biology

country: Germany

institute: Max Planck Institute for Molecular Biomedicine

summary: Recent data suggests that COVID-19 is a systemic disease affecting multiple organs including the central nervous system. Retinal involvement in COVID-19 has been indicated by several studies, yet many questions remain regarding the ability of SARS-CoV-2 to infect and replicate retinal cells and its effect on the retina. Here we have used human stem cell derived retinal organoids to study retinal infection by SARS-CoV-2. Indeed, SARS-CoV-2 can infect and replicate in retinal organoids, as it is shown to be able to infect different retinal lineages, including retinal ganglion cells and photoreceptors which are the targets of many retinal diseases leading to blindness. SARS-CoV-2 infection of retinal organoids also induces the expression of several inflammatory genes, including Interleukin 33, which is known to be associated with acute COVID-19 disease and with retinal degeneration. Finally, we show that blocking the ACE2 receptor using antibody treatment significantly reduces retinal organoid infection, indicating that SARS-CoV-2 infects retinal cells in an ACE2 dependent manner. These results suggest a direct retinal involvement in COVID-19, and emphasize the need to monitor retinal pathologies as a possible element of “long COVID”.

sample_organism: Severe acute respiratory syndrome coronavirus 2

overall_design: Retinal organoids were infected with SARS-CoV-2 or mock infected as control. Sampling was done at 24 and 96 hours post infection, every condition in triplicate.

first: Yotam

last: Menuchin Lasowski

first: André

last: Schreiber

first: Aaron

last: Lecanda

first: Angeles

middle: E

last: Mecate Zambrano

first: Olympia

last: Pasthaki

first: Thomas

last: Rauen

first: Stephan

last: Ludwig

last: Schöler

first: Linda

last: Brunotte


title: Integration sites sequencing of HIV infection on ZNF417 ZNF587 dKO 293T cells

summary: We infected ZNF417 ZNF587 dKO 293T or control 293T with NL4.3 virus for 48 hours. Then genomic DNA was purified from cell lysis with by phenol chloroform. DNA was sheared into 300 bp-500 bp random fragments using Covaris Adaptive Focused Acoustics (Covaris, Woburn, MA). Sheared DNA was ligated with a linker and PCR with primers targeting HIV LTR and linker. PCR products were performed second PCR with Illumina adaptor primers. Libraries were so constructed and sequenced on Novaseq 6000.

overall_design: ZNF417 and ZNF587 dKO 293T cells were infected with HIV virus for 48 hours and genomic DNA was harvested for integration sites deep sequencing.


title: MadR transcriptional regulation of mycolic acid biosynthesis in mycobacteria

city: Seattle

first: Eliza

last: Peterson

state: WA

address: 401 Terry Ave N

institute: Institute for Systems Biology

summary: Mycobacterium tuberculosis has a complex cell envelope that is remodelled throughout infection to respond and survive the hostile and variable intracellular conditions within the host. Despite the importance of cell wall homeostasis in pathogenicity, little is known about the environmental signals and regulatory networks controlling cell wall biogenesis in mycobacteria. The mycolic acid desaturase regulator (MadR) is a transcriptional repressor responsible for regulation of the essential aerobic desaturases desA1 and desA2 that are differentially regulated throughout infection along with mycolate modification genes and thus, likely involved in mycolic acid remodelling. Here we generated a madR null mutant in M. smegmatis that exhibited traits of an impaired cell wall with increased permeability, susceptibility to rifampicin and cell surface disruption as a consequence of desA1/desA2 dysregulation. Analysis of mycolic acids revealed the presence of a highly desaturated mycolate in the null mutant that exists in relative trace amounts in the wildtype, but increases in abundance upon cell surface disruption as a result of relieved repression on the desA1/desA2 promoters. Transcriptomic profiling confirmed MadR as a cell surface disruption responsive regulator of desA1/desA2 and further implicating it in the control of bespoke β-oxidation pathways and transport evolutionarily diversified subnetworks associated with virulence. In vitro characterisation of MadR using electromobility shift assays and analysis of binding affinities is suggestive of a unique acyl-CoA pool sensing mechanism, whereby MadR is able to bind a range of acyl-CoA but MadR repression of desA1/desA2 promoters is only relieved upon binding of saturated acyl-CoA of chain length C16-C24. We propose this acyl effector ligand mechanism as distinct to other regulators of mycolic acid biosynthesis or fatty acid desaturases and places MadR as the key regulatory checkpoint that coordinates mycolic acid remodelling in response to host derived cell surface perturbation

overall_design: Cultures of Mycobacterium smegmatis mc2155, M. smegmatis ΔmadR and M. smegmaits  ΔmadR_C (complemented strain) were grown in the presence or absence of 0.025% SDS for 90 minutes and harvested for mRNA expression profiling by Illumina sequencing, in triplicate. The sequence reads that passed quality filters were aligned with Bowtie2 and processed using the R package DuffyNGS.

first: Nitin

last: Baliga


title: On the discovery of ADRAM, an experience-dependent long noncoding RNA that drives fear extinction though direct interaction with the chaperone protein 14-3-3

city: St Lucia

first: Qiongyi

last: Zhao

state: Queensland

address: Building 79, QBI, The University of Queensland

department: Queensland Brain Institute

country: Australia

institute: The University of Queensland

summary: Long-noncoding RNA (lncRNA) comprise a new class of genes that have been assigned key roles in development and disease. Many lncRNAs are specifically transcribed in the brain where they regulate the expression of protein-coding genes that underpin neuronal function; however, their role in learning and memory remains largely unexplored. We used RNA Capture-Seq to identify a large population of lncRNAs that are expressed in the infralimbic cortex of adult male mice in response to fear-related learning, with 14.5% of these annotated in the GENCODE database as lncRNAs with no known function. We combined these data with cell-type-specific ATAC-seq on neurons that had been selectively activated by fear-extinction learning, and discovered 434 lncRNAs derived from enhancer regions in the vicinity of protein-coding genes. We found a novel experience-induced lncRNA called ADRAM that acts as both a scaffold and a combinatorial guide to recruit the brain-enriched chaperone protein 14-3-3 to the promoter of the memory-associated immediate early gene Nr4a2. This leads to the expulsion of histone deactylases 3 and 4, and the recruitment of the histone acetyltransferase creb binding protein, which drives learning-induced Nr4a2 expression. Knockdown of ADRAM disrupts this interaction, blocks the expression of Nr4a2, and ultimately impairs the formation of fear-extinction memory. This study expands the lexicon of experience-dependent lncRNA activity in the brain, highlights enhancer-derived RNAs (eRNAs) as key players in the epigenetic regulation of gene expression associated with fear extinction, and suggests eRNAs, such as ADRAM, may constitute viable targets in developing novel treatments for fear-related anxiety disorders.

overall_design: lncRNA capture sequencing and ATAC-seq for RC and EXT mice

first: Xiang

last: Li

first: Wei

last: Wei

first: Timothy

middle: W

last: Bredy


title: Genome-wide R-loop distribution in Drosophila photoreceptor neurons throughout aging

city: West Lafayette

first: Hana

last: Hall

state: Indiana

address: 175 S University St, Biochemistry Builiding, room 320

department: Biochemistry

institute: Purdue University

summary: We report the application of R-loop mapping in a cell-type specific context using photoreceptor neurons at different ages, as measured by days post eclosion (Day 10, Day 30 and Day 50). To validate our approach, we also performed R-loop mapping in 10 day old nuclei that were treated with RNAse H1 for 3 hours prior to application of MapR.

overall_design: Photoreceptor neuron-specific genome-wide profiling of R-loops using MapR

first: Juan

middle: P

last: Jauregui-Lozano


title: Genome-wide H3K27me3 profile in in vitro produced bovine blastocysts

city: Kyoto

first: Shuntaro

last: Ikeda

address: Kitashirakawa Oiwake-cho, Sakyo-ku

department: Graduate School of Agriculture

institute: Kyoto University

summary: We aimed to obtain genome-wide profiles of histone H3 lysine 27 trimethylation (H3K27me3) in bovine blastocysts

overall_design: In vitro produced bovine blastocysts were processed to ChIP-Seq using H3K27me3 antibody. The experiments were duplicated using two biological replicates.

first: Satomi

last: Yamazaki

first: Naojiro

last: Minami


title: Exosome mediated genetic reprogramming of tumor associated macrophages by exoASO-STAT6 leads to potent monotherapy anti-tumor activity

city: Cambridge

first: Kelvin

last: Zhang

state: Massachusetts

address: 35 CambridgePark Drive, Suite 500

institute: Codiak Biosciences

summary: The M2 phenotype is controlled by key transcription factors such as STAT6. We describe an engineered exosome therapeutic candidate delivering an antisense oligonucleotide (ASO) targeting STAT6 (exoASO-STAT6), which preferentially silences STAT6 expression in tumor-associated macrophages (TAMs). We demonstrate that administration of exoASO-STAT6 reprograms TAMs to an M1 phenotype, leading to the induction of nitric oxide synthase 2 (NOS2) and remodeling of the TAMs.

overall_design: Examination of mRNA profile in CT26 tumors after exoASO-STAT6 treatment

first: Su-Chul

last: Jang

first: Sushrut

last: Karmerkar

first: Olga

last: Burenkova

first: Kyriakos

last: Economides

first: Dalia

last: Burzyn

first: Sriram

last: Sathyanarayanan


title: Comparision of LPS/GalN-induced gene expression in Rack1F/F and Rack1F/F;Alb-cre livers.

city: Beijing

first: Jiyan

state: China

address: 27 Taiping Road, East Building, Rm0749

institute: Institute of Basic Medical Sciences

summary: In order to further discover the resistance mechanism of Rack1F/F;Alb-cre mice to LPS/ GalN-induced fulfulant hepatitis, we used genome-wide microarray expression profiling as a discovery platform to identify potential genes associated with resistance to LPS/ GalN-induced in Rack1F/F;Alb-cre mice. Fulminant hepatitis was induced by LPS/GalN in Rack1F/F;Alb-cre mice and Rack1F/F  mice for 0, 1,3 and 6 hours, respectively.

overall_design: LPS/GalN induced gene expression in Rack1F/F;Alb-cre mice and Rack1F/F  mice were measured at 0,1,3,6 hours after exposure to LPS (35ug/kg) and GalN (1000mg/kg).  Two independent experiments were performed at each time point and each used different donors for each experiment.

first: Qingyang

last: Wang


title: Whole-transcriptome analysis in breast cancer with or without lymph node metastasis

city: JINAN

first: XIANGYU

last: GUO

address: WENHUAXI ROAD 44

institute: SHANDONG UNIVERSITY

summary: To identify the mRNAs, long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) involved in the pathogenesis of breast cancer, we performed the whole transcriptome sequencing. The whole transcriptome sequencing was performed with three breast cancer tissues with or without lymph node metastasis in each group, respectively. The results showed that 728 mRNAs, 131 lncRNAs, and 144 circRNAs were differentially expressed in lymph node metastasis group and no lymph node metastasis group (fold change≥2, p<0.05). These data indicate that dysregulation of mRNAs, lncRNAs, and circRNAs may contribute to breast cancer progression.

overall_design: We performed the whole-transcriptome sequencing in six cases of breast cancers, including three cases with lymph node metastasis(M) and three cases without lymph node metastasis(N), and identified the expression profiles of mRNAs, lncRNAs, and circRNAs.

first: X

last: Guo

first: P

last: Gao


title: Expression data from Stx2-treated human renal proximal tubular epithelial cell (RPTEC)

first: Fumiko

last: Obata

summary:
Shiga toxin type 2 (Stx2) from Escherichia coli is thought to be a main factor to casue renal dysfunction in Enterohemorrhagic E. coli (EHEC) infection. The renal dysfunction caused by the proximal tubular defects can be detected in the earlier EHEC infection. However, the precise information of gene expression from proximal tubular epithelial cells has yet to be clarified.
We performed microarray experiments using Stx2-injected mouse kidney and Stx2-treated human renal proximal tubular epithelial cells (RPTEC), and extracted common genes that were differentially expressed.

overall_design:
Stx2-treated RPTEC from various time points were used for RNA extraction and hybridization on Agilent microarrays.
time course
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Authors state Raw data files are not attached because they have been lost.

first: Ryo

last: Ozuru

first: Jun

last: Fujii


title: Zinc finger protein Zfp335 controls thymocyte differentiation and survival through b-selection-dependent and -independent mechanisms [ChIP-seq]

status: Public on Dec 30 2021

city: xi'an

first: 昕

last: 王

address: yanta west road 76

institute: xi'an jiaotong university

summary: T cell development proceeds in a series of developmental stages, which is precisely orchestrated by multiple signaling and molecular networks. Here we found a zinc finger protein Zfp335 intrinsically controls DN to DP transition, as T cell-specific deficiency in Zfp335 leads to a substantial accumulation of DN3 along with reduction of DP, CD4+ and CD8+ thymocytes. This developmental blockade at DN stage results from the impaired intracellular TCRβ expression as well as increased susceptibility to apoptosis in thymocytes. Transcriptomic and ChIP-seq analyses revealed a direct regulation of transcription factors Bcl6 and Rorc by Zfp335. Importantly, enhanced expression of TCRβ and Bcl6/RorγT restores the developmental defect during DN3 to DN4 transition and improves thymocytes survival, respectively. These findings identify a critical role of Zfp335 in controlling T cell development by maintaining intracellular TCR expression-mediated β-selection and independently activating cell survival signaling.

overall_design: Examination of DN3 and DN4 thymocytes from the thymic of WT and Zfp335 deficient mice

first: Baojun

first: Xin


title: Cleavage of histone H2A during embryonic stem cell differentiation destabilizes nucleosomes to counteract gene activation

city: Philadelphia

first: Yemin

last: Lan

state: PA

address: 3400 Civic Center Blvd

institute: University of Pennsylvania


title: Crypt and villus transcriptomic responses in mouse small intestine following oral exposure to hexavalent chromium

city: Austin

first: Grace

last: Chappell

address: 9390 Research Blvd, Bldg 2, Ste. #100

department: Health Sciences

institute: ToxStrategies, Inc.

summary: Transcriptomic analyses were conducted separately on crypt and villus tissue from formalin-fixed paraffin-embedded transverse duodenal sections from the same study in which microarray-based analyses were previously conducted. A total of 28 groups (7 dose groups x 2 timepoints x 2 tissue compartments) were analyzed for differential gene expression, dose-response, and gene set enrichment. Tissue compartment isolation was confirmed by differences in expression of typical markers of crypt and villus compartments. Fewer than 21 genes were altered in the crypt compartment of mice exposed to 0.1-5 ppm Cr(VI) for 7 or 90 days, which increased to hundreds or thousands of genes at ≥20 ppm Cr(VI). Consistent with histological evidence for crypt proliferation, a significant, dose-dependent increase in genes that regulate mitotic cell cycle was prominent in the crypt, while subtle in the villus, when compared to samples from time-matched controls. Minimal transcriptomic evidence of DNA damage response in either the crypts or the villi is consistent with published in vivo genotoxicity data.

overall_design: Examination of whole transcriptome in the villus and the crypt tissue compartments of FFPE duedenal tissue from mice from six dose groups (plus vehicle controls) orally exposed to Cr(VI) for 7 or 90 days.

middle: A

first: Jeff

middle: C

last: Wolf

first: Chad

middle: M

last: Thompson


title: Zinc finger protein Zfp335 controls thymocyte differentiation and survival through b-selection-dependent and -independent mechanisms

overall_design: Examination of DN4 thymocytes from the thymus of WT and Zfp335 deficient mice

last: Zhang

first: Anjun

last: Jiao

first: Yanhong

last: Su


title: Gene expression changes caused by expression of histone 3.3(K9M) in A375 cells

status: Public on Dec 29 2021

city: New York

first: Yariv

last: Houvras

state: NY

address: 1300 York Ave

department: Surgery

institute: Weill Cornell Medical College

summary: We use RNAseq to examine gene expression changes in A375 cells after expression of mScarlet tagged H3.3(wildtype) vs. H3.3(K9M)

overall_design: FACS was used to sort mScarlet positive cells after transfection with constructs driving expression of H3.3 (wild-type) versus H3.3 (K9M)

first: Raul

last: Martinez-McFaline


title: Mitochondrial C5aR1 activity in macrophages controls IL-1β production underlying sterile inflammation

first: Majid

last: Kazemian

address: 201 S. University Street

institute: Purdue university

summary: While serum-circulating complement destroys invading pathogens, intracellularly active complement, termed the ‘complosome’, functions as a vital orchestrator of cell-metabolic events underlying T cell effector responses. Whether intracellular complement is also non-redundant for the activity of myeloid immune cells is currently unknown. Here, we show that monocytes and macrophages constitutively express complement component (C) 5 and can generate autocrine C5a via formation of an intracellular C5 convertase. Further, cholesterol crystal-sensing by macrophages induces C5aR1 signaling on mitochondrial membranes, which shifts ATP production via reverse electron chain flux towards reactive oxygen species (ROS) production and anaerobic glycolysis to favor IL-1β production. Consequently, atherosclerosis-prone mice lacking macrophage-specific C5ar1 had ameliorated cardiovascular disease on a high-cholesterol diet. Conversely, inflammatory gene signatures and IL-1β produced by cells in unstable atherosclerotic plaques of patients were normalized by a specific cell-permeable C5aR1 antagonist. Deficiency of the macrophage cell autonomous C5 system also protected mice from crystal nephropathy mediated by folic acid. These data demonstrate unexpected intracellular formation of a C5 convertase and identify C5aR1 as a direct modulator of mitochondrial function and inflammatory output from myeloid cells. Together, these findings suggest that the complosome is a novel contributor to the biologic processes underlying sterile inflammation and indicate that targeting this system could be beneficial in macrophage-dependent diseases, such as atherosclerosis.

overall_design: Four atherosclerosis patients are recuited in this study. The biopsies containing atherosclerotic plaques of each patient were split into macroscopically equal pieces and treated with non-cell permeable C5aR1 antagonist PMX53 (10 μg/ml), cell permeable C5aR1 antagonist JEP1375 (10 μg/ml) or PBS for 6 hrs.

first: Nathalie

last: Niyonzima

first: Claudia

last: Kemper

first: Bingyu

last: Yan


title: Colon Transcriptome is Modified by a Dietary Pattern/Atorvastatin Interaction in the Ossabaw Pig

city: Boston

first: Alice

middle: H

last: Lichtenstein

state: MA

address: 711 Washington St

department: Jean Mayer USDA Human Nutrition Research Center on Aging

institute: Tufts University

summary: Overall diet quality and statin therapy are important modulators of inflammation and CAD progression, yet their effects on colon is not well understood. Our objective was to examine the effects and interaction of  dietary patterns and statin therapy on colonic mucosa gene expression in the Ossabaw pig.

overall_design: Thirty-two Ossabaw pigs (16 boars, 16 gilts) were randomized using a 2x2 factorial design into four groups: Hearty Healthy diet (HHD); Heart Healthy diet + atorvastatin (HHD+S); Western diet (WD, also noted as Atherigenic [ARST] below); and Western diet + atorvastatin (WD+S). Heart Healthy diet (high in unsaturated fat, unrefined grain, fruits/vegetables [HHD]) or Western diet (high in saturated fat, cholesterol, refined grain [WD]), with or without atorvastatin.

first: Shumao

last: Ye

first: Nirupa

last: Matthan

first: Stefania

last: Lamon-Fava

first: Gloria

last: Solano-Aguilar

first: Jerrold

last: Turner

first: Maura

last: Walker

first: Zhi

last: Chai

first: Sukla

last: Lakshman

first: Celine

last: Chen

first: Harry

last: Dawson

first: Joseph

middle: F

last: Urban Jr


title: Impact of miR-143-3p and miR-145-5p overexpression on human M238P melanoma cells

city: Valbonne - Sophia-Antipolis

first: Kevin

last: Lebrigand

address: 660 route des lucioles

institute: IPMC/CNRS

summary: To identify putative novel specific targets of miR-143-3p and miR-145-5p, we overexpressed these miRNAs in BRAFV600E mutant human M238P melanoma cells using synthetic mimics or a synthetic “negative” control mimic (miR-Neg). RNA samples were harvested 48 hours post-transfection and 2 independent experiments were carried out.

overall_design: 2 independent experiments performed in a one color design, corresponding to 3 conditions (miR-Neg, miR-143-3p and miR-145-5p) for a total of 6 samples.

first: Serena

last: Diazzi

first: Bernard

last: Mari

first: Marin

last: Truchi


title: RNA-Seq analysis (long-reads) of human parental M238P and BRAF inhibitors-resistant M238R melanoma cells

summary: M238P BRAFV600E mutant melanoma cells sensitive to the BRAF inhibitor Vemurafenib (PLX4032) and the corresponding resistant M238R cell line were cultured in standard conditions. RNA samples were then harvested and sequenced with the Nanopore long-reads protocol.

overall_design: 2 samples: M238P versus M238R

first: Diazzi

last: Serena

first: Lebrigand

last: Kevin


title: Blockade of the pro-fibrotic reaction mediated by the miR-143/-145 cluster enhances the responses to targeted therapy in melanoma

summary: Lineage dedifferentiation towards a mesenchymal-like state displaying myofibroblast and fibrotic features is a common mechanism of adaptive and resistance to targeted therapy in melanoma. Here we show that the anti-fibrotic drug Nintedanib is active to normalize the fibrous ECM network, enhance the efficacy of MAPK-targeted therapy and delay tumor relapse in a pre-clinical model of melanoma. Acquisition of this resistant phenotype and its reversion by Nintedanib pointed to miR-143/-145 pro-fibrotic cluster as a driver of this mesenchymal-like phenotype. Upregulation of the miR-143/-145 cluster under BRAFi/MAPKi therapy was observed in melanoma cells in vitro and in vivo and was associated with an invasive/undifferentiated profile. The 2 mature miRNAs generated from this cluster, miR-143-3p and miR-145-5p collaborated to mediate transition towards a drug resistant undifferentiated mesenchymal-like state by targeting Fascin actin-bundling protein 1 (FSCN1), modulating the dynamic crosstalk between the actin cytoskeleton and the ECM through the regulation of focal adhesion dynamics and mechanotransduction pathways. Our study brings insights into a novel miRNA-mediated regulatory network that contributes to non-genetic adaptive drug resistance and provides proof-of-principle that preventing MAPKi-induced pro-fibrotic stromal response is a viable therapeutic opportunity for patients on targeted therapy.

overall_design: Refer to individual Series


city: Princeton

first: Prakriti

last: Paul

state: NJ

address: South Drive

institute: Lewis-Sigler Institute, Princeton University

summary:
Purpose: Understand how the misexpression of key sensory cell determinant POU IV affects cell type specification of various sensory neurons in the peripheral nervous system in Ciona.
Methods: CesA>POU IV (2.5ng/l) + CesA>GFP (5.0ng/l) injected eggs and control eggs were fertilized side by side, and allowed to develop to the late tailbud stage (13.5h after fertilization at 18°C).  For each sample, 120 morphologically normal embryos were used for single cell RNA-seq assays. Dissociation of the embryos and single cell RNA-seq assays by 10x Genomics chromium system were done as described in 9. Horie R. et al., Shared evolutionary origin of vertebrate neural crest and cranial placodes. Nature 560, 228-232 (2018).
Results: Misexpression of POU IV leads to the formation of Synthetic Cell Types that exhibit properties of other sensory neurons, especially those of bipolar tail neurons (BTNs) and a synthetic gene battery that is not found in any wildtype sensory neuron.

overall_design: Single-cell RNA-seq data for control and POU IV-misexpressed Ciona intestinalis embryos at late tailbud (LTB) stage.

first: Horie

last: Ryoko

last: Takeo

first: Chen

last: Kai


title: Dissecting the Epithelial and Immune Evolution during Colorectal Carcinogenesis by Single-Cell RNA Sequencing

city: ChengDu

first: Jinen

last: Song

address: Renmin road

institute: Sichuan university

summary: Adenoma-carcinoma sequence is a well-accepted roadmap for the development of sporadic colorectal carcinoma. However, cellular heterogeneity in aberrant epithelia and the complexity of tumor microenvironment limited the understanding of carcinogenesis. Here we performed a scRNA-seq survey from patient-matched samples, including blood, normal, para-cancer, polyp, and cancer tissues.

overall_design:
scRNA-Seq of the patient-matched blood, normal, para-cancer, polyp, and cancer tissues
The raw data are under evaluation by human genetic resources office. They will be uploaded to GEO once they are approved.

first: Xiaobo

last: Zheng

first: Xiaowei

last: Liu

first: Chune

last: Yu

first: Zongguang

last: Zhou

first: Hubing

last: Shi


title: Liver-specific overexpression of GBP5 induces liver injury and inflammation

city: Harbin

first: Zheng

last: Chen

state: Heilongjiang

address: 92 Xidazhijie  Harbin Institute of Technology

institute: Harbin Institute of Technology

summary:
Purpose: The goal of this study is to compare transcriptome profilings of livers in which GBP5 and βGal were overexpressed, respectively.

Methods: Liver-specific GBP5-overexpressing and βGal–overexpressing mice were generated by tail-vein injection of individual purified adenovirus(6 × 1011 viral particles per mouse), respectively. RNA-seq was performed by using HiSeq X Ten platform in a single test. Paired-end clean reads were aligned to the mouse reference genome (Ensemble_GRCm38.89) with TopHat (version 2.0.12), and the aligned reads were used to quantify mRNA expression by using HTSeq-count (version 0.6.1).

Conclusion: Our study represents the first detailed analysis of liver transcriptomes from GBP5- and βGal-overexpressing livers, generated by RNA-seq technology. Our results show that 633 genes were significantly up-regulated in the GBP5-overexpressing livers, whereas 586 genes were dramatically down-regulated in the GBP5-overexpressing livers. GO analysis showed that some up-regulated genes were related to the immune system process, response to stress, cell cycle, and cell death in GBP5-overexpressing livers.

overall_design: mRNA profiles of βGal- and GBP5-overexpressing livers were generated by deep sequencing, in a single test, using HiSeq X Ten platform.

first: Kaixin

last: Ding

first: Xinzhi

last: Li

first: Xiaomeng

last: Ren


title: Gene expression changes detected in mouse melanomas after conditional genetic inactivation of ezh2 using tyr:Cre-ERT2 on a braf;pten genetic background

first: Yariv

last: Houvras

summary: We use RNAseq to examine gene expression changes in braf;pten mouse melanomas after arising after conditional genetic inactivation of ezh2

overall_design: We compare gene expression in braf;pten melanomas (TBP) versus braf;pten melanomas with ezh2 genetic inactivation (TBPE)

first: Sara

last: Dinapoli


title: Expression of histone 3 wildtype vs. histone 3 (K9M) using MiniCoopR in zebrafish

status: Public on Dec 28 2021

summary: We use RNAseq to examine gene expression changes in H3.3(wildtype) vs. H3.3(K9M) in zebrafish melanomas

overall_design: MiniCoopR used to express transgenes in a BRAF(V600E);p53 zebrafish melanoma model

first: Raul

last: Martinez-McFaline


title: RNA-Seq analysis of human M238P melanoma cells transduced with a lentivirus expressing the miR-143/145 cluster or a control lentivirus

first: Kevin

last: Lebrigand

summary: To assess the transcriptomic response associated with upregulation of the miR-143/-145 cluster, BRAFV600E mutant human melanoma cells M238P cells were transduced for stable expression of miR-143/145 cluster or with a control vector. Forty-eight hours after transduction, cells were treated with 1 μg/mL of puromycin for one week. RNA samples were then harvested. Two independent experiments were carried out.

overall_design: 2 samples in duplicates: control versus miR-143/-145

first: Diazzi

last: Serena

first: Marin

last: Truchi

first: Bernard

last: Mari

first: Lebrigand

last: Kevin


title: Treatment of a metabolic liver disease by in vivo prime editing in mice

city: Zurich

first: Tanja

last: Rothgangl

address: Winterthurerstrasse, 190

department: Pharmacology and Toxicology

country: Switzerland

institute: UZH Zurich

summary: Prime editing is a highly versatile CRISPR-based genome editing technology with the potential to correct the vast majority of genetic defects1. However, correction of a disease phenotype in vivo in somatic tissues has not been achieved yet. Here, we establish proof-of-concept for in vivo prime editing, that resulted in rescue of a metabolic liver disease. We first develop a size-reduced prime editor (PE) lacking the RNaseH domain of the reverse transcriptase (SpCas9-PERnH), and a linker- and NLS-optimized intein-split PE construct (SpCas9-PE p.1153) for delivery by adeno-associated viruses (AAV). Systemic dual AAV-mediated delivery of this variant in neonatal mice enables installation of a transversion mutation at the Dnmt1 locus with 15% efficiency on average. Next, we targeted the disease-causing mutation in the phenylalanine hydroxylase (Pah)enu2 mouse model for phenylketonuria (PKU). Correction rates of 1.5% using the dual AAV approach could be increased to up to 14% by delivery of full-length SpCas9-PE via adenoviral vector 5 (AdV5), leading to full restoration of physiological blood phenylalanine (L-Phe) levels below 120 µmol/L. Our study demonstrates in vivo prime editing in the liver at two independent loci, emphasizing the potential of PEs for future therapeutic applications.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: NGS data from >= 3 biological replicates

first: Desiree

last: Boeck

first: Gerald

last: Schwank


title: The expression profile of PIWI-interacting RNAs regulated by Trypanosoma cruzi during early infection of primary human cardiac myocytes

city: Nashville

first: Pius

middle: N

last: Nde

state: TN

address: 1005 Dr. D.B. Todd Jr. Blvd

department: Microbiology and Immunology

institute: Meharry Medical College

summary: Trypanosoma cruzi dysregulates the gene expression profile of primary human cardiomyocytes (PHCM) during the early phase of infection through a mechanism which remains to be elucidated. The role that small non-coding RNAs (sncRNA) including PIWI-interacting RNA (piRNA) play in regulating gene expression during the early phase of infection is unknown. To understand how T. cruzi dysregulate gene expression in the heart, we challenged PHCM with T. cruzi trypomastigotes and analyzed sncRNA, especially piRNA, by RNA-sequencing. The parasite induced significant differential expression of host piRNAs. An average of 21,595,866 (88.40%) of clean reads mapped to the human reference genome. The parasite induced 217 unique piRNAs that were significantly differentially expressed (q ≥ 0.8). Of these differentially expressed piRNAs, 6 were known and 211 were novel piRNAs. In silico analysis showed that some of the dysregulated known and novel piRNAs could target and potentially regulate the expression of genes reported to play important roles during T. cruzi infection. This is the first report showing that T. cruzi can induce differential expression of piRNAs in PHCM, advancing our knowledge about the involvement of piRNAs in an infectious disease model.

overall_design: piRNA profiles of primary human cardiac myocytes challenged with T. cruzi

first: Kayla

last: Rayford

first: Ayorinde

last: Cooley

first: Ashutosh

last: Arun

first: Girish

last: Rachakonda

first: Yulia

last: Kleschenko

first: Fernando

last: Villalta

first: Siddharth

last: Pratap

first: Maria

last: Lima


title: Profiling the transcriptional and translational response to BRAF targeted therapy in melanoma

status: Public on Dec 25 2021

city: Melbourne

first: Lorey

last: Smith

state: VIC

address: 4/30 Mater St

institute: Peter MacCallum Cancer Centre

summary: We examined the transcriptional and translational response to BRAF targeted therapy in BRAF mutant melanoma by applying RNA-seq to polysome profiling, a method that isolates mRNA bound to actively translating ribosomes. By applying a published data analysis pipeline, anota2seq, we determined differentially expressed genes in total RNA and polysome-bound RNAseq datasets. We performed the analysis on two treatment timepoints, allowing determination of changes in expression that occur across different treatment times. These analyses identified extensive translational reprogramming during the BRAF targeted therapy response.

overall_design: Examination of total mRNA and polysome-bound mRNA in BRAF mutant melanoma cells treated with the BRAF inhibitor vemurafenib (0, 24h, 40h).


title: Expression of histone 3 wildtype vs. histone 3 (K27M) using MiniCoopR in zebrafish

first: Yariv

last: Houvras

summary: We use RNAseq to examine gene expression changes in H3.3(wildtype) vs. H3.3(K27M) in zebrafish melanomas

first: Sara

last: Dinapoli


title: Age-related gene signatures in brain tumors of young and old mice

city: Toon

first: Daisuke

last: Yamashita

address: Shitsukawa

department: Neurosurgery

institute: Ehime University Graduate School of Medicine

summary: The rise in population aging worldwide is causing an unparalleled increase in death from many cancers, including glioblastoma (GBM). Here, we have explored the impact of aging and rejuvenation on GBM tumorigenesis. Compared with old GBM, young GBM displayed elevated neuronal/synaptic signaling via brain-derived neurotrophic factor (BDNF) and SLIT and NTRK like-family member 6 (SLITRK6), promoting favorable survival rates. These effects were attributed to the rise in nicotinamide adenine dinucleotide (NAD+) levels, as brain rejuvenation by parabiosis or administration of nicotinamide mononucleotide (NMN) in mice elicited a younger phenotype with activated neuronal/synaptic signaling and improved outcomes. Our data indicate that age-associated NAD+ loss contributes to the highly aggressive GBM in the elderly. These findings have therapeutic implications in GBM and provide mechanistic insights into the exacerbation of GBM tumorigenesis with age.

overall_design: Mouse GICs was intracranially injected in young and old mouse brain. Tumor tissues were collected when mice had symptoms. GICs were extablished from these tumor tissues.

first: Ichiro

last: Nakano


title: D-galactosamine (GalN) and lipopolysaccharide (LPS) effect on GBP5 deficient liver

first: Zheng

last: Chen

summary:
Purpose: The goal of this study is to compare transcriptome profilings of liver from GBP5 knockout and WT control mice treated with GalN/LPS.
Methods: GBP5 knockout and WT control mice were treated with GalN (800 μg/g body weight) and LPS (100 ng/g body weight) for 6 h to induce liver inflammation and injury. RNA samples were pooled from livers of GBP5 KO and WT mice (n=4 for each group). RNA-seq was performed by using HiSeq X Ten platform. Paired-end clean reads were aligned to the mouse reference genome (Ensembl_GRCm38.89) with TopHat (version 2.0.12), and the aligned reads were used to quantify mRNA expression by using HTSeq-count (version 0.6.1).
Conclusion: Our study represents the first detailed analysis of liver transcriptomes from GBP5 KO and WT mice treated with GalN/LPS, generated by RNA-seq technology. The RNA-seq analysis showed that 405 genes were down-regulated and 33 genes were up-regulated in the liver of GBP5 KO mice. GO analysis showed that the down-regulated genes were primarily related to the immune system process and response to stress. KEGG pathway enrichment analysis showed that phagocytosis and Jak-STAT signaling pathway were significantly decreased in the liver of GBP5 KO mice.

overall_design: RNA samples were pooled from livers of GBP5 KO and WT mice (n=4 for each group). mRNA profiles of GBP5 KO and WT livers treated with GalN/LPS were generated by deep sequencing, in a single test, using HiSeq X Ten platform.

first: Xiaomeng

last: Ren


title: KLF15 Cistromes Reveal a Hepatocyte Pathway Governing Plasma Corticosteroid Transport and Systemic Inflammation

city: Houston

first: Charles

middle: Yang

last: Lin

state: TX

address: 1 Baylor Plaza

department: Molecular and Human Genetics

institute: Baylor College of Medicine

summary: Circulating corticosteroids orchestrate stress adaptation, including inhibition of inflammation.  While pathways governing corticosteroid biosynthesis and intracellular signaling are understood, less is known about mechanisms controlling plasma corticosteroid transport.  Here, we show hepatocyte KLF15 (Kruppel-like factor 15) controls plasma corticosteroid transport and inflammatory responses through direct transcriptional activation of Serpina6, which encodes corticosteroid binding globulin (CBG).  Klf15-deficient mice have profoundly low CBG, reduced plasma corticosteroid binding capacity, and heightened mortality during inflammatory stress. These defects are completely rescued by reconstituting CBG, supporting that KLF15 works primarily through CBG to control plasma corticosterone homeostasis. To understand transcriptional mechanisms, we generated the first KLF15 cistromes using newly engineered Klf153xFLAG mice.  Unexpectedly, liver KLF15 is predominantly promoter-enriched, including Serpina6, where it binds a palindromic GC-rich motif, opens chromatin, and transactivates genes with minimal associated gene repression. Overall, we provide new mechanistic insight into KLF15 function and identify a hepatocyte-intrinsic transcriptional module that potently regulates systemic corticosteroid transport and inflammation.

overall_design: ChIP-seq (n=2) was used to define KLF15 binding map while RNA-seq (control vs KLF15-LKO, n=4) was used for gene expression and ATAC-seq (control vs KLF15-LKO, n=2) for open chromatin profiling.

first: Saptarsi

last: Haldar

middle: Y

first: Zhen

last: Jiang

first: Selma

middle: Z

last: Elsarrag


title: CircRNA expression profiling in human ovarian cancer

city: Nanjing

first: Shanliang

last: Zhong

address: Baiziting 42

institute: The Affiliated Cancer Hospital of Nanjing Medical University

summary: We collected 3 paired ovarian tumor tissues and ovarian normal tissues. Total RNA was extracted from tissues using Trizol reagent. Then total RNA was processed and circRNAs were profiled using RNA-seq technique. Raw read counts were filtered to remove the genes with a zero read count in 3 or more samples, and then normalized with DESeq2 in the statistical program R (version 4.0.1). Differentially expressed genes were screened using following criterions: fold change was larger than 2 and adjusted P value was less than 0.05.

overall_design: The circRNA expression in ovarian tumors was compared to circRNA expression in normal ovarian tissues.

first: Mengmeng

last: Lyu


title: Ischemia-reperfusion injury and immunosuppressants promote polyomavirus replication through common molecular mechanisms

status: Public on Dec 27 2021

city: Guangzhou

first: Xu-Tao

address: 58# Zhongshan Road 2

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

summary:
Background
Immunosuppressants and renal ischemia-reperfusion injury (IRI) are risk factors for BK polyomavirus infection after kidney transplantation. However, the mechanism remains unclear.
Methods
We used a model of mouse polyomavirus (MPyV) infection to investigate the mechanism of IRI and immunosuppressants to promote polyomavirus replication.
Results
After primary infection, MPyV established persistent infection in the kidneys until week 9 and subsequently were significantly increased by IRI or immunosuppressants treatment individually. In IRI group, viral loads peaked on day 3 in the left kidney, which were significantly higher than that in the right kidney and the control group, and then gradually decreased. In immunosuppressants group, viral loads increased on day 3 without significant difference between left and right kidney, which were significantly higher than that in the control group, and then maintained at high levels. Protein-protein interaction network analysis screened complement C3, EGFR, and FN1 as core genes. Pathway enrichment analysis based on the IRI or immunosuppressants related genes selected by WGCNA indicated that NF-?B signaling pathway was the main pathway involved in promoting MPyV replication. We further confirmed our findings using published datasets GSE47199 and GSE75693.
Conclusions
Our study demonstrated that IRI and immunosuppressants promote polyomavirus replication through common molecular mechanisms.

overall_design: The mice were randomly divided into 3 groups as follows (1) ischemia-reperfusion injury (IRI) group (N = 20), (2) immunosuppressant (IS) group (N = 20), and (3) control group (N = 20). On 3, 7, 10, 14, and 21 days post-treatment, four mice were randomly sacrificed at each time point, and blood and kidney specimens were collected. Kidney specimens for MPyV-DNA testing were stored at -20C. Kidney tissues from IRI group (day 3, left kidneys N=4, right kidneys N=4, day 7, left kidneys N=4, right kidneys N=4) and IS group (day 3, left kidneys, N=4) were stored in liquid nitrogen for high-throughput RNA sequencing (RNA-seq).

first: Yang

last: Huang

first: Lei

last: Zhang

first: Gang


title: A comprehensive temporal patterning gene network in Drosophila medulla neuroblasts revealed by single-cell RNA sequencing

city: Urbana

first: Xin

last: Li

state: IL

address: 601 S. Goodwin Avenue, B107 CLSL

department: CDB

institute: University of Illinois Urbana-Champaign

summary: During development, neural stem cells are temporally patterned to sequentially generate a variety of neural types before exiting the cell cycle. Temporal patterning is well-studied in Drosophila, where neural stem cells called neuroblasts sequentially express cascades of Temporal Transcription Factors (TTFs) to control the birth-order dependent neural specification. However, currently known TTFs were mostly identified through candidate antibody screening and may not be complete. In addition, many fundamental questions remain concerning the TTF cascade initiation, progression, and termination. It is also not known why temporal progression only happens in neuroblasts but not in their differentiated progeny.  In this work, we performed single-cell RNA sequencing of Drosophila medulla neuroblasts of all ages to study the temporal patterning process with single-cell resolution. Our scRNA-seq data revealed that sets of genes involved in different biological processes show high to low or low to high gradients as neuroblasts age. We also identified a list of novel TTFs, and experimentally characterized their roles in the temporal progression and neural fate specification. Our study revealed a comprehensive temporal gene network that patterns medulla neuroblasts from start to end. Furthermore, we found that the progression and termination of this temporal cascade also require transcription factors differentially expressed along the differentiation axis (neuroblasts -> -> neurons). Lola proteins function as a speed modulator of temporal progression in neuroblasts; while Nerfin-1, a factor required to suppress de-differentiation in post-mitotic neurons,  acts at the final temporal stage together with the last TTF  of the cascade, to promote the switch to gliogenesis and the cell cycle exit.  Our comprehensive study of the medulla neuroblast temporal cascade illustrated mechanisms that might be conserved in the temporal patterning of neural stem cells.

overall_design: Single-cell RNA sequencing of Drosophila medulla neuroblasts of all ages

first: Hailun

last: Zhu

first: Sihai

last: Zhao

first: Yu

first: Alokananda

last: Ray


title: Cellular trajectory analysis links tissue maturation to cellular specialization in the plant meristem II

status: Public on Dec 24 2021

city: Cambridge

first: Bernhard

last: Blob

address: 47 Bateman Street

country: United Kingdom

institute: Sainsbury Laboratory, Cambridge University

summary: The mechanisms that allow cells in the plant meristem to coordinate tissue-wide maturation gradients with specialized cell networks are critical for indeterminate growth. Here, we combine long-term imaging, single-cell transcriptomics, genetic analysis, and network modeling to reconstruct the differentiation trajectory of the 20 protophloem cells in the Arabidopsis root. We found that lineage bifurcation and cellular specification are mediated near the stem cell niche by PEAR regulators. However, many PEAR downstream effects are repressed by a broad gradient of PLETHORA transcription factors, which directly inhibit PEARs’ own direct target APL until dissipating seven cells from the stem cell niche. Modeling and molecular epistasis suggest a so-called “seesaw” cascade of sharp transitions in which transcription factors regulating consecutive steps mutually inhibit each other’s targets, ending in programmed enucleation. This analysis provides a mechanistic understanding of how morphogen-like maturation gradients interface with cell-type specific transcriptional regulators to stage cellular differentiation

overall_design: RNA seq profiling of mutant and overexpressor lines

first: Jung-ok

last: Heo

first: Pawel

last: Roszak

first: Yrjo

last: Helariutta


title: Fine tuning of epithelial taste bud organoid to promote functional recapitulation of taste reactivity

city: Seoul

first: Han-Sung

last: Jung

address: 50, Yonsei-ro, Seodaemun-gu

department: Dental School

country: South Korea

institute: Yonsei University

summary: Taste stem/progenitor cells from the mouse posterior tongue have been recently used to generate taste bud organoids. However, the inaccessible location of the taste receptor cells is observed in conventional organoids. Here, we established a suspension culture method for fine tuning of taste bud organoid by apicobasal polarity alteration to form the accessible localization of taste receptor cells in organoid. Compared to conventional Matrigel-embedded organoids, suspension-cultured organoids showed comparable differentiation and renewal rates to those of taste buds in vivo and exhibited functional taste receptor cells and cycling progenitor cells. Accessible taste receptor cells on the outer region of taste bud organoids enabled the direct application of calcium imaging for evaluating the taste response. Moreover, suspension-cultured organoids could be genetically altered using gene editing methods. Suspension-cultured taste bud organoid harmoniously integrated with the recipient lingual epithelium; maintained the taste receptor cells and gustatory innervation capacity. Thus, we propose that suspension-cultured organoids may provide efficient model for taste research including taste bud development, regeneration and transplantation

overall_design: Examination of the gene expression between Matrigel-embedded taste organoid and Suspension-cultured taste organoid derived from the mice tongue

first: Anish

last: Adpaikar

first: Sushan

first: Hyun-Yi

last: Kim

first: Ki

first: Seok

last: Moon

first: Jong-Min

last: Lee


title: Cardiac transcriptome analysis of RKIP-transgenic and GRK2-transgenic mice by NGS

first: Ursula

last: Quitterer

address: Winterthurerstrasse 190

department: Molecular Pharmacology

institute: ETH Zurich

summary: The RAF kinase inhibitor protein, RKIP, is a dual inhibitor of the RAF1 kinase and the G-protein-coupled receptor kinase 2 (GRK2). By inhibition of the proto-oncogenic and pro-survival RAF1-MAPK pathway, the RAF kinase inhibitor protein, RKIP, acts as a tumor suppressor, which enhances cardiomyocyte death and promotes the development of symptoms of heart failure. To elucidate pathomechanisms of heart failure induced by RKIP, the study determined the cardiac transcriptomes of  eight-month-old, male, transgenic mice with cardiac-specific expression of RKIP (PEBP1) under control of the myocardium-specific, alpha-MHC promoter. In addition, the study determined the cardiac transcriptomes of GRK2-transgenic mice. Tg-GRK2 mice have a slightly increased transgenic expression of GRK2. According to NGS data, cardiac GRK2-Grk2 transcript levels of Tg-GRK2 mice are 1.59±0.10-fold higher than those of non-transgenic FVB hearts. In Tg-GRK2 mice, transgenic GRK2 is expressed under control of the ubiquitous CMV immediate-early promoter/enhancer. The non-transgenic control group are age-matched, male, nontransgenic FVB/N mice. NGS data of this study document transcriptome changes underlying the heart failure phenotype induced by transgenic RKIP expression and cardiac degeneration induced by GRK2 expression.

overall_design: Cardiac transcriptomes were determined by NGS of hearts isolated from three male RKIP-transgenic (Tg-RKIP) mice and three male GRK2-transgenic (Tg-GRK2) mice at an age of 8 months. Tg-RKIP mice and Tg-GRK2 mice have an FVB/N background. The non-transgenic control group are three, age-matched, non-transgenic, male FVB/N mice. Hearts were isolated from male mice at the end of the observation period at an age of 8 months. The group size was n=3 male mice at an age of 8 months per group. Tg-RKIP mice showed symptoms of heart failure. NGS data of the cardiac transcriptome confirmed the heart heart failure phenotype induced by an increased cardiac RKIP (PEBP1) transcript level. NGS data also showed altered transcript levels of selected heart failure-promoting genes in hearts of Tg-GRK2 mice with slightly (1.59-fold) increased cardiac GRK2 levels. All mice of the study were weaned at an age of 3 to 4 weeks, housed in groups of 2-5 mice in individually ventilated cages under SPF conditions with a 12h dark cycle, and had free access to food and water until the end of the observation period at an age of 8 months.

first: Joshua

last: Abd Alla


title: Crosstalk with astrocytes establishes tumor edge in glioblastoma

first: Daisuke

last: Yamashita

summary: Clinical outcomes for patients with glioblastoma (GBM) are extremely poor due to inevitable tumor recurrence even after extensive treatments. These recurrences are thought to manifest from cells located within the tumor edge. Despite this, the precise molecular mechanism governing GBM spatial phenotypic heterogeneity (e.g. edge vs. core) and subsequent tumor recurrence remains poorly elucidated. Here, using patient-derived GBM core and edge tissues, we analyzed transcriptional and metabolic signatures in an effort to determine how GBM facilitates the edge phenotype and its associated recurrence-initiating cells (RICs). In so doing, we unexpectedly identified CD38 as an essential protein in the formation of the edge phenotype and found a CD38-driven interaction between edge GBM cells and neighboring astrocytes that communally develops a GBM edge that is unresectable by surgery and retains RICs.

overall_design: Human clones were established from GBM core and edge tissues from two patients. For analysis of normal brain tissues, cortical tissues were collected from WT and CD38KO mice. Next, mouse GBM cells were intracranially injected in the brain of WT and CD38KO mice and tumor tissues were collected when mice had symptoms.

first: Ichiro

last: Nakano


title: Cellular trajectory analysis links tissue maturation to cellular specialization in the plant meristem

sample_organism:
Arabidopsis thaliana
blank sample

platform_organism:
Arabidopsis thaliana
blank sample

overall_design: Isolation and RNA seq of developing phloem cells in the root meristem of Arabidopsis thaliana

first: Winnie

last: Lau

first: Fiona

last: Hamey

first: Kenneth

last: Birnbaum

first: Jacopo

last: Cirrone

first: Sebastian

last: Ahnert

first: Berthold

last: Göttgens


title: Specific mesoderm subset derived from human pluripotent stem cells ameliorates microvascular pathology in type 2 diabetic mice

city: Birmingham

first: Maria

middle: B

last: Grant

state: Alabama

address: 1670 University Blvd, VH490

department: Ophthalmology

institute: University of Alabama at Birmingham (UAB)

summary: Human iPSCs were differentiated into a specific mesoderm subset characterized by KDR+CD56+APLNR+ (KNA+) expression. KNA+ cells possessed high clonal proliferative potential and specification into endothelial colony forming cell (ECFC) phenotype. KNA+ cells differentiated into perfused blood vessels when implanted subcutaneously into the flank of NOD/SCID mice and when injected into the vitreous of type 2 diabetic mice (db/db mice). Transcriptomic analysis showed that differentiation of hiPSCs derived from diabetics into KNA+ cells was sufficient to change baseline differences in gene expression caused by the diabetic status and reprogram diabetic cells to a pattern similar to KNA+ cells derived from nondiabetic hiPSCs. Proteomic array studies performed on retinas of db/db mice injected with either control or diabetic donor derived KNA+ cells showed correction of aberrant signaling in db/db retinas towards normal healthy retina. These data provide “proof of principle” that KNA+ cells restore perfusion and correct vascular dysfunction in db/db mice.

overall_design: Transcriptomic assessment of KNA and iPSC cells

first: Mervin

last: Yoder


title: Effect of cyclic tensile stretch on meniscus cells in the presence and absence of IL-1

status: Public on Dec 23 2021

city: Durham

first: Amy

last: McNulty

state: NC

address: 203 Research Dr

department: Orthopaedic Surgery

institute: Duke University

summary: Meniscus injuries are highly prevalent and are linked to the development of post-traumatic osteoarthritis (PTOA). The inflammatory cytokine IL-1 is elevated in synovial fluid following knee injuries, causes degradation of meniscus tissue, and inhibits meniscus repair. Cyclic tensile stretch has been shown to modulate the expression of pro-inflammatory cytokines and degradative enzymes induced by IL-1; however, there remains a dearth of knowledge on global effects of loading on meniscus cell phenotype and transcriptomic profiles. In this study, we performed mRNA-Seq on meniscus cells isolated from inner and outer zone regions of porcine menisci subjected to cyclic tensile strain in the presence and absence of IL-1 to identify cellular responses to mechanical load, identify differences in response to load based on zonal characteristics, and identify IL-1 induced inflammatory responses modulated by load.

overall_design: RNA-Seq of inner and outer zone porcine meniscus cells subjected to 5% cyclic tensile strain at 0.5Hz in the presence or absence of IL-1 (n=3/group)

first: Rebecca

last: Irwin

middle: L

first: Benjamin

last: Andress


title: Stromal HIF2 Regulates Immune Suppression in the Pancreatic Cancer Microenvironment

first: Yanqing

last: Huang

address: 6565 MD Anderson Blvd

department: Experimental Radiation Oncology

institute: MD Anderson Cancer Center

summary:
Background & Aims. Pancreatic ductal adenocarcinoma (PDAC) has a hypoxic, immunosuppressive stroma, which contributes to its resistance to immune checkpoint blockade therapies. The hypoxia-inducible factors (HIFs) mediate the cellular response to hypoxia, but their role within the PDAC tumor microenvironment remains unknown.
Methods. We used a dual recombinase mouse model to delete Hif1a or Hif2a in α-smooth muscle actin (αSMA)-expressing cancer-associated fibroblasts (CAFs) arising within spontaneous pancreatic tumors. The effects of CAF-Hif2a  expression on tumor progression and composition of the tumor microenvironment were evaluated by Kaplan-Meier analysis, quantitative real-time polymerase chain reaction, histology, immunostaining, and by both bulk and single-cell RNA sequencing. CAF-macrophage crosstalk was modeled ex vivo using conditioned media from CAFs after treatment with hypoxia and PT2399, a HIF2 inhibitor currently in clinical trials. Syngeneic flank and orthotopic PDAC models were used to assess whether HIF2 inhibition improves response to immune checkpoint blockade.
Results. CAF-specific deletion of HIF2, but not HIF1, suppressed PDAC tumor progression and growth, and improved survival of mice by 50% (n = 21-23 mice/group, Log-rank P = 0.0009). Deletion of CAF-HIF2 modestly reduced tumor fibrosis and significantly decreased the intratumoral recruitment of immunosuppressive M2 macrophages and regulatory T cells. Treatment with the clinical HIF2 inhibitor PT2399 significantly reduced in vitro macrophage chemotaxis and M2 polarization, and improved tumor responses to immunotherapy in both syngeneic PDAC mouse models.
Conclusions. Together, these data suggest that stromal HIF2 is an essential component of PDAC pathobiology and is a druggable therapeutic target that could relieve tumor microenvironment immunosuppression and enhance immune responses in this disease.

overall_design: Eight samples were analyzed, four replicates for KPF CAF-HIF2 Ctrl and four replicates for KPF CAF-HIF2 KO tumors.

first: Cullen

last: Taniguchi


title: Zinc finger protein 277 is an intestinal transit-amplifying cell marker and colon cancer oncogene

city: Baltimore

first: Guofeng

last: Xie

state: MD

address: 22 South Greene St

institute: University of Maryland Baltimore

summary: To identify the target genes of transcription factor zinc finger protein (ZNF) 277, we performed RNAseq experiments using RNAs from human colon cancer cell line HT29 and its clones with ZNF277 knockout by CRISPR.

overall_design: Differntial transcripts from 3 control and 3 ZNF277 KO cell lines

first: Zhongsheng

last: Peng


title: E-cigarette Aerosol Reduces Left Ventricular Function in Adolescent Mice

city: Columbus

first: Matthew

middle: William

last: Gorr

state: OH

address: 473 W 12th Ave

institute: The Ohio State University

summary: Purpose: to determine the cardiac complications of vaping in adult (3 month old) and juvenile (3 week old) FVB mice. Methods: mice were exposed to filtered air or vape with or without nicotine for 4 hrs/day, 5 days/week for a total of 3 months time. RNA-seq. was conducted on RNA isolated from the left ventricle of mice from each group.

overall_design: mRNA profiles of cardiac tissue from mice exposed to e-cigarette vapor

first: Evan

last: Neczypor

first: Ty

last: Saldana

middle: J

last: Mears

first: David

last: Aslaner

first: Yael

last: Escobar

first: Loren

last: Wold


title: Age-related gene signatures in brain tumors of young and old mice [dataset 2]

first: Daisuke

last: Yamashita

overall_design: Mouse tumor cells were intracranially injected in the striatum of young or old female C57BL/6 mice, either a control group or old mice rejuvenated by parabiosis or by administration of nicotinamide mononucleotide (NMN, 300 mg/kg/day) (total 12 mice; 3 mice/group). Tumor tissues were collected when mice had symptoms.

first: Rachel

last: Munk

first: Myriam

last: Gorospe

first: Ichiro

last: Nakano


title: Immune phenotype of the CD4+ T cells in the aged lymphoid organs and lacrimal glands

first: Cintia

middle: S.

last: de Paiva

address: 6565 Fannin Street NC505G

summary:
Aging is associated with a massive infiltration of T lymphocytes in the lacrimal gland. . Among the non-Tregs, we also found a significant increase in the levels of EOMESmed/high, TbetnegIFN-𝛄+ and CD62L+CD44neg CD4+ T cells with aging, which are associated with cell exhaustion, immunopathology and the generation of tertiary lymphoid tissue. More importantly, human lacrimal glands (age range 55-81 years) also showed presence of CD4+Foxp3+ cells. Further studies are needed to propose potential molecular targets to avoid immune-mediated lacrimal gland dysfunction with aging.
Here we aimed to characterize the immune phenotype of aged CD4+ T cells in this tissue as compared with lymphoid organs. To perform this, we sorted regulatory T cells (Tregs, CD4+CD25+GITR+) and non-Tregs (CD4+CD25negGITRneg) cells and subjected these cells to an immunology NanoStringⓇ panel in lymphoid organs. These results were confirmed by flow cytometry, live imaging and tissue immunostaining in the lacrimal gland. Importantly, effector T helper 1 (Th1) genes were highly upregulated on aged Tregs, including the master regulator Tbx21

overall_design: Cells from eye and lacrimal gland draining lymph nodes were pooled with splenocytes. Cells were stained and sorted based on the expression of CD4+CD25+GITR+ (Tregs, or DP) and compared to CD4+CD25-GITR- (non-Tregs). After sorting, cells were lysed in RNA, total RNA was isolated and NanoString was performed.

first: Claudia

last: Trujillo-Vargas

first: Humberto

last: Hernandez

first: Rodrigo

middle: G

last: de Souza

first: Zhiyuan

last: Yu

first: Jeremías

last: Galletti

first: Jana

last: Dietrich

first: Friedrich

last: Paulsen

middle: S


title: Synthesis of immunogenicity-free RNA circles as potent PKR inhibitors

city: Shanghai

first: Li

last: Yang

address: 131 Dong-An Road

department: Institutes of Biological Sciences

institute: Fudan University

summary: Exon(s) back-splicing-generated circular RNAs as a group can suppress the double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize immunogenicity-free, short dsRNA-containing circular RNAs as potent PKR inhibitors. Here, we report that circular RNAs synthesized by permuted td introns from T4 bacteriophage and by Anabeana pre-tRNA group I intron induce an immune response. Mechanistically, autocatalytic splicing introduces extra ~74 nt and ~186 nt fragments (E1+E2+spacer) that form additional stable loop structures that likely provoke innate immune responses in circular RNA products. In contrast, circular RNAs produced by T4 RNA ligase without unwanted fragment exhibit little immunogenicity. Importantly, directly-ligated circular RNAs form short dsRNA-regions exhibit 103~106 fold higher efficiency than the reported chemical compounds C16 and 2-AP in suppressing PKR activation, highlighting the use of circular RNAs as novel inhibitors for PKR over-reaction diseases.

overall_design: circSHAPE-MaP of in vitro circRNA with/without protein.

first: Chu

middle: X

last: Liu

first: Si

middle: K

last: Guo

first: Fang

first: Yi

last: Xu

first: Ling

last: Chen


title: LKB1 drives stasis and C/EBP-mediated reprogramming to an alveolar type II fate in lung cancer [scRNA-seq]

city: Stanford

first: Christopher

last: Murray

state: California

address: 1291 Welch Rd. B261, Beckman Center

department: Genetics

institute: Stanford University

summary: Background: LKB1 is among the most frequently altered tumor suppressors in lung adenocarcinoma. Inactivation of Lkb1 accelerates the growth and progression of oncogenic KRAS-driven lung tumors in mouse models. However, the molecular mechanisms by which LKB1 constrains lung tumorigenesis and whether the aggressive cancer state that stems from Lkb1 deficiency can be reverted remains unknown.  Purpose: To determine whether the restoration of Lkb1 drives changes in cell state and/or abundance within established oncogenic KRAS-driven lung tumors.  Approach: To control LKB1 function in vivo, we generated an Lkb1XTR allele, which enables Cre-mediated disruption of Lkb1 expression during tumor development and subsequent FLPo-ERT2-mediated reactivation of Lkb1 within established tumors. Lung tumors were initiated in KT;Lkb1XTR/XTR (non-restorable) and KT;Lkb1XTR/XTR;FLPo-ERT2 (restorable) mice with Lenti-Cre. Following tumor development, lung tumor-bearing were treated with either corn oil vehicle or tamoxifen for two weeks prior to isolating total viable cells by FACS for single cell RNA-seq.  Results: Lkb1 restoration did not result in any dramatic changes in the relative abundance of stromal cell types beyond a significant increase in a rare population of putative mast cells. There was a marked shift in the epithelial compartment from an indeterminate state to an alveolar type II epithelial-like identity in response to Lkb1 restoration.  Conclusions: The acute transcriptional response to Lkb1 restoration is largely limited to the neoplastic epithelial compartment.

overall_design: Lung tumors were initiated in KT;Lkb1XTR/XTR (non-restorable), and KT;Lkb1XTR/XTR;FLPo-ERT2 (restorable) mice with Lenti-Cre. Following tumor development and two weeks of treatment with either corn oil vehicle or tamoxifen, total viable cells FACS-isolated from lung tumors and subjected to single cell RNA-seq.

first: Monte

last: Winslow


title: LKB1 drives stasis and C/EBP-mediated reprogramming to an alveolar type II fate in lung cancer [Secondary Tuba-seq]

summary: Background: LKB1 is among the most frequently altered tumor suppressors in lung adenocarcinoma. Inactivation of Lkb1 accelerates the growth and progression of oncogenic KRAS-driven lung tumors in mouse models. However, the molecular mechanisms by which LKB1 constrains lung tumorigenesis and whether the aggressive cancer state that stems from Lkb1 deficiency can be reverted remains unknown.  Purpose: To ascertain the tumor-suppressive capacity C/EBP family members, including Cebpa, Cebpb, and Cebpd, through CRISPR/Cas9-mediated targeting in a genetically engineered mouse model of oncogenic KRAS-driven lung adenocarcinoma.  Approach: Measurements of tumor size across genetic perturbations were obtained by tumor barcode sequencing (Tuba-seq; Rogers et al., 2017 Nature Methods). Briefly, lung tumors were initiated in KrasLSL-G12D/+;R26LSL-tdTomato (KT) and KT;H11LSL-Cas9 mice using a pool of Lenti-sgRNA/Cre vectors that are each modified with two-component barcodes composed of sgRNA-specific and clonal identifiers. This particular pool of Lenti-sgRNA/Cre vectors was composed of vectors targeting each of the three C/ebp paralogs individually, in pairwise combinations, and all three simultaneously. Following tumor development, the two-component lentiviral barcodes integrated within transduced populations were amplified from genomic DNA of whole-lung homogenates. The resulting amplicons were then subjected to next-generation sequencing. From the resulting reads, barcode pileups were generated and filtered prior to conversion to absolute numbers of neoplastic cells via normalization to spiked-in samples of known quantities of barcoded neoplastic cells. The resulting tumor size distributions were then analyzed by multiple statistical measures as described previously (Rogers et al., 2017 Nature Methods).  Results: The targeting of Cebpa significantly increased tumor size relative to tumors initiated with vectors encoding inert sgRNAs (oncogenic KRAS only tumors). The targeting of Cebpb and Cebpd had no significant impact on tumor growth. There were no additive effects of knocking out additional paralogs on top of Cebpa.   Conclusions: C/EBPa is the dominant tumor-suppressive paralog and there was no evidence of functional redundancy among C/EBPa, C/EBPb, and C/EBPd.

overall_design: Lung tumors were initiated in 10 KrasLSL-G12D/+;R26LSL-tdTomato;H11LSL-Cas9 mice using a pool of Lenti-sgRNA/Cre vectors targeting C/EBP family members Cebpa, Cebpb, and Cebpd individually, in pairwise combinations, and all three simultaneously, in addition to five inert sgRNAs (to generate tumors driven by oncogenic KRAS only). Five KrasLSL-G12D/+;R26LSL-tdTomato mice (lacking Cas9) were also transduced with the same lentiviral pool to control for differences in virus titer. Lentiviral vectors were diversified with two-component barcodes composed of sgRNA-specific and clonal identifiers to uniquely tag each tumor and label with a genotype-specific identifier. The size of each clonal outgrowth was determined by deep sequencing of amplicons derived from the lentiviral barcodes integrated within the genomic DNA of tumor-bearing lungs. Comparisons of tumor size relative to populations of tumors initiated with inert sgRNA-encoding vectors were performed across genotypes by stratifying clones according to sgRNA-specific identifiers.


title: Effect of dynamic compression on meniscus tissue explants in the presence and absence of IL-1

first: Amy

last: McNulty

summary: Meniscus injuries are highly prevalent and are linked to the development of post-traumatic osteoarthritis (PTOA). The inflammatory cytokine IL-1 is elevated in synovial fluid following knee injuries, causes degradation of meniscus tissue, and inhibits meniscus repair. Dynamic mechanical compression of meniscus tissue has been shown to improve integrative repair in the presence of IL-1; however, there remains a dearth of knowledge on global effects of loading on meniscus cell phenotype and transcriptomic profiles. In this study, we performed mRNA-Seq on meniscus tissue explants from inner and outer zone regions of porcine menisci subjected to dynamic compression in the presence and absence of IL-1 to identify cellular responses to mechanical load, identify differences in response to load based on zonal characteristics, and identify IL-1 induced inflammatory responses modulated by load.

overall_design: RNA-Seq of inner and outer zone porcine meniscus tissue explants subjected to 10% dynamic compression at 1Hz in the presence or absence of IL-1 (n=3/group)

first: Benjamin

last: Andress


title: LKB1 drives stasis and C/EBP-mediated reprogramming to an alveolar type II fate in lung cancer [Primary Tuba-seq]

summary: Background: LKB1 is among the most frequently altered tumor suppressors in lung adenocarcinoma. Inactivation of Lkb1 accelerates the growth and progression of oncogenic KRAS-driven lung tumors in mouse models. However, the molecular mechanisms by which LKB1 constrains lung tumorigenesis and whether the aggressive cancer state that stems from Lkb1 deficiency can be reverted remains unknown.  Purpose: To assess the impact of CRISPR/Cas9-mediated targeting of a subset of LKB1-dependent genes and C/EBP factors on tumor size in the context of a genetically engineered mouse model of oncogenic KRAS-driven lung adenocarcinoma.  Approach: Measurements of tumor size across genetic perturbations were obtained by tumor barcode sequencing (Tuba-seq; Rogers et al., 2017 Nature Methods). Briefly, lung tumors were initiated in KrasLSL-G12D/+;R26LSL-tdTomato (KT) and KT;H11LSL-Cas9 mice using a pool of Lenti-sgRNA/Cre vectors that are each modified with two-component barcodes composed of sgRNA-specific and clonal identifiers. This particular pool of Lenti-sgRNA/Cre vectors was composed of vectors targeting a series of LKB1-dependent genes as well a subset of C/EBP transciription factors. Following tumor development, the two-component lentiviral barcodes integrated within transduced populations were amplified from genomic DNA of whole-lung homogenates. The resulting amplicons were then subjected to next-generation sequencing. From the resulting reads, barcode pileups were generated and filtered prior to conversion to absolute numbers of neoplastic cells via normalization to spiked-in samples of known quantities of barcoded neoplastic cells. The resulting tumor size distributions were then analyzed by multiple statistical measures as described previously (Rogers et al., 2017 Nature Methods).  Results: Targeting of Lkb1 dramatically increased lung tumor size relative to inert sgRNAs. Little to no impact on tumor size was observed upon targeting LKB1-dependent genes. However, simultaneous targeting of Cebpa/b/d resulted in a significant increase in tumor size.  Conclusions: A subset of C/EBP transcription factors constrain oncogenic KRAS-driven lung tumor growth.

overall_design: Lung tumors were initiated in 15 KrasLSL-G12D/+;R26LSL-tdTomato;H11LSL-Cas9 mice using a pool of Lenti-sgRNA/Cre vectors targeting a series of LKB1-dependent genes, C/EBP family members Cebpa, Cebpb, and Cebpd in addition to five inert sgRNAs (to generate tumors driven by oncogenic KRAS only). Nine KrasLSL-G12D/+;R26LSL-tdTomato mice (lacking Cas9) were also transduced with the same lentiviral pool to control for differences in virus titer. Lentiviral vectors were diversified with two-component barcodes composed of sgRNA-specific and clonal identifiers to uniquely tag each tumor and label with a genotype-specific identifier. The size of each clonal outgrowth was determined by deep sequencing of amplicons derived from the lentiviral barcodes integrated within the genomic DNA of tumor-bearing lungs.


title: RNA-sequencing comparison of murine cardiac endothelials from ketogenic and control diet mice

city: Heidelberg

first: Andreas

last: Fischer

address: Im Neuenheimer Feld 280

institute: DKFZ

summary: RNA-sequencing was used to compare the transcriptome in cardiac endothelial cells from mice fed a ketogenic diet or control diet.

overall_design: Week 8 wild type C57BL/6J mice were randomly assigned to control diet (11.6 kcal % fat) or the ketogenic diet (89.5 kcal% fat) groups. Mice were kept on either diet with ad libitum access to food and drinking water until the end of the experiment. Endothelial cells (CD31+ cells) were isolated from heart tissue of mice.

first: Eva-Maria

last: Weis


title: Cellular trajectory analysis links tissue maturation to cellular specialization in the plant meristem [protophloem sieve element cells]

first: Bernhard

last: Blob

overall_design: RNA seq profiling of bulk-isolated (FACS) protophloem sieve element cells

first: Jung-ok

last: Heo

first: Maria

last: Angels de Luis Balaguer

first: Rosangela

last: Sozzani

first: Yrjo

last: Helariutta


title: LKB1 drives stasis and C/EBP-mediated reprogramming to an alveolar type II fate in lung cancer [XTR Bulk RNA-seq]

summary: Background: LKB1 is among the most frequently altered tumor suppressors in lung adenocarcinoma. Inactivation of Lkb1 accelerates the growth and progression of oncogenic KRAS-driven lung tumors in mouse models. However, the molecular mechanisms by which LKB1 constrains lung tumorigenesis and whether the aggressive cancer state that stems from Lkb1 deficiency can be reverted remains unknown.Purpose: To assess the acute transcriptional response to Lkb1 restoration within established lung tumors in a genetically engineered mouse model of oncogenic KRAS-driven lung adenocarcinoma.  Approach: To control LKB1 function in vivo, we generated an Lkb1XTR allele, which enables Cre-mediated disruption of Lkb1 expression during tumor development and subsequent FLPo-ERT2-mediated reactivation of Lkb1 within established tumors. Lung tumors were initiated in KrasLSL-G12D/+;R26LSL-tdTomato (KT; Lkb1 wild-type), KT;Lkb1XTR/XTR (non-restorable), and KT;Lkb1XTR/XTR;FLPo-ERT2 (restorable) mice with Lenti-Cre. Prior isolating neoplastic cells by FACS for gene expression profiling by RNA-seq, lung tumor-bearing were treated with either corn oil vehicle or tamoxifen for two weeks following tumor development.  Results: Lkb1 restoration resulted in higher expression of markers of alveolar type II epithelial cells as well as gene sets relating to immunomodulation and lipid metabolism export, which are important functions of mature alveolar type II epithelial cells.   Conclusions: LKB1 promotes the expression of C/EBP target genes and consequently drives features of alveolar type II epithelial cell differentiation.

overall_design: Lung tumors were initiated in KrasLSL-G12D/+;R26LSL-tdTomato (KT; Lkb1 wild-type), KT;Lkb1XTR/XTR (non-restorable), and KT;Lkb1XTR/XTR;FLPo-ERT2 (restorable) mice with Lenti-Cre. Following tumor development and two weeks of treatment with either corn oil vehicle or tamoxifen, neoplastic cells were FACS-isolated from lung tumors and subjected to bulk RNA-seq.


title: Parathyroid tissue transcriptome over 24h of PTHcre;Bmal1 flox/flox mice and of Bmal1 flox/flox mice

city: Copenhagen

first: Søren

last: Egstrand

address: Blegdamsvej 9

department: Depart. of Nephrology

country: Denmark

institute: Rigshospitalet

summary: The objective is to elucidate the role of the circadian clock in the parathyroid glands. We investigate the parathyroid transcriptome of wildtype mice (Bmal1 flox/flox) and of mice with parathyroid-specific excision of the circadian clock gene Bmal1 (PTHcre;Bmal1 flox/flox) harvested at 4 hour interval for 24 hours. Rhythmic genes were identified by JTK_CYCLE analysis and revealed 1455 rhythmic genes of the Bmal1 flox/flox and 1055 rhythmic genes of the PTHcre;Bmal1 flox/flox. We found global damepning of circadian clock genes with abolished rhythmicity of Cry1, Cry2, Clock, Npas2, Rorc, and Usp2. We used GSEA analysis to investigate differential expression at each timepount and found downregulation of genes involved in ATP synthesis in PTHcre;Bmal1 flox/flox mice at 4 consecutive timepoints during the active period of the mouse.

overall_design: Samples are pooled parathyroid glands from two mice of either Bmal1 flox/flox (WT) or PTHcre;Bmal1 flox/flox (KO) genotype. Samples were collected at 6 timepoints with 4 hour interval starting when lights turn on at zeitgebertime (=ZT) 0. In total 21 samples from WT and 18 samples from KO were collected.

last: Mace

first: Marya

last: Morevati

first: Anders

last: Nordholm

first: Lars

last: Engelholm

first: Jesper

last: Thomsen

first: Annemarie

last: Brüel

first: Tally

last: Naveh-Many

first: Yuliu

first: Klaus

last: Olgaard

first: Ewa

last: Lewin


title: PRBM1 inactivation promotes upregulation of human endogenous retroviruses in a HIF-dependent manner

city: Chapel Hill

first: William

last: Kim

address: 450 West Dr

department: Lineberger Comprehensive Cancer Center

institute: UNC Chapel Hill

summary: By analyzing RNAseq data derived from TCGA and clinical trial in ccRCC we observed that PBRM1 mutation is significantly associated with expression changes of hERVs (human endogenous retroviruses). We validated this observation in UMRC2 cell line and further demonstrated that PBRM1 specifically regulated members of HERVERI superfamily. We found that the increased expression of hERVs by loss of PBRM1 reversed by further knocking down HIF complex. Our data indicated a role of PBRM1 in negative regulation of selective hERVs in a HIF-dependent manner

sample_organism:
Homo sapiens
Human endogenous retroviruses

overall_design: PBRM1 was permanently knocking down by shRNA in UMRC2 cell line. siRNA of HIF1a and HIF2a was used to further knock down HIF1a and HIF1b in the PBRM1 kd cells. With two biological replicates for each condition, there are 24 samples in total to be collected for RNA extraction and subsequently RNA sequencing.

first: Mi

last: Zhou

first: Janet

last: Leung


title: LKB1 drives stasis and C/EBP-mediated reprogramming to an alveolar type II fate in lung cancer [C/EBP Bulk RNA-seq]

first: Christopher

last: Murray

summary: Background: LKB1 is among the most frequently altered tumor suppressors in lung adenocarcinoma. Inactivation of Lkb1 accelerates the growth and progression of oncogenic KRAS-driven lung tumors in mouse models. However, the molecular mechanisms by which LKB1 constrains lung tumorigenesis and whether the aggressive cancer state that stems from Lkb1 deficiency can be reverted remains unknown. Restoration of Lkb1 in established lung tumors promotes the expression of C/EBP target genes as well as features of alveolar type II cell differentiation, which requires the activity of C/EBP transcription factors in the developmental setting.  Purpose: To determine the extent to which the disruption of C/EBP transcription factors recapitulates the transcriptional changes induced by the inactivation of Lkb1.Approach: To assess the changes gene expression induced by CRISPR/Cas9-mediated disruption of either C/EBP transcription factors or Lkb1, we induced lung tumors in KrasLSL-G12D/+;R26LSL-tdTomato;H11LSL-Cas9 mice using Lenti-sgNeo1/sgNT/sgNeo2/Cre (sgInert), Lenti-sgLkb1/Cre (sgLkb1), or Lenti-sgCebpa/sgCebpb/sgCebpd/Cre (sgCebpa/b/d). Neoplastic cells were then isolated from lung tumors by FACS for gene expression profiling by RNA-seq.  Results: The disruption of C/EBP transcription factors partially recapitulates the gene expression changes induced by Lkb1 inactivation. Among the genes that are jointly dependent upon C/EBP transcription factors and LKB1 is an enrichment of NKX2-1-dependent target genes.  Conclusions: C/EBP transcription factors likely operate downstream of LKB1 in an indirect manner, collaborating with another key developmental regulator, NKX2-1, to enforce alveolar type II cell differentiation to constrain tumor growth.

overall_design: Lung tumors were initiated in KrasLSL-G12D/+;R26LSL-tdTomato;H11LSL-Cas9 mice with Lenti-sgNeo1/sgNT/sgNeo2/Cre (sgInert), Lenti-sgLkb1/Cre (sgLkb1), or Lenti-sgCebpa/sgCebpb/sgCebpd/Cre (sgCebpa/b/d). Following tumor development, neoplastic cells were FACS-isolated from lung tumors and subjected to bulk RNA-seq.

first: Monte

last: Winslow


title: LKB1 drives stasis and C/EBP-mediated reprogramming to an alveolar type II fate in lung cancer [Sorted scRNA-seq]

summary: Background: LKB1 is among the most frequently altered tumor suppressors in lung adenocarcinoma. Inactivation of Lkb1 accelerates the growth and progression of oncogenic KRAS-driven lung tumors in mouse models. However, the molecular mechanisms by which LKB1 constrains lung tumorigenesis and whether the aggressive cancer state that stems from Lkb1 deficiency can be reverted remains unknown. By bulk gene expression profiling, Lkb1 restoration promotes the expression of markers and functions of alveolar type II cells, suggesting that LKB1 may govern a cell-state transition within the neoplastic epithelial compartment.  Purpose: To determine whether the restoration of Lkb1 drives changes in cell state and/or abundance within established oncogenic KRAS-driven lung tumors.  Approach: To control LKB1 function in vivo, we generated an Lkb1XTR allele, which enables Cre-mediated disruption of Lkb1 expression during tumor development and subsequent FLPo-ERT2-mediated reactivation of Lkb1 within established tumors. Lung tumors were initiated in KT;Lkb1XTR/XTR (non-restorable) and KT;Lkb1XTR/XTR;FLPo-ERT2 (restorable) mice with Lenti-Cre. Following tumor development, lung tumor-bearing were treated with either corn oil vehicle or tamoxifen for two weeks prior to isolating specifically neoplastic cells by FACS for single cell RNA-seq.  Results: Single cell analysis revealed that the neoplastic epithelial compartment was composed of alveolar type I- and type II-like subpopulations, a Krt8+ transitional state, an actively proliferating subpopulations, and an indeterminate subpopulation partially resembling the alveolar type II-like identity. Dynamic inference analyses indicated that the indeterminate population represents an intermediate state along the alveolar type II to alveolar type I trans-differentiation trajectory. Notably, the indeterminate cluster was more proliferative than the alveolar type II-like subpopulation and exhibited higher expression of Sox9, which is a marker of distal lung epithelial progenitors. There was a marked shift in the epithelial compartment from the indeterminate state to alveolar type II epithelial-like identity in response to Lkb1 restoration.  Conclusions: LKB1 governs the transition between a proliferative progenitor-like population and mature alveolar type II-like identity within oncogenic KRAS-driven lung tumors

overall_design: Lung tumors were initiated in KT;Lkb1XTR/XTR (non-restorable), and KT;Lkb1XTR/XTR;FLPo-ERT2 (restorable) mice with Lenti-Cre. Following tumor development and two weeks of treatment with either corn oil vehicle or tamoxifen, neoplastic cells were FACS-isolated from lung tumors and subjected to single cell RNA-seq.


title: Bacterial Heterochromatin Formation Through Liquid-Liquid Phase Separation

city: Ann Arbor

first: Lydia

last: Freddolino

state: MI

address: 1150 W. Medical Center Dr., MSRB 3 room 3315

department: Biological Chemistry

institute: University of Michigan

summary: Heterochromatin is most often associated with eukaryotic organisms. Yet, bacteria also contain areas with densely protein-occupied chromatin that silences gene expression. One nucleoid-associated silencing factor is Hfq, which is strongly enriched at prophages and mobile genetic elements. Here we demonstrate that polyphosphate, an ancient and highly conserved polyanion, is essential for the specific binding of Hfq to these regions. Lack of either polyphosphate or Hfq causes unsolicited prophage and transposon mobilization, which drastically increases mutagenesis. We discovered that Hfq and polyphosphate form high molecular weight complexes that interact with DNA in phase-separated viscous condensates. We propose that polyP provides a scaffold that promotes Hfq binding to DNA regions with high intrinsic curvature, and thus serves as one hitherto unknown driver of heterochromatin formation in bacteria.

overall_design: We present two major types of data here: First, a series of RNA-seq samples comparing the behavior of WT and ppk E. coli cells, either under basal growth or in the presence of cisplatin treatment (overall 2-4 replicates for each of three conditions for each genotype). Second, a series of ChIP-seq samples tracking the location of Hfq binding to the chromosome of wild type vs. ppk cells (3 genotypes, 2 replicates per genotype for ChIP and 2 replicates per genotype for matched input samples)

first: Francois

last: Beaufay

first: Haley

last: Amemiya

first: Jian

last: Guan

first: Rishav

last: Mitra

first: Benjamin

last: Meinen

first: Joseph

last: Basalla

first: Anthony

last: Vecchiarelli

first: James

last: Bardwell

first: Peter

first: Ursula

last: Jakob


title: The evolution, evolvability, and engineering of gene regulatory DNA [GPRA]

first: Carl

last: de Boer

address: 415 Main St

institute: The Broad Institute

summary: In order to generate data produce a generalizable sequence-to-expression model, we randomly sampled ~21 million 80 bp sequences and tested their activity as promoters (in yeast) by measuring expression level by FACS (sorting into 18 bins).

overall_design: Here, all libraries tested have the following sequences flanking the random 80 bp oligo: the pTpA distal region was (pT) GCTAGCAGGAATGATGCAAAAGGTTCCCGATTCGAACTGCATTTTTTTCACATC and proximal region (pA) was GGTTACGGCTGTTTCTTAATTAAAAAAAGATAGAAAACATTAGGAGTGTAACACAAGACTTTCGGATCCTGAGCAGGCAAGATAAACGA (up to the theoretical TSS).  80 Ns were inserted in between proximal and distal regions. Here, both experiments represent yeast grown in SD-Ura (Sunrise Sciences), and the designed-sequence library used a strain of S288C with Ura3 deleted, while the large-scale random promoter library used strain Y8205 (Boone Lab).

first: Eeshit Dhaval

last: Vaishnav