QuantSeq 3’ mRNA-Seq FWD for Illumina
CDK12 controls G1/S progression by regulating RNAPII processivity at core DNA replication genes
Anil Paul Chirackal Manavalan, Kveta Pilarova, Michael Kluge, Koen Bartholomeeusen, Michal Rajecky, Jan Oppelt, Prashant Khirsariya, Kamil Paruch, Lumir Krejci, Caroline C Friedel, Dalibor Blazek
CDK12 is a kinase associated with elongating RNA polymerase II (RNAPII) and is frequently mutated in cancer. CDK12 depletion reduces the expression of homologous recombination (HR) DNA repair genes, but comprehensive insight into its target genes and cellular processes is lacking. We use a chemical genetic approach to inhibit analog‐sensitive CDK12, and find that CDK12 kinase activity is required for transcription of core DNA replication genes and thus for G1/S progression. RNA‐seq and ChIP‐seq reveal that CDK12 inhibition triggers an RNAPII processivity defect characterized by a loss of mapped reads from 3′ends of predominantly long, poly(A)‐signal‐rich genes. CDK12 inhibition does not globally reduce levels of RNAPII‐Ser2 phosphorylation. However, individual CDK12‐dependent genes show a shift of P‐Ser2 peaks into the gene body approximately to the positions where RNAPII occupancy and transcription were lost. Thus, CDK12 catalytic activity represents a novel link between regulation of transcription and cell cycle progression. We propose that DNA replication and HR DNA repair defects as a consequence of CDK12 inactivation underlie the genome instability phenotype observed in many cancers.
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Ribonucleotide reductase from Fusarium oxysporum does not Respond to DNA replication stress
Rotem Cohen, Shira Milo, Sushma Sharma, Alon Savidor, Shay Covo
Ribonucleotide reductase (RNR) catalyzes the rate limiting step in dNTP biosynthesis and is tightly regulated at the transcription and activity levels. One of the best characterized responses of yeast to DNA damage is up-regulation of RNR transcription and activity and consequently, elevation of the dNTP pools. Hydroxyurea is a universal inhibitor of RNR that causes S phase arrest. It is used in the clinic to treat certain types of cancers. Here we studied the response of the fungal plant pathogen Fusarium oxysporum to hydroxyurea in order to generate hypotheses that can be used in the future in development of a new class of pesticides. F. oxysporum causes severe damage to more than 100 agricultural crops and specifically threatens banana cultivation world-wide. Although the recovery of F. oxysporum from transient hydroxyurea exposure was similar to the one of Saccharomyces cerevisiae, colony formation was strongly inhibited in F. oxysporum in comparison with S. cerevisiae. As expected, genomic and phosphoproteomic analyses of F. oxysporum conidia (spores) exposed to hydroxyurea showed hallmarks of DNA replication perturbation and activation of recombination. Unexpectedly and strikingly, RNR was not induced by either hydroxyurea or the DNA-damaging agent methyl methanesulfonate as determined at the RNA and protein levels. Consequently, dNTP concentrations were significantly reduced, even in response to a low dose of hydroxyurea. Methyl methanesulfonate treatment did not induce dNTP pools in F. oxysporum, in contrast to the response of RNR and dNTP pools to DNA damage and hydroxyurea in several tested organisms. Our results are important because the lack of a feedback mechanism to increase RNR expression in F. oxysporum is expected to sensitize the pathogen to a fungal-specific ribonucleotide inhibitor. The potential impact of our observations on F. oxysporum genome stability and genome evolution is discussed.
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Association analysis between quantitative MRI features and hypoxia-related genetic profiles in prostate cancer: a pilot study
Yu Sun, Scott Williams, David Byrne, Simon Keam, Hayley M. Reynolds, Catherine Mitchell, Darren Wraith, Declan Murphy, and Annette Haworth
Objective:
To investigate the association between multiparametric MRI (mpMRI) imaging features and hypoxia-related genetic profiles in prostate cancer.
Methods:
In vivo mpMRI was acquired from six patients prior to radical prostatectomy. Sequences included T2 weighted (T2W) imaging, diffusion-weighted imaging, dynamic contrast enhanced MRI and blood oxygen-level dependent imaging. Imaging data were co-registered with histology using three-dimensional deformable registration methods. Texture features were extracted from T2W images and parametric maps from functional MRI. Full transcriptome genetic profiles were obtained using next generation sequencing from the prostate specimens. Pearson correlation coefficients were calculated between mpMRI data and hypoxia-related gene expression levels. Results were validated using glucose transporter one immunohistochemistry (IHC).
Results:
Correlation analysis identified 34 candidate imaging features (six from the mpMRI data and 28 from T2W texture features). The IHC validation showed that 16 out of the 28 T2W texture features achieved weak but significant correlations (p < 0.05).
Conclusions:
Weak associations between mpMRI features and hypoxia gene expressions were found. This indicates the potential use of MRI in assessing hypoxia status in prostate cancer. Further validation is required due to the low correlation levels.
Advances in knowledge:
This is a pilot study using radiogenomics approaches to address hypoxia within the prostate, which provides an opportunity for hypoxia-guided selective treatment techniques.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
4‐1BB Delineates Distinct Activation Status of Exhausted Tumor‐Infiltrating CD8+ T Cells in Hepatocellular Carcinoma
Hyung‐Don Kim, Seongyeol Park, Seongju Jeong, Yong Joon Lee, Hoyoung Lee, Chang Gon Kim, Kyung Hwan Kim, Seung‐Mo Hong, Jung‐Yun Lee, Sunghoon Kim, Hong Kwan Kim, Byung Soh Min, Jong Hee Chang, Young Seok Ju, Eui‐Cheol Shin, Gi‐Won Song, Shin Hwang, Su‐Hyung Park
Background and Aims
Targeting costimulatory receptors with agonistic antibodies is a promising cancer immunotherapy option. We aimed to investigate costimulatory receptor expression, particularly 4‐1BB (CD137 or tumor necrosis factor receptor superfamily member 9), on tumor‐infiltrating CD8+ T cells (CD8+ tumor‐infiltrating lymphocytes [TILs]) and its association with distinct T‐cell activation features among exhausted CD8+ TILs in hepatocellular carcinoma (HCC).
Approach and Results
Tumor tissues, adjacent nontumor tissues, and peripheral blood were collected from HCC patients undergoing surgical resection (n = 79). Lymphocytes were isolated and used for multicolor flow cytometry, RNA‐sequencing, and in vitro functional restoration assays. Among the examined costimulatory receptors, 4‐1BB was most prominently expressed on CD8+ TILs. 4‐1BB expression was almost exclusively detected on CD8+ T cells in the tumor—especially on programmed death 1 (PD‐1)high cells and not PD‐1int and PD‐1neg cells. Compared to PD‐1int and 4‐1BBnegPD‐1high CD8+ TILs, 4‐1BBposPD‐1high CD8+ TILs exhibited higher levels of tumor reactivity and T‐cell activation markers and significant enrichment for T‐cell activation gene signatures. Per‐patient analysis revealed positive correlations between percentages of 4‐1BBpos cells among CD8+ TILs and levels of parameters of tumor reactivity and T‐cell activation. Among highly exhausted PD‐1high CD8+ TILs, 4‐1BBpos cells harbored higher proportions of cells with proliferative and reinvigoration potential. Our 4‐1BB–related gene signature predicted survival outcomes of HCC patients in the The Cancer Genome Atlas cohort. 4‐1BB agonistic antibodies enhanced the function of CD8+ TILs and further enhanced the anti‐PD‐1–mediated reinvigoration of CD8+ TILs, especially in cases showing high levels of T‐cell activation.
Conclusion
4‐1BB expression on CD8+ TILs represents a distinct activation state among highly exhausted CD8+ T cells in HCC. 4‐1BB costimulation with agonistic antibodies may be a promising strategy for treating HCCs exhibiting prominent T‐cell activation.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Divergent gene expression networks underlie morphological diversity of abscission zones in grasses
Yunqing Yu, Hao Hu, Andrew N. Doust, Elizabeth A. Kellogg
- Abscission is a process in which plants shed their parts, and is mediated by a particular set of cells, the abscission zone (AZ). In grasses (Poaceae), the position of the AZ differs among species, raising the question of whether its anatomical structure and genetic control are conserved.
- The ancestral position of the AZ was reconstructed. A combination of light microscopy, transmission electron microscopy, RNA‐Seq analyses and RNA in situ hybridisation were used to compare three species, two (weedy rice and Brachypodium distachyon ) with the AZ in the ancestral position and one (Setaria viridis ) with the AZ in a derived position below a cluster of flowers (spikelet).
- Rice and Brachypodium are more similar anatomically than Setaria . However, the cell wall properties and the transcriptome of rice and Brachypodium are no more similar to each other than either is to Setaria . The set of genes expressed in the studied tissues is generally conserved across species, but the precise developmental and positional patterns of expression and gene networks are almost entirely different.
- Transcriptional regulation of AZ development appears to be extensively rewired among the three species, leading to distinct anatomical and morphological outcomes.
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Gas6 is a reciprocal regulator of mitophagy during mammalian oocyte maturation
Kyeoung-Hwa Kim, Eun-Young Kim, Jung-Jae Ko & Kyung-Ah Lee
Previously, we found that the silencing of growth arrest-specific gene 6 (Gas6) expression in oocytes impairs cytoplasmic maturation through mitochondrial overactivation with concurrent failure of pronuclear formation after fertilization. In this study, we report that Gas6 regulates mitophagy and safeguards mitochondrial activity by regulating mitophagy-related genes essential to the complete competency of oocytes. Based on RNA-Seq and RT-PCR analysis, in Gas6-silenced MII oocytes, expressions of mitophagy-related genes were decreased in Gas6-silenced MII oocytes, while mitochondrial proteins and Ptpn11, the downstream target of Gas6, was increased. Interestingly, GAS6 depletion induced remarkable MTOR activation. Gas6-depleted MII oocytes exhibited mitochondrial accumulation and aggregation caused by mitophagy inhibition. Gas6-depleted MII oocytes had a markedly lower mtDNA copy number. Rapamycin treatment rescued mitophagy, blocked the increase in MTOR and phosphorylated-MTOR, and increased the mitophagy-related gene expression in Gas6-depleted MII oocytes. After treatment with Mdivi-1, a mitochondrial division/mitophagy inhibitor, all oocytes matured and these MII oocytes showed mitochondrial accumulation but reduced Gas6 expression and failure of fertilization, showing phenomena very similar to the direct targeting of Gas6 by RNAi. Taken together, we conclude that the Gas6 signaling plays a crucial role in control of oocytes cytoplasmic maturation by modulating the dynamics and activity of oocyte mitochondria.
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Fibro-adipogenic progenitors of dystrophic mice are insensitive to NOTCH regulation of adipogenesis
Milica Marinkovic, Claudia Fuoco, Francesca Sacco, Andrea Cerquone Perpetuini, Giulio Giuliani, Elisa Micarelli, Theodora Pavlidou, Lucia Lisa Petrilli, Alessio Reggio, Federica Riccio, Filomena Spada, Simone Vumbaca, Alessandro Zuccotti, Luisa Castagnoli, Matthias Mann, Cesare Gargioli Gianni Cesareni
Fibro-adipogenic progenitors (FAPs) promote satellite cell differentiation in adult skeletal muscle regeneration. However, in pathological conditions, FAPs are responsible for fibrosis and fatty infiltrations. Here we show that the NOTCH pathway negatively modulates FAP differentiation both in vitro and in vivo. However, FAPs isolated from young dystrophin-deficient mdx mice are insensitive to this control mechanism. An unbiased mass spectrometry–based proteomic analysis of FAPs from muscles of wild-type and mdx mice suggested that the synergistic cooperation between NOTCH and inflammatory signals controls FAP differentiation. Remarkably, we demonstrated that factors released by hematopoietic cells restore the sensitivity to NOTCH adipogenic inhibition in mdx FAPs. These results offer a basis for rationalizing pathological ectopic fat infiltrations in skeletal muscle and may suggest new therapeutic strategies to mitigate the detrimental effects of fat depositions in muscles of dystrophic patients.
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FOXA2 Is Required for Enhancer Priming during Pancreatic Differentiation
Kihyun Lee, Hyunwoo Cho, Robert W. Rickert, Qing V. Li, Julian Pulecio, Christina S. Leslie, Danwei Huangfu
Transcriptional regulatory mechanisms of lineage priming in embryonic development are largely uncharacterized because of the difficulty of isolating transient progenitor populations. Directed differentiation of human pluripotent stem cells (hPSCs) combined with gene editing provides a powerful system to define precise temporal gene requirements for progressive chromatin changes during cell fate transitions. Here, we map the dynamic chromatin landscape associated with sequential stages of pancreatic differentiation from hPSCs. Our analysis of chromatin accessibility dynamics led us to uncover a requirement for FOXA2, known as a pioneer factor, in human pancreas specification not previously shown from mouse knockout studies. FOXA2 knockout hPSCs formed reduced numbers of pancreatic progenitors accompanied by impaired recruitment of GATA6 to pancreatic enhancers. Furthermore, FOXA2 is required for proper chromatin remodeling and H3K4me1 deposition during enhancer priming. This work highlights the power of combining hPSC differentiation, genome editing, and computational genomics for discovering transcriptional mechanisms during development.
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Gain of 20q11.21 in Human Pluripotent Stem Cells Impairs TGF-β-Dependent Neuroectodermal Commitment
C. Markouli, E. Couvreu De Deckersberg, M. Regin, H.T. Nguyen, F. Zambelli, A. Keller, D. Dziedzicka, J. De Kock, L. Tilleman, F. Van Nieuwerburgh, L. Franceschini, K. Sermon, M. Geens, C. Spits
Gain of 20q11.21 is one of the most common recurrent genomic aberrations in human pluripotent stem cells. Although it is known that overexpression of the antiapoptotic gene Bcl-xL confers a survival advantage to the abnormal cells, their differentiation capacity has not been fully investigated. RNA sequencing of mutant and control hESC lines, and a line transgenically overexpressing Bcl-xL, shows that overexpression of Bcl-xL is sufficient to cause most transcriptional changes induced by the gain of 20q11.21. Moreover, the differentially expressed genes in mutant and Bcl-xL overexpressing lines are enriched for genes involved in TGF-β- and SMAD-mediated signaling, and neuron differentiation. Finally, we show that this altered signaling has a dramatic negative effect on neuroectodermal differentiation, while the cells maintain their ability to differentiate to mesendoderm derivatives. These findings stress the importance of thorough genetic testing of the lines before their use in research or the clinic.
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TGF-β signaling promotes tube-structure-forming growth in pancreatic duct adenocarcinoma
Takashi Yamaguchi, Sanae Ikehara, Yoshihiro Akimoto, Hayao Nakanishi, Masahiko Kume, Kazuo Yamamoto, Osamu Ohara & Yuzuru Ikehara
Tube-forming growth is an essential histological feature of pancreatic duct adenocarcinoma (PDAC) and of the pancreatic duct epithelium; nevertheless, the nature of the signals that start to form the tubular structures remains unknown. Here, we showed the clonal growth of PDAC cell lines in a three-dimensional (3D) culture experiment that modeled the clonal growth of PDAC. At the beginning of this study, we isolated the sphere- and tube-forming clones from established mouse pancreatic cancer cell lines via limiting dilution culture using collagen gel. Compared with cells in spherical structures, the cells in the formed tubes exhibited a lower CK19 expression in 3D culture and in the tumor that grew in the abdominal cavity of nude mice. Conversely, the expression of the transforming growth factor β (TGF-β)-signaling target mRNAs was higher in the formed tube vs the spherical structures, suggesting that TGF-β signaling is more active in the tube-forming process than the sphere-forming process. Treatment of sphere-forming clones with TGF-β1 induced tube-forming growth, upregulated the TGF-β-signaling target mRNAs, and yielded electron microscopic findings of a fading epithelial phenotype. In contrast, the elimination of TGF-β-signaling activation by treatment with inhibitors diminished the tube-forming growth and suppressed the expression of the TGF-β-signaling target mRNAs. Moreover, upregulation of the Fn1, Mmp2, and Snai1 mRNAs, which are hallmarks of tube-forming growth in PDAC, was demonstrated in a mouse model of carcinogenesis showing rapid progression because of the aggressive invasion of tube-forming cancer. Our study suggests that the tube-forming growth of PDAC relies on the activation of TGF-β signaling and highlights the importance of the formation of tube structures.
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Genomic and Transcriptomic Determinants of Therapy Resistance and Immune Landscape Evolution during Anti-EGFR Treatment in Colorectal Cancer
Andrew Woolston, Khurum Khan, Georgia Spain, Louise J. Barber, Beatrice Griffiths, Reyes Gonzalez-Exposito, Lisa Hornsteiner, Marco Punta, Yatish Patil, Alice Newey, Sonia Mansukhani, Matthew N. Davies, Andrew Furness, Francesco Sclafani, Clare Peckitt, Mirta Jimenez, Kyriakos Kouvelakis, Romana Ranftl, Ruwaida Begum, Isma Rana, Janet Thomas, Annette Bryant, Sergio Quezada, Andrew Wotherspoon, Nasir Khan, Nikolaos Fotiadis, Teresa Marafioti, Thomas Powles, Stefano Lise, Fernando Calvo, Sebastian Guettler, Katharina von Loga, Sheela Rao, David Watkins, Naureen Starling, Ian Chau, Anguraj Sadanandam, David Cunningham and Marco Gerlinger
Despite biomarker stratification, the anti-EGFR antibody cetuximab is only effective against a subgroup of colorectal cancers (CRCs). This genomic and transcriptomic analysis of the cetuximab resistance landscape in 35 RAS wild-type CRCs identified associations of NF1 and non-canonical RAS/RAF aberrations with primary resistance and validated transcriptomic CRC subtypes as non-genetic predictors of benefit. Sixty-four percent of biopsies with acquired resistance harbored no genetic resistance drivers. Most of these had switched from a cetuximab-sensitive transcriptomic subtype at baseline to a fibroblast- and growth factor-rich subtype at progression. Fibroblast-supernatant conferred cetuximab resistance in vitro, confirming a major role for non-genetic resistance through stromal remodeling. Cetuximab treatment increased cytotoxic immune infiltrates and PD-L1 and LAG3 immune checkpoint expression, potentially providing opportunities to treat cetuximab-resistant CRCs with immunotherapy.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
YAP nuclear translocation through dynein and acetylated microtubule controls fibroblast activation
Eunae You, Panseon Ko, Jangho Jeong, Seula Keum, Jung-Woong Kim, Young-Jin Seo, Woo Keun Song, Sangmyung Rhee
Myofibroblasts are the major cell type that are responsible for increase the mechanical stiffness in fibrotic tissues. It has well documented that the TGF-β/Smad axis is required for myofibroblast differentiation under the rigid substrate condition. However, the mechanism driving myofibroblast differentiation in soft substrates remains unknown. In this research, we demonstrated that interaction of yes-associated protein (YAP) and acetylated microtubule via dynein, a microtubule motor protein drives nuclear localization of YAP in soft matrix, which in turn increased TGF-β1 induced transcriptional activity of Smad for myofibroblast differentiation. Pharmacological and genetical disruption of dynein impaired the nuclear translocation of YAP and decreased the TGF-β1 induced Smad activity even though phosphorylation and nuclear localization of Smad occurred normally in α-tubulin acetyltransferase (α-TAT1) knockout cell. Moreover, microtubule acetylation prominently appeared in the fibroblast-like cells nearby the blood vessel in the fibrotic liver induced by CCl4 administration which were conversely decreased by TGF-β receptor inhibitor. As a result, quantitative inhibition of microtubule acetylation may be suggested as a new target for overcome the fibrotic diseases.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Selective Killing of RAS-Malignant Tissues by Exploiting Oncogene-Induced DNA Damage
Lada Murcia, Marta Clemente-Ruiz, Priscillia Pierre-Elies, Anne Royou, Marco Milán
Several oncogenes induce untimely entry into S phase and alter replication timing and progression, thereby generating replicative stress, a well-known source of genomic instability and a hallmark of cancer. Using an epithelial model in Drosophila, we show that the RAS oncogene, which triggers G1/S transition, induces DNA damage and, at the same time, silences the DNA damage response pathway. RAS compromises ATR-mediated phosphorylation of the histone variant H2Av and ATR-mediated cell-cycle arrest in G2 and blocks, through ERK, Dp53-dependent induction of cell death. We found that ERK is also activated in normal tissues by an exogenous source of damage and that this activation is necessary to dampen the pro-apoptotic role of Dp53. We exploit the pro-survival role of ERK activation upon endogenous and exogenous sources of DNA damage to present evidence that its genetic or chemical inhibition can be used as a therapeutic opportunity to selectively eliminate RAS-malignant tissues.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Indazole-Cl inhibits hypoxia-induced cyclooxygenase-2 expression in vascular smooth muscle cells
Choa Park, Joonwoo Park, Myeong Kuk Shim, Mee-Ra Rhyu, Byung-Koo Yoon, Kyung Sook Kim and YoungJoo Lee
Atherosclerosis is the most common root cause of arterial disease, such as coronary artery disease and carotid artery disease. Hypoxia is associated with the formation of macrophages and increased inflammation and is known to be present in lesions of atherosclerotic. Vascular smooth muscle cells (VSMCs) are one of the major components of blood vessels, and hypoxic conditions affect VSMC inflammation, proliferation and migration, which contribute to vascular stenosis and play a major role in the atherosclerotic process. Estrogen receptor (ER)-β is thought to play an important role in preventing the inflammatory response in VSMCs. In this report, we studied the anti-inflammatory effect of indazole (In)-Cl, an ERβ-specific agonist, under conditions of hypoxia. Expression of cyclooxygenase-2 reduced by hypoxia was inhibited by In-Cl treatment in VSMCs, and this effect was antagonized by an anti-estrogen compound. Additionally, the production of reactive oxygen species induced under conditions of hypoxia was reduced by treatment with In-Cl. Increased cell migration and invasion by hypoxia were also dramatically decreased following treatment with In-Cl. The increase in cell proliferation following treatment with platelet-derived growth factor was attenuated by In-Cl in VSMCs. RNA sequencing analysis was performed to identify changes in inflammation-related genes following In-Cl treatment in the hypoxic state. Our results suggest that ERβ is a potential therapeutic target for the suppression of hypoxia-induced inflammation in VSMCs.
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Nicotinamide riboside augments the human skeletal muscle NAD+ metabolome and induces transcriptomic and anti-inflammatory signatures in aged subjects: a placebo-controlled, randomized trial
Yasir S Elhassan, Katarina Kluckova, Rachel S Fletcher, Mark Schmidt, Antje Garten, Craig L Doig, David M Cartwright, Lucy Oakey, Claire V Burley, Ned Jenkinson, Martin Wilson, Samuel J E Lucas, Ildem Akerman, Alex Seabright, Yu-Chiang Lai, Daniel A Tennant, Peter Nightingale, Gareth A Wallis, Konstantinos N Manolopoulos, Charles Brenner, Andrew Philp, Gareth G Lavery
NAD+ is modulated by conditions of metabolic stress and has been reported to decline with aging, but human data are sparse. Nicotinamide riboside (NR) supplementation ameliorates metabolic dysfunction in rodents. We aimed to establish whether oral NR supplementation in aged participants can increase the skeletal muscle NAD+ metabolome, and questioned if tissue NAD+ levels are depressed with aging. We supplemented 12 aged men with NR 1g per day for 21-days in a placebo-controlled, randomized, double-blind, crossover trial. Targeted metabolomics showed that NR elevated the muscle NAD+ metabolome, evident by increased nicotinic acid adenine dinucleotide and nicotinamide clearance products. Muscle RNA sequencing revealed NR-mediated downregulation of energy metabolism and mitochondria pathways. NR also depressed levels of circulating inflammatory cytokines. In an additional study, 31P magnetic resonance spectroscopy-based NAD+ measurement in muscle and brain showed no difference between young and aged individuals. Our data establish that oral NR is available to aged human muscle and identify anti-inflammatory effects of NR, while suggesting that NAD+ decline is not associated with chronological aging per se in human muscle or brain.
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Plasticity and evolution are two processes individuals to respond to environmental change, but how both are related and impact each other is still controversial. We studied plastic and evolutionary responses in gene expression of Tribolium castaneum after exposure to new environments that differed from ancestral conditions in temperature, humidity or both. Using experimental evolution with ten replicated lines per condition, we were able to demonstrate adaptation after 20 generations. We measured gene expression in each condition in adapted selection lines and control lines to infer evolutionary and plastic changes. We found more evidence for changes in mean expression (shift in the intercept of reaction norms) in adapted lines than for changes in plasticity (shifts in slopes). Plasticity was mainly preserved and was responsible for a large part of the phenotypic divergence in expression between ancestral and new conditions. However, we found that genes with the largest evolutionary changes in expression also evolved reduced plasticity and often showed expression levels closer to the ancestral stage. Results obtained in the three different conditions were similar suggesting that restoration of ancestral expression levels during adaptation is a general evolutionary pattern. We increased the sample size in the most stressful condition and were then able to detect a positive correlation between proportion of genes with reversion of the ancestral plastic response and mean fitness per selection line.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Chronically stimulated human MAIT cells are unexpectedly potent IL‐13 producers
Jason Kelly, Yosuke Minoda, Tobias Meredith, Garth Cameron, Marie‐Sophie Philipp, Daniel G Pellicci, Alexandra J Corbett, Christian Kurts, Daniel HD Gray, Dale I Godfrey, George Kannourakis, Stuart P Berzins
Mucosal‐associated invariant T (MAIT) cells are unconventional T cells that recognize antigens derived from riboflavin biosynthesis. In addition to anti‐microbial functions, human MAIT cells are associated with cancers, autoimmunity, allergies and inflammatory disorders, although their role is poorly understood. Activated MAIT cells are well known for their rapid release of Th1 and Th17 cytokines, but we have discovered that chronic stimulation can also lead to potent interleukin (IL)‐13 expression. We used RNA‐seq and qRT‐PCR to demonstrate high expression of the IL‐13 gene in chronically stimulated MAIT cells, and directly identify IL‐13 using intracellular flow cytometry and multiplex bead analysis of MAIT cell cultures. This unexpected finding has important implications for IL‐13‐dependent diseases, such as colorectal cancer (CRC), that occur in mucosal areas where MAIT cells are abundant. We identify MAIT cells near CRC tumors and show that these areas and precancerous polyps express high levels of the IL‐13 receptor, which promotes tumor progression and metastasis. Our data suggest that MAIT cells have a more complicated role in CRC than currently realized and that they represent a promising new target for immunotherapies where IL‐13 can be a critical factor.
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Deletion of organic cation transporter Oct3 promotes hepatic fibrosis via upregulation of TGFβ
Johanna Vollmar, Yong Ook Kim, Jens U. Marquardt, Diana Becker, Peter R. Galle, Detlef Schuppan, and Tim Zimmermann
American journal of physiology. Gastrointestinal and liver physiology, doi:10.1152/ajpgi.00088.2019
Organic cation transporters (OCT) are responsible for the intracellular uptake and detoxification of a broad spectrum of endogenous and exogenous substrates. OCTs are downregulated in cholestasis, fibrosis, and hepatocellular carcinoma, but the underlying molecular mechanisms and downstream effects of OCT deletion are unknown. Oct3-knockout (Oct3−/−; FVB.Slc22a3tm10pb) and wild-type (WT; FVB) mice were subject to escalating doses of carbon tetrachloride (CCl4) or thioacetamide (TAA) for 6 wk to induce advanced parenchymal liver fibrosis. Secondary biliary fibrosis was generated by bile duct ligation. Liver fibrosis was assessed by hydroxyproline determination, quantitative Sirius red morphometry, and quantitative real-time PCR for fibrosis and inflammation-related genes. Ductular reaction was assessed by bile duct count per field of view in hematoxylin and eosin staining. General gene expression analyses were performed in liver tissue from untreated Oct3−/− and WT mice. Finally, primary murine hepatocytes were treated with the nonselective OCT inhibitor quinine, and transforming growth factor-β1 (Tgfβ1) protein expression was quantified by quantitative real-time PCR and Western blot. Oct3−/− mice developed significantly more fibrosis after bile duct ligation and CCl4 treatment compared with WT mice. Ductular reaction was enhanced in the long-term model. Concomitantly, Oct1 mRNA expression was downregulated during cholestatic and chemically (TAA and CCl4) induced fibrogenesis. The downregulation of Oct1 mRNA in fibrotic liver tissue reversed within 4 wk after TAA cessation. Gene expression analysis by next-generation sequencing revealed an enrichment of Tgfβ1 target genes in Oct3−/− mice. Tgfβ1 mRNA expression was significantly upregulated after chemically induced fibrosis (P < 0.001) in Oct3−/− compared with WT mice. Accordingly, in primary murine hepatocytes functional inhibition of OCT led to an upregulation of Tgfβ1 mRNA expression. Loss of Oct3 promotes fibrogenesis by affecting Tgfβ-mediated homeostasis in mice with chronic biliary and parenchymal liver damage and fibrosis.
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Transcriptomic analysis of 3D Cardiac Differentiation of Human Induced Pluripotent Stem Cells Reveals Faster Cardiomyocyte Maturation Compared to 2D Culture
Mariana A. Branco, João P. Cotovio, Carlos A. V. Rodrigues, Sandra H. Vaz, Tiago G. Fernandes, Leonilde M. Moreira, Joaquim M. S. Cabral & Maria Margarida Diogo
Human induced pluripotent stem cells (hiPSCs) represent an almost limitless source of cells for disease modelling and drug screening applications. Here we established an efficient and robust 3D platform for cardiomyocyte (CMs) production from hiPSCs, solely through small-molecule-based temporal modulation of the Wnt signalling, which generates more than 90% cTNT+ cells. The impact of performing the differentiation process in 3D conditions as compared to a 2D culture system, was characterized by transcriptomic analysis by using data collected from sequential stages of 2D and 3D culture. We highlight that performing an initial period of hiPSC aggregation before cardiac differentiation primed hiPSCs towards an earlier mesendoderm lineage differentiation, via TGF-β/Nodal signaling stabilization. Importantly, it was also found that CMs in the 3D microenvironment mature earlier and show an improved communication system, which we suggested to be responsible for a higher structural and functional maturation of 3D cardiac aggregates.
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Identification of monocyte-like precursors of granulocytes in cancer as a mechanism for accumulation of PMN-MDSCs
Jérôme Mastio, Thomas Condamine, George Dominguez, Andrew V. Kossenkov, Laxminarasimha Donthireddy, Filippo Veglia, Cindy Lin, Fang Wang, Shuyu Fu, Jie Zhou, Patrick Viatour, Sergio Lavilla-Alonso, Alexander T. Polo, Evgenii N. Tcyganov, Charles Mulligan, Jr., Brian Nam, Joseph Bennett, Gregory Masters, Michael Guarino, Amit Kumar, Yulia Nefedova, Robert H. Vonderheide, Lucia R. Languino, Scott I. Abrams, Dmitry I. Gabrilovich
The Journal of Experimental Medicine, doi:10.1084/jem.20181952
We have identified a precursor that differentiates into granulocytes in vitro and in vivo yet belongs to the monocytic lineage. We have termed these cells monocyte-like precursors of granulocytes (MLPGs). Under steady state conditions, MLPGs were absent in the spleen and barely detectable in the bone marrow (BM). In contrast, these cells significantly expanded in tumor-bearing mice and differentiated to polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Selective depletion of monocytic cells had no effect on the number of granulocytes in naive mice but decreased the population of PMN-MDSCs in tumor-bearing mice by 50%. The expansion of MLPGs was found to be controlled by the down-regulation of Rb1, but not IRF8, which is known to regulate the expansion of PMN-MDSCs from classic granulocyte precursors. In cancer patients, putative MLPGs were found within the population of CXCR1+CD15−CD14+HLA-DR−/lo monocytic cells. These findings describe a mechanism of abnormal myelopoiesis in cancer and suggest potential new approaches for selective targeting of MDSCs.
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Antisense oligonucleotide treatment ameliorates IFN-γ–induced proteinuria in APOL1-transgenic mice
Mariam Aghajan, Sheri L. Booten, Magnus Althage, Christopher E. Hart, Anette Ericsson, Ingela Maxvall, Joseph Ochaba, Angela Menschik-Lundin, Judith Hartleib, Steven Kuntz, Danielle Gattis, Christine Ahlström, Andrew T. Watt, Jeffery A. Engelhardt, Brett P. Monia, Maria Chiara Magnone, and Shuling Guo
African Americans develop end-stage renal disease at a higher rate compared with European Americans due to 2 polymorphisms (G1 and G2 risk variants) in the apolipoprotein L1 (APOL1) gene common in people of African ancestry. Although this compelling genetic evidence provides an exciting opportunity for personalized medicine in chronic kidney disease, drug discovery efforts have been greatly hindered by the fact that APOL1 expression is lacking in rodents. Here, we describe a potentially novel physiologically relevant genomic mouse model of APOL1-associated renal disease that expresses human APOL1 from the endogenous human promoter, resulting in expression in similar tissues and at similar relative levels as humans. While naive APOL1-transgenic mice did not exhibit a renal disease phenotype, administration of IFN-γ was sufficient to robustly induce proteinuria only in APOL1 G1 mice, despite inducing kidney APOL1 expression in both G0 and G1 mice, serving as a clinically relevant “second hit.” Treatment of APOL1 G1 mice with IONIS-APOL1Rx, an antisense oligonucleotide (ASO) targeting APOL1 mRNA, prior to IFN-γ challenge robustly and dose-dependently inhibited kidney and liver APOL1 expression and protected against IFN-γ–induced proteinuria, indicating that the disease-relevant cell types are sensitive to ASO treatment. Therefore, IONIS-APOL1Rx may be an effective therapeutic for APOL1 nephropathies and warrants further development.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Targeting enhancer switching overcomes non-genetic drug resistance in acute myeloid leukaemia
Charles C. Bell, Katie A. Fennell, Yih-Chih Chan, Florian Rambow, Miriam M. Yeung, Dane Vassiliadis, Luis Lara, Paul Yeh, Luciano G. Martelotto, Aljosja Rogiers, Brandon E. Kremer, Olena Barbash, Helai P. Mohammad, Timothy M. Johanson, Marian L. Burr, Arindam Dhar, Natalie Karpinich, Luyi Tian, Dean S. Tyler, Laura MacPherson, Junwei Shi, Nathan Pinnawala, Chun Yew Fong, Anthony T. Papenfuss, Sean M. Grimmond, Sarah-Jane Dawson, Rhys S. Allan, Ryan G. Kruger, Christopher R. Vakoc, David L. Goode, Shalin H. Naik, Omer Gilan, Enid Y. N. Lam, Jean-Christophe Marine, Rab K. Prinjha & Mark A. Dawson
Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Differences between dorsal root and trigeminal ganglion nociceptors in mice revealed by translational profiling
Salim Megat, Pradipta R. Ray, Diana Tavares-Ferreira, Jamie K. Moy, Ishwarya Sankaranarayanan, Andi Wanghzou, Tzu Fang Lou, Paulino Barragan-Iglesias, Zachary T. Campbell, Gregory Dussor, Theodore J. Price
The Journal of Neuroscience, doi:10.1523/JNEUROSCI.2663-18.2019
Nociceptors located in the TG and DRG are the primary sensors of damaging or potentially damaging stimuli for the head and body, respectively, and are key drivers of chronic pain states. While nociceptors in these two tissues show a high degree of functional similarity, there are important differences in their development lineages, their functional connections to the central nervous system, and recent genome-wide analyses of gene expression suggest that they possess some unique genomic signatures. Here, we used translating ribosome affinity purification (TRAP) to comprehensively characterize and compare mRNA translation in Scn10a-positive nociceptors in the TG and DRG of male and female mice. This unbiased method independently confirms several findings of differences between TG and DRG nociceptors described in the literature but also suggests preferential utilization of key signaling pathways. Most prominently, we provide evidence that translational efficiency in mechanistic target of rapamycin (mTOR)-related genes is higher in the TG compared to DRG while several genes associated with the negative regulator of mTOR, AMPK activated protein kinase (AMPK), have higher translational efficiency in DRG nociceptors. Using capsaicin as a sensitizing stimulus we show that behavioral responses are greater in the TG region and this effect is completely reversible with mTOR inhibition. These findings have implications for the relative capacity of these nociceptors to be sensitized upon injury. Altogether, our data provide a comprehensive, comparative view of transcriptome and translatome activity in TG and DRG nociceptors that enhances our understanding of nociceptor biology.
SIGNIFICANCE STATEMENT
The DRG and TG provide sensory information from the body and head, respectively. Nociceptors in these tissues are critical first neurons in the pain pathway. Injury to peripheral neurons in these tissues can cause chronic pain. Interestingly, clinical and preclinical findings support the conclusion that injury to TG neurons is more likely to cause chronic pain and chronic pain in the TG area is more intense and more difficult to treat. We used TRAP technology to gain new insight into potential differences in the translatomes of DRG and TG neurons. Our findings demonstrate previously unrecognized differences between TG and DRG nociceptors that provide new insight into how injury may differentially drive plasticity states in nociceptors in these two tissues.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina and RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat)
Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia
Gerard Minuesa, Steven K. Albanese, Wei Xie, Yaniv Kazansky, Daniel Worroll, Arthur Chow, Alexandra Schurer, Sun-Mi Park, Christina Z. Rotsides, James Taggart, Andrea Rizzi, Levi N. Naden, Timothy Chou, Saroj Gourkanti, Daniel Cappel, Maria C. Passarelli, Lauren Fairchild, Carolina Adura, J. Fraser Glickman, Jessica Schulman, Christopher Famulare, Minal Patel, Joseph K. Eibl, Gregory M. Ross, Shibani Bhattacharya, Derek S. Tan, Christina S. Leslie, Thijs Beuming, Dinshaw J. Patel, Yehuda Goldgur, John D. Chodera & Michael G. Kharas
The MUSASHI (MSI) family of RNA binding proteins (MSI1 and MSI2) contribute to a wide spectrum of cancers including acute myeloid leukemia. We find that the small molecule Ro 08–2750 (Ro) binds directly and selectively to MSI2 and competes for its RNA binding in biochemical assays. Ro treatment in mouse and human myeloid leukemia cells results in an increase in differentiation and apoptosis, inhibition of known MSI-targets, and a shared global gene expression signature similar to shRNA depletion of MSI2. Ro demonstrates in vivo inhibition of c-MYC and reduces disease burden in a murine AML leukemia model. Thus, we identify a small molecule that targets MSI’s oncogenic activity. Our study provides a framework for targeting RNA binding proteins in cancer.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
The Role of Transcription Factors and Nuclear Pore Proteins in Controlling the Spatial Organization of the Yeast Genome
Donna Garvey Brickner, Carlo Randise-Hinchliff, Marine Lebrun Corbin, Julie Ming Liang, Stephanie Kim, Bethany Sump, Agustina D’Urso, Seo Hyun Kim, Atsushi Satomura, Heidi Schmit, Robert Coukos, Subin Hwang, Raven Watson, Jason H. Brickner,
Loss of nuclear pore complex (NPC) proteins, transcription factors (TFs), histone modification enzymes, Mediator, and factors involved in mRNA export disrupts the physical interaction of chromosomal sites with NPCs. Conditional inactivation and ectopic tethering experiments support a direct role for the TFs Gcn4 and Nup2 in mediating interaction with the NPC but suggest an indirect role for factors involved in mRNA export or transcription. A conserved “positioning domain” within Gcn4 controls interaction with the NPC and inter-chromosomal clustering and promotes transcription of target genes. Such a function may be quite common; a comprehensive screen reveals that tethering of most yeast TFs is sufficient to promote targeting to the NPC. While some TFs require Nup100, others do not, suggesting two distinct targeting mechanisms. These results highlight an important and underappreciated function of TFs in controlling the spatial organization of the yeast genome through interaction with the NPC.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Fall armyworm (Spodoptera frugiperda Smith) feeding elicits differential defense responses in upland and lowland switchgrass
Nathan A. Palmer, Saumik Basu, Tiffany Heng-Moss, Jeffrey D. Bradshaw, Gautam Sarath, Joe Louis
Switchgrass (Panicum virgatum L.) is a low input, high biomass perennial grass being developed for the bioenergy sector. Upland and lowland cultivars can differ in their responses to insect herbivory. Fall armyworm [FAW; Spodoptera frugiperda JE Smith (Lepidoptera: Noctuidae)] is a generalist pest of many plant species and can feed on switchgrass as well. Here, in two different trials, FAW larval mass were significantly reduced when fed on lowland cultivar Kanlow relative to larvae fed on upland cultivar Summer plants after 10 days. Hormone content of plants indicated elevated levels of the plant defense hormone jasmonic acid (JA) and its bioactive conjugate JA-Ile although significant differences were not observed. Conversely, the precursor to JA, 12-oxo-phytodienoic acid (OPDA) levels were significantly different between FAW fed Summer and Kanlow plants raising the possibility of differential signaling by OPDA in the two cultivars. Global transcriptome analysis revealed a stronger response in Kanlow plant relative to Summer plants. Among these changes were a preferential upregulation of several branches of terpenoid and phenylpropanoid biosynthesis in Kanlow plants suggesting that enhanced biosynthesis or accumulation of antifeedants could have negatively impacted FAW larval mass gain on Kanlow plants relative to Summer plants. A comparison of the switchgrass-FAW RNA-Seq dataset to those from maize-FAW and switchgrass-aphid interactions revealed that key components of plant responses to herbivory, including induction of JA biosynthesis, key transcription factors and JA-inducible genes were apparently conserved in switchgrass and maize. In addition, these data affirm earlier studies with FAW and aphids that the cultivar Kanlow can provide useful genetics for the breeding of switchgrass germplasm with improved insect resistance.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
MITF controls the TCA cycle to modulate the melanoma hypoxia response
Pakavarin Louphrasitthiphol, Ioanna Ledaki, Jagat Chauhan, Paola Falletta, Robert Siddaway, Francesca M. Buffa, David R. Mole, Tomoyoshi Soga, Colin R. Goding
In response to the dynamic intra‐tumor microenvironment, melanoma cells adopt distinct phenotypic states associated with differential expression of the microphthalmia‐associated transcription factor (MITF). The response to hypoxia is driven by hypoxia‐inducible transcription factors (HIFs) that reprogram metabolism and promote angiogenesis. HIF1α indirectly represses MITF that can activate HIF1α expression. Although HIF and MITF share a highly related DNA‐binding specificity, it is unclear whether they co‐regulate subset of target genes. Moreover, the genomewide impact of hypoxia on melanoma and whether melanoma cell lines representing different phenotypic states exhibit distinct hypoxic responses is unknown. Here we show that three different melanoma cell lines exhibit widely different hypoxia responses with only a core 23 genes regulated in common after 12 hr in hypoxia. Surprisingly, under hypoxia MITF is transiently up‐regulated by HIF1α and co‐regulates a subset of HIF targets including VEGFA . Significantly, we also show that MITF represses itself and also regulates SDHB to control the TCA cycle and suppress pseudo‐hypoxia. Our results reveal a previously unsuspected role for MITF in metabolism and the network of factors underpinning the hypoxic response in melanoma.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
CYFIP1 overexpression increases fear response in mice but does not affect social or repetitive behavioral phenotypes
Catherine Fricano-Kugler, Aaron Gordon, Grace Shin, Kun Gao, Jade Nguyen, Jamee Berg, Mary Starks & Daniel H. Geschwind
Background
CYFIP1, a protein that interacts with FMRP and regulates protein synthesis and actin dynamics, is overexpressed in Dup15q syndrome as well as autism spectrum disorder (ASD). While CYFIP1 heterozygosity has been rigorously studied due to its loss in 15q11.2 deletion, Prader-Willi and Angelman syndrome, the effects of CYFIP1 overexpression, as is observed in patients with CYFIP1 duplication, are less well understood.
Methods
We developed and validated a mouse model of human CYFIP1 overexpression (CYFIP1 OE) using qPCR and western blot analysis. We performed a large battery of behavior testing on these mice, including ultrasonic vocalizations, three-chamber social assay, home-cage behavior, Y-maze, elevated plus maze, open field test, Morris water maze, fear conditioning, prepulse inhibition, and the hot plate assay. We also performed RNA sequencing and analysis on the basolateral amygdala.
Results
Extensive behavioral testing in CYFIP1 OE mice reveals no changes in the core behaviors related to ASD: social interactions and repetitive behaviors. However, we did observe mild learning deficits and an exaggerated fear response. Using RNA sequencing of the basolateral amygdala, a region associated with fear response, we observed changes in pathways related to cytoskeletal regulation, oligodendrocytes, and myelination. We also identified GABA-A subunit composition changes in basolateral amygdala neurons, which are essential components of the neural fear conditioning circuit.
Conclusion
Overall, this research identifies the behavioral and molecular consequences of CYFIP1 overexpression and how they contribute to the variable phenotype seen in Dup15q syndrome and in ASD patients with excess CYFIP1.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Comprehensive Profiling of Primary and Metastatic ccRCC Reveals a High Homology of the Metastases to a Subregion of the Primary Tumour
Paranita Ferronika, Joost Hof, Gursah Kats-Ugurlu, Rolf H. Sijmons, Martijn M. Terpstra, Kim de Lange, Annemarie Leliveld-Kors, Helga Westers and Klaas Kok
While intratumour genetic heterogeneity of primary clear cell renal cell carcinoma (ccRCC) is well characterized, the genomic profiles of metastatic ccRCCs are seldom studied. We profiled the genomes and transcriptomes of a primary tumour and matched metastases to better understand the evolutionary processes that lead to metastasis. In one ccRCC patient, four regions of the primary tumour, one region of the thrombus in the inferior vena cava, and four lung metastases (including one taken after pegylated (PEG)-interferon therapy) were analysed separately. Each sample was analysed for copy number alterations and somatic mutations by whole exome sequencing. We also evaluated gene expression profiles for this patient and 15 primary tumour and 15 metastasis samples from four additional patients. Copy number profiles of the index patient showed two distinct subgroups: one consisted of three primary tumours with relatively minor copy number changes, the other of a primary tumour, the thrombus, and the lung metastases, all with a similar copy number pattern and tetraploid-like characteristics. Somatic mutation profiles indicated parallel clonal evolution with similar numbers of private mutations in each primary tumour and metastatic sample. Expression profiling of the five patients revealed significantly changed expression levels of 57 genes between primary tumours and metastases, with enrichment in the extracellular matrix cluster. The copy number profiles suggest a punctuated evolution from a subregion of the primary tumour. This process, which differentiated the metastases from the primary tumours, most likely occurred rapidly, possibly even before metastasis formation. The evolutionary patterns we deduced from the genomic alterations were also reflected in the gene expression profiles.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Critical role of the fibroblast growth factor signalling pathway in Ewing’s sarcoma octamer‐binding transcription factor 4‐mediated cell proliferation and tumorigenesis
Junghoon Kim, Hyo Sun Kim, Jung‐Jae Shim, Jungwoon Lee, Ah‐young Kim, Jungho Kim
Certain bone and soft tissue (BST ) tumours harbour a chromosomal translocation [t(6;22)(p21;q12)], which fuses the Ewing’s sarcoma (EWS ) gene at 22q12 with the octamer‐binding transcription factor 4 (Oct‐4 ) gene at 6p21, resulting in the chimeric EWS ‐Oct‐4 protein that possesses high transactivation ability. Although abnormal activation of signalling pathways can lead to human cancer development, the pathways underlying these processes in human BST tumours remain poorly explored. Here, we investigated the functional significance of fibroblast growth factor (FGF ) signalling in human BST tumours. To identify the gene(s) involved in the FGF signalling pathway and potentially regulated by EWS ‐Oct‐4 (also called EWS ‐POU 5F1), we performed RNA ‐Seq analysis, electrophoretic mobility shift assays, chromatin immunoprecipitation assays, and xenograft assays. Treating GBS 6 or ZHBT c4 cells‐expressing EWS ‐Oct‐4 with the small molecule FGF receptor (FGFR ) inhibitors PD 173074, NVPBGJ 398, ponatinib, and dovitinib suppressed cellular proliferation. Gene expression analysis revealed that, among 22 Fgf and four Fgfr family members, Fgf‐4 showed the highest upregulation (by 145‐fold) in ZHBT c4 cells‐expressing EWS ‐Oct‐4. Computer‐assisted analysis identified a putative EWS ‐Oct‐4‐binding site at +3017/+3024, suggesting that EWS ‐Oct‐4 regulates Fgf‐4 expression in human BST tumours. Fgf‐4 enhancer constructs showed that EWS ‐Oct‐4 transactivated the Fgf‐4 gene reporter in vitro , and that overexpression of EWS ‐Oct‐4 stimulated endogenous Fgf‐4 gene expression in vivo . Finally, PD 173074 significantly decreased tumour volume in mice. Taken together, these data suggest that FGF ‐4 signalling is involved in EWS ‐Oct‐4‐mediated tumorigenesis, and that its inhibition impairs tumour growth in vivo significantly.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Quantitative global studies reveal differential translational control by start codon context across the fungal kingdom
Edward W J Wallace, Corinne Maufrais, Jade Sales-Lee, Laura R Tuck, Luciana de Oliveira, Frank Feuerbach, Frédérique Moyrand, Prashanthi Natarajan, Hiten D Madhani, Guilhem Janbon
Eukaryotic protein synthesis generally initiates at a start codon defined by an AUG and its surrounding Kozak sequence context, but the quantitative importance of this context in different species is unclear. We tested this concept in two pathogenic Cryptococcus yeast species by genome-wide mapping of translation and of mRNA 5′ and 3′ ends. We observed thousands of AUG-initiated upstream open reading frames (uORFs) that are a major contributor to translation repression. uORF use depends on the Kozak sequence context of its start codon, and uORFs with strong contexts promote nonsense-mediated mRNA decay. Transcript leaders in Cryptococcus and other fungi are substantially longer and more AUG-dense than in Saccharomyces. Numerous Cryptococcus mRNAs encode predicted dual-localized proteins, including many aminoacyl-tRNA synthetases, in which a leaky AUG start codon is followed by a strong Kozak context in-frame AUG, separated by mitochondrial-targeting sequence. Analysis of other fungal species shows that such dual-localization is also predicted to be common in the ascomycete mould, Neurospora crassa. Kozak-controlled regulation is correlated with insertions in translational initiation factors in fidelity-determining regions that contact the initiator tRNA. Thus, start codon context is a signal that quantitatively programs both the expression and the structures of proteins in diverse fungi.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Bisphenol A Promotes the Invasive and Metastatic Potential of Ductal Carcinoma In Situ and Protumorigenic Polarization of Macrophages
Hyelim Kim, Hoe Suk Kim, Yin Ji Piao, Woo Kyung Moon
Increased cancer risk and immune disorders linked with exposure to environmental endocrine disruptors like bisphenol A (BPA) have been steadily reported. Nevertheless, the impacts of BPA on the breast ductal carcinoma in situ (DCIS) progression and macrophage polarization remain to be elucidated. Here, we analyzed the differentially expressed genes in BPA-exposed DCIS cells and explored BPA effects on DCIS progression and macrophage polarization in vitro and in vivo. Two hundred and ninety-one genes were differentially expressed in 10−8 M BPA-exposed DCIS cells, in which the gene ontology terms of biological processes associated with negative regulation of cell death, cell adhesion, and immune response was enriched. 10−8 M BPA promoted the proliferation and migration of DCIS cells and the migration of macrophages, and upregulated the expression of M1 (NOS2) or M2 markers (Arg-1 and CD206) in macrophages. In coculture system, the migratory capacity of both cells and the expression levels of NOS2, Arg-1, and CD206 in macrophages were significantly enhanced upon 10−8 M BPA. In a DCIS xenograft model, oral exposure to an environmentally human-relevant low dose of 2.5 µg/l BPA for 70 days via drinking water led to an approximately 2-fold promotion in the primary tumor growth rate and a significant enhancement of lymph node metastasis along with increased protumorigenic CD206+ M2 polarization of macrophages. These results demonstrate that BPA acts as an accelerator to promote DCIS progression to invasive breast cancer by affecting DCIS cell proliferation and migration as well macrophage polarization toward a protumorigenic phenotype.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
KRASG12C inhibition produces a driver-limited state revealing collateral dependencies
Kevin Lou, Veronica Steri, Alex Y. Ge, Y. Christina Hwang, Christopher H. Yogodzinski, Arielle R. Shkedi, Alex L. M. Choi, Dominique C. Mitchell, Danielle L. Swaney, Byron Hann, John D. Gordan, Kevan M. Shokat, and Luke A. Gilbert
Inhibitors targeting KRASG12C, a mutant form of the guanosine triphosphatase (GTPase) KRAS, are a promising new class of oncogene-specific therapeutics for the treatment of tumors driven by the mutant protein. These inhibitors react with the mutant cysteine residue by binding covalently to the switch-II pocket (S-IIP) that is present only in the inactive guanosine diphosphate (GDP)–bound form of KRASG12C, sparing the wild-type protein. We used a genome-scale CRISPR interference (CRISPRi) functional genomics platform to systematically identify genetic interactions with a KRASG12C inhibitor in cellular models of KRASG12C mutant lung and pancreatic cancer. Our data revealed genes that were selectively essential in this oncogenic driver–limited cell state, meaning that their loss enhanced cellular susceptibility to direct KRASG12C inhibition. We termed such genes “collateral dependencies” (CDs) and identified two classes of combination therapies targeting these CDs that increased KRASG12C target engagement or blocked residual survival pathways in cells and in vivo. From our findings, we propose a framework for assessing genetic dependencies induced by oncogene inhibition.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Targeting IDH1 as a Prosenescent Therapy in High-grade Serous Ovarian Cancer
Erika S. Dahl, Raquel Buj, Kelly E. Leon, Jordan M. Newell, Yuka Imamura, Benjamin G. Bitler, Nathaniel W. Snyder and Katherine M. Aird
Molecular Cancer Research, doi:10.1158/1541-7786.MCR-18-1233
Epithelial ovarian cancer (EOC) is the deadliest gynecologic cancer. High-grade serous carcinoma (HGSC) is the most frequently diagnosed and lethal histosubtype of EOC. A significant proportion of patients with HGSC relapse with chemoresistant disease. Therefore, there is an urgent need for novel therapeutic strategies for HGSC. Metabolic reprogramming is a hallmark of cancer cells, and targeting metabolism for cancer therapy may be beneficial. Here, we found that in comparison with normal fallopian tube epithelial cells, HGSC cells preferentially utilize glucose in the TCA cycle and not for aerobic glycolysis. This correlated with universally increased TCA cycle enzyme expression in HGSC cells under adherent conditions. HGSC disseminates as tumor cell spheroids within the peritoneal cavity. We found that wild-type isocitrate dehydrogenase I (IDH1) is the only TCA cycle enzyme upregulated in both adherent and spheroid conditions and is associated with reduced progression-free survival. IDH1 protein expression is also increased in patients with primary HGSC tumors. Pharmacologic inhibition or knockdown of IDH1 decreased proliferation of multiple HGSC cell lines by inducing senescence. Mechanistically, suppression of IDH1 increased the repressive histone mark H3K9me2 at multiple E2F target gene loci, which led to decreased expression of these genes. Altogether, these data suggest that increased IDH1 activity is an important metabolic adaptation in HGSC and that targeting wild-type IDH1 in HGSC alters the repressive histone epigenetic landscape to induce senescence.
Implications: Inhibition of IDH1 may act as a novel therapeutic approach to alter both the metabolism and epigenetics of HGSC as a prosenescent therapy.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Background/Aim: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cancer-selective, cell-death-inducing agent with little toxicity to normal cells. However, various human cancers and cancer cell lines have been reported to be resistant to TRAIL. Molecular clarification of resistance mechanism is needed. Materials and Methods: Compound screening, proliferation assays, western blotting, and flow cytometry were used to examine the sensitizer activity of methyl transferase inhibitor BIX-01294 in combination with TRAIL, in hepatocellular carcinoma (HCC) cells. RNA sequencing analysis and single guide (sg)RNA-mediated gene deletion were used to investigate the role of survivin in sensitization. Results: In HCC cells, BIX-01294 enhanced TRAIL sensitivity by reducing survivin expression at the RNA level. Small interference RNA-mediated gene knockdown demonstrated the mechanism of sensitization to be via the reduction of survivin. Conclusion: Euchromatin histone methyltransferase 2 (EHMT2) inhibition by BIX-01294 may be a potent anti-tumor therapeutic strategy for human HCC.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Differential gene expression profile analysis of RIG-I- versus type I IFN-stimulated primary human nasal epithelial cells and protection from influenza virus replication
Evelyn Hartmann, Vanessa von Lilien Waldau, Jan Phillip Stümpel, Janos Ludwig, Martin Schlee, Christoph Coch and Gunther Hartmann
Nasal mucosa is a key barrier against upper airway infection including influenza. Trained innate antiviral immunity has the potential to broadly protect the host from a variety of respiratory viruses. Although exposure of cells to type I IFN confers some degree of protection, direct stimulation of innate immune sensors may induce a more effective and longer lasting antiviral immunity.
In the present study we analysed the gene expression profile and functional consequence of direct activation of RIG-I by its specific ligand 5′-triphosphate RNA (3pRNA) in primary human nasal epithelial cells, and compared these effects to stimulation of other innate receptors and stimulation by type I IFN. Gene expression analysis was carried out by Lexogen QuantSeq 3′mRNA sequencing. For functional analysis, epithelial cells were exposed to H1N1 influenza virus strain A/PR/8/34. Viral replication was analysed by qPCR.
Gene set enrichment analysis revealed upregulation of pathways related to antiviral immune response and type I Interferon signaling. Activation of RIG-I in epithelial cells induced a broad panel of antiviral defense mechanisms which extends beyond those stimulated by recombinant type I IFN. RIG-I-stimulated cells were completely protected from influenza viral replication, while viability of the cells was not reduced. Recombinant IFN-b resulted in lower protection against viral replication compared to 3pRNA, demonstrating that RIG-I induces additional functional antiviral activities not induced by exogenous type I IFN acting alone.
Thus, our findings support the concept that stimulation of RIG in epithelial cells is a promising strategy to protect cells from viruses including emerging viral infections of the respiratory tract.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Rhythmic Food Intake Drives Rhythmic Gene Expression More Potently than the Hepatic Circadian Clock in Mice
Ben J. Greenwell, Alexandra J. Trott, Joshua R. Beytebiere, Shanny Pao, Alexander Bosley, Erin Beach, Patrick Finegan, Christopher Hernandez, Jerome S. Menet
Every mammalian tissue exhibits daily rhythms in gene expression to control the activation of tissue-specific processes at the most appropriate time of the day. Much of this rhythmic expression is thought to be driven cell autonomously by molecular circadian clocks present throughout the body. By manipulating the daily rhythm of food intake in the mouse, we here show that more than 70% of the cycling mouse liver transcriptome loses rhythmicity under arrhythmic feeding. Remarkably, core clock genes are not among the 70% of genes losing rhythmic expression, and their expression continues to exhibit normal oscillations in arrhythmically fed mice. Manipulation of rhythmic food intake also alters the timing of key signaling and metabolic pathways without altering the hepatic clock oscillations. Our findings thus demonstrate that systemic signals driven by rhythmic food intake significantly contribute to driving rhythms in liver gene expression and metabolic functions independently of the cell-autonomous hepatic clock.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Selective Roles of Vertebrate PCF11 in Premature and Full-Length Transcript Termination
Kinga Kamieniarz-Gdula, Michal R. Gdula, Karin Panser, Takayuki Nojima, Joan Monks, Jacek R. Wiśniewski, Joey Riepsaame, Neil Brockdorff, Andrea Pauli, Nick J. Proudfoot
The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including mNET-seq, 3′ mRNA-seq, chromatin RNA-seq, and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and consequent gene downregulation. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina and QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina
Diphenyl Phosphate-Induced Toxicity During Embryonic Development
Constance A. Mitchell, Aalekhya Reddam, Subham Dasgupta, Sharon Zhang, Heather M. Stapleton, and David C. Volz
Environmental science & technology, doi:10.1021/acs.est.8b07238
Diphenyl phosphate (DPHP) is an aryl phosphate ester (APE) used as an industrial catalyst and chemical additive and is the primary metabolite of flame retardant APEs, including triphenyl phosphate (TPHP). Minimal DPHP-specific toxicity studies have been published despite ubiquitous exposure within human populations following metabolism of TPHP and other APEs. Therefore, the objective of this study was to determine the potential for DPHP-induced toxicity during embryonic development. Using zebrafish as a model, we found that DPHP significantly increased the distance between the sinus venosus and bulbus arteriosis (SV-BA) at 72 h postfertilization (hpf) following initiation of exposure before and after cardiac looping. Interestingly, pretreatment with d-mannitol mitigated DPHP-induced effects on SV-BA length despite the absence of DPHP effects on pericardial area, suggesting that DPHP-induced cardiac defects are independent of pericardial edema formation. Using mRNA-sequencing, we found that DPHP disrupts pathways related to mitochondrial function and heme biosynthesis; indeed, DPHP significantly decreased hemoglobin levels in situ at 72 hpf following exposure from 24 to 72 hpf. Overall, our findings suggest that, similar to TPHP, DPHP impacts cardiac development, albeit the potency of DPHP is significantly less than TPHP within developing zebrafish.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Synthetic Lethality Screening Identifies FDA-Approved Drugs that Overcome ATP7B-Mediated Tolerance of Tumor Cells to Cisplatin
Marta Mariniello, Raffaella Petruzzelli, Luca G. Wanderlingh, Raffaele La Montagna, Annamaria Carissimo, Francesca Pane, Angela Amoresano, Ekaterina Y. Ilyechova, Michael M. Galagudza, Federico Catalano, Roberta Crispino, Ludmila V. Puchkova, Diego L. Medina 1 and Roman S. Polishchuk
Tumor resistance to chemotherapy represents an important challenge in modern oncology. Although platinum (Pt)-based drugs have demonstrated excellent therapeutic potential, their effectiveness in a wide range of tumors is limited by the development of resistance mechanisms. One of these mechanisms includes increased cisplatin sequestration/efflux by the copper-transporting ATPase, ATP7B. However, targeting ATP7B to reduce Pt tolerance in tumors could represent a serious risk because suppression of ATP7B might compromise copper homeostasis, as happens in Wilson disease. To circumvent ATP7B-mediated Pt tolerance we employed a high-throughput screen (HTS) of an FDA/EMA-approved drug library to detect safe therapeutic molecules that promote cisplatin toxicity in the IGROV-CP20 ovarian carcinoma cells, whose resistance significantly relies on ATP7B. Using a synthetic lethality approach, we identified and validated three hits (Tranilast, Telmisartan, and Amphotericin B) that reduced cisplatin resistance. All three drugs induced Pt-mediated DNA damage and inhibited either expression or trafficking of ATP7B in a tumor-specific manner. Global transcriptome analyses showed that Tranilast and Amphotericin B affect expression of genes operating in several pathways that confer tolerance to cisplatin. In the case of Tranilast, these comprised key Pt-transporting proteins, including ATOX1, whose suppression affected ability of ATP7B to traffic in response to cisplatin. In summary, our findings reveal Tranilast, Telmisartan, and Amphotericin B as effective drugs that selectively promote cisplatin toxicity in Pt-resistant ovarian cancer cells and underscore the efficiency of HTS strategy for identification of biosafe compounds, which might be rapidly repurposed to overcome resistance of tumors to Pt-based chemotherapy.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Transcriptome-wide effect of DE-ETIOLATED1 (DET1) suppression in embryogenic callus of Carica papaya
Nur Diyana Jamaluddin, Emelda Rosseleena Rohani, Normah Mohd Noor & Hoe-Han Goh
Papaya is one of the most nutritional fruits, rich in vitamins, carotenoids, flavonoids and other antioxidants. Previous studies showed phytonutrient improvement without affecting quality in tomato fruit and rapeseed through the suppression of DE-ETIOLATED-1 (DET1), a negative regulator in photomorphogenesis. This study is conducted to study the effects of DET1 gene suppression in papaya embryogenic callus. Immature zygotic embryos were transformed with constitutive expression of a hairpin DET1 construct (hpDET1). PCR screening of transformed calli and reverse transcription quantitative PCR (RT-qPCR) verified that DET1 gene downregulation in two of the positive transformants. High-throughput cDNA 3′ ends sequencing on DET1-suppressed and control calli for transcriptomic analysis of global gene expression identified a total of 452 significant (FDR < 0.05) differentially expressed genes (DEGs) upon DET1 suppression. The 123 upregulated DEGs were mainly involved in phenylpropanoid biosynthesis and stress responses, compared to 329 downregulated DEGs involved in developmental processes, lipid metabolism, and response to various stimuli. This is the first study to demonstrate transcriptome-wide relationship between light-regulated pathway and secondary metabolite biosynthetic pathways in papaya. This further supports that the manipulation of regulatory gene involved in light-regulated pathway is possible for phytonutrient improvement of tropical fruit crops.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
In Vivo CRISPRa Decreases Seizures and Rescues Cognitive Deficits in a Rodent Model of Epilepsy
Gaia Colasante, Yichen Qiu, Luca Massimino, Claudia Di Berardino, Jonathan H Cornford, Albert Snowball, Mikail Weston, Steffan P Jones, Serena Giannelli, Andreas Lieb, Stephanie Schorge, Dimitri M Kullmann, Vania Broccoli, Gabriele Lignani
Epilepsy is a major health burden, calling for new mechanistic insights and therapies. CRISPR-mediated gene editing shows promise to cure genetic pathologies, although hitherto it has mostly been applied ex vivo. Its translational potential for treating non-genetic pathologies is still unexplored. Furthermore, neurological diseases represent an important challenge for the application of CRISPR, because of the need in many cases to manipulate gene function of neurons in situ. A variant of CRISPR, CRISPRa, offers the possibility to modulate the expression of endogenous genes by directly targeting their promoters. We asked if this strategy can effectively treat acquired focal epilepsy, focusing on ion channels because their manipulation is known be effective in changing network hyperactivity and hypersynchronziation. We applied a doxycycline-inducible CRISPRa technology to increase the expression of the potassium channel gene Kcna1 (encoding Kv1.1) in mouse hippocampal excitatory neurons. CRISPRa-mediated Kv1.1 upregulation led to a substantial decrease in neuronal excitability. Continuous video-EEG telemetry showed that AAV9-mediated delivery of CRISPRa, upon doxycycline administration, decreased spontaneous generalized tonic-clonic seizures in a model of temporal lobe epilepsy, and rescued cognitive impairment and transcriptomic alterations associated with chronic epilepsy. The focal treatment minimizes concerns about off-target effects in other organs and brain areas. This study provides the proof-of-principle for a translational CRISPR-based approach to treat neurological diseases characterized by abnormal circuit excitability.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
The role of the miR-17-92 cluster in macrophage driven innate immunity * PhD Thesis
H. James Stunden
Research into miRNA, discovered in 1993, has exploded and revolutionized our understanding of molecular regulation in biological systems. MicroRNAs are now a well established as a regulatory mechanism of many pathways and functions within cells of eukaryotic organisms, though much needs to be learnt about the intricacies of such regulation. In recent years, targeting this system of post-translational regulation has been a goal of many therapeutics, but requires much greater insight into how miRNA work, and the broadness of their activity.
The innate immune system is critical for mounting an effective response against invading pathogens and protecting the host from damage. But being such a powerful system, unchecked it can wreak havoc on the host itself. While the innate immune system is tightly regulated by many mechanisms, further understanding could lead to major advances in therapeutics of autoimmune diseases. As the miR-17-92 cluster has already been identified as a regulator of innate immune functions, and continued research in animal models is necessary for therapeutics to become a reality.
This thesis focuses on the role and function of the miR-17-92 cluster within macrophages, which are a major component of the innate immune system. It highlights the complexity and often subtle nature of microRNA regulation in biological systems. It describes the generation of a mouse line with a myeloid-specific deletion of the miR-17-92 cluster is described, and shown that despite this deletion, there is no change both to the innate immune response of these mice, or to the TLR signalling cascade. It is postulated that while the miR-17-92 cluster affects innate immune signaling in some other cell types, it is unlikely to have a similar role in macrophages.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Cardiomyocyte Differentiation of hiPSCs in 2D and 3D Culture Systems:
A Comparative Study and Transcriptomic Analysis * MSc Thesis
João Pedro Brinquete Cotovio
Cardiovascular diseases (CVDs) are the number one human cause of death worldwide, representing 31% of global death with unprecedented costs associated. Accordingly, cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) represent a promising cell source for heart regeneration, disease modelling and drug screening. However, similarities and differences between different culture platforms for cardiomyocyte differentiation, either two-dimensional (2D) or threedimensional (3D), are still poorly explored. To unveil how the particular microenvironment of each platform can be translated into distinct cardiomyocyte properties, in this work, a comparative study between cardiomyocyte differentiation of hiPSCs cultured as 2D adherent monolayer and 3D aggregates was developed. In addition, a transcriptomic analysis throughout sequential stages of cardiomyocyte differentiation for 3D culture system was performed. Although both platforms originate cardiomyocytelike features, distinct molecular signatures between them were found. The results support a faster progression in cardiac development for 3D aggregates that ultimately exhibit a maturation status that closely resembles that of adult cardiomyocytes. It is proposed that stronger Wnt signalling induction in 3D culture is due to larger amounts of E-cadherin (E-CAD) on 3D aggregates, leading to higher pools of β-catenin upon adherens junction dissociation during epithelial-to-mesenchymal transition (EMT). These findings elevate the considerable potential of 3D culture system for cardiomyocyte differentiation and aid in the basic understanding of mechanisms governing either pluripotent spheroids or monolayer during mesoderm commitment, demonstrating that cardiomyocyte differentiation is indeed culture shape-dependent.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Preeclampsia is Associated with Sex-Specific Transcriptional and Proteomic Changes in Fetal Erythroid Cells
Zahra Masoumi, Gregory E. Maes, Koen Herten, Álvaro Cortés-Calabuig, Abdul Ghani Alattar, Eva Hanson, Lena Erlandsson, Eva Mezey, Mattias Magnusson, Joris R Vermeesch, Mary Familari and Stefan R Hansson
International journal of molecular sciences, doi:10.3390/ijms20082038
Preeclampsia (PE) has been associated with placental dysfunction, resulting in fetal hypoxia, accelerated erythropoiesis, and increased erythroblast count in the umbilical cord blood (UCB). Although the detailed effects remain unknown, placental dysfunction can also cause inflammation, nutritional, and oxidative stress in the fetus that can affect erythropoiesis. Here, we compared the expression of surface adhesion molecules and the erythroid differentiation capacity of UCB hematopoietic stem/progenitor cells (HSPCs), UCB erythroid profiles along with the transcriptome and proteome of these cells between male and female fetuses from PE and normotensive pregnancies. While no significant differences were observed in UCB HSPC migration/homing and in vitro erythroid colony differentiation, the UCB HSPC transcriptome and the proteomic profile of the in vitro differentiated erythroid cells differed between PE vs. normotensive samples. Accordingly, despite the absence of significant differences in the UCB erythroid populations in male or female fetuses from PE or normotensive pregnancies, transcriptional changes were observed during erythropoiesis, particularly affecting male fetuses. Pathway analysis suggested deregulation in the mammalian target of rapamycin complex 1/AMP-activated protein kinase (mTORC1/AMPK) signaling pathways controlling cell cycle, differentiation, and protein synthesis. These results associate PE with transcriptional and proteomic changes in fetal HSPCs and erythroid cells that may underlie the higher erythroblast count in the UCB in PE.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Diagnosing enterovirus meningitis via blood transcriptomics: an alternative for lumbar puncture?
Esther Bartholomeus, Nicolas De Neuter, Annelies Lemay, Luc Pattyn, David Tuerlinckx, David Weynants, Koen Van Lede, Gerlant van Berlaer, Dominique Bulckaert, Tine Boiy, Ann Vander Auwera, Marc Raes, Dimitri Van der Linden, Helene Verhelst, Susanne Van Steijn, Tijl Jonckheer, Joke Dehoorne, Rik Joos, Hilde Jansens, Arvid Suls, Pierre Van Damme, Kris Laukens, Geert Mortier, Pieter Meysman & Benson Ogunjimi
Journal of Translational Medicinevolume, doi:10.1186/s12967-019-2037-6
Background
Meningitis can be caused by several viruses and bacteria. Identifying the causative pathogen as quickly as possible is crucial to initiate the most optimal therapy, as acute bacterial meningitis is associated with a significant morbidity and mortality. Bacterial meningitis requires antibiotics, as opposed to enteroviral meningitis, which only requires supportive therapy. Clinical presentation is usually not sufficient to differentiate between viral and bacterial meningitis, thereby necessitating cerebrospinal fluid (CSF) analysis by PCR and/or time-consuming bacterial cultures. However, collecting CSF in children is not always feasible and a rather invasive procedure.
Methods
In 12 Belgian hospitals, we obtained acute blood samples from children with signs of meningitis (49 viral and 7 bacterial cases) (aged between 3 months and 16 years). After pathogen confirmation on CSF, the patient was asked to give a convalescent sample after recovery. 3′ mRNA sequencing was performed to determine differentially expressed genes (DEGs) to create a host transcriptomic profile.
Results
Enteroviral meningitis cases displayed the largest upregulated fold change enrichment in type I interferon production, response and signaling pathways. Patients with bacterial meningitis showed a significant upregulation of genes related to macrophage and neutrophil activation. We found several significantly DEGs between enteroviral and bacterial meningitis. Random forest classification showed that we were able to differentiate enteroviral from bacterial meningitis with an AUC of 0.982 on held-out samples.
Conclusions
Enteroviral meningitis has an innate immunity signature with type 1 interferons as key players. Our classifier, based on blood host transcriptomic profiles of different meningitis cases, is a possible strong alternative for diagnosing enteroviral meningitis.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina and Globin Block Module for QuantSeq
Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition
Miha Modic, Markus Grosch, Gregor Rot, Silvia Schirge, Tjasa Lepko, Tomohiro Yamazaki, Flora C.Y. Lee, Ejona Rusha, Dmitry Shaposhnikov, Michael Palo, Juliane Merl-Pham, Davide Cacchiarelli, Boris Rogelj, Stefanie M. Hauck, Christian von Mering, Alexander Meissner, Heiko Lickert, Tetsuro Hirose, Jernej Ule, Micha Drukker
RNA-binding proteins (RBPs) and long non-coding RNAs (lncRNAs) are key regulators of gene expression, but their joint functions in coordinating cell fate decisions are poorly understood. Here we show that the expression and activity of the RBP TDP-43 and the long isoform of the lncRNA Neat1, the scaffold of the nuclear compartment “paraspeckles,” are reciprocal in pluripotent and differentiated cells because of their cross-regulation. In pluripotent cells, TDP-43 represses the formation of paraspeckles by enhancing the polyadenylated short isoform of Neat1. TDP-43 also promotes pluripotency by regulating alternative polyadenylation of transcripts encoding pluripotency factors, including Sox2, which partially protects its 3′ UTR from miR-21-mediated degradation. Conversely, paraspeckles sequester TDP-43 and other RBPs from mRNAs and promote exit from pluripotency and embryonic patterning in the mouse. We demonstrate that cross-regulation between TDP-43 and Neat1 is essential for their efficient regulation of a broad network of genes and, therefore, of pluripotency and differentiation.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina and QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina
A Non-Dicer RNase III and Four Other Novel Factors Required for RNAi-Mediated Transposon Suppression in the Human Pathogenic Yeast Cryptococcus neoformans
Jordan E. Burke, Adam D. Longhurst, Prashanthi Natarajan, Beiduo Rao, John Liu, Jade Sales-Lee, Yasaman Mortensen, James J. Moresco, Jolene K. Diedrich, John R. Yates and Hiten D. Madhani
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina and Small RNA-Seq Library Prep Kit for Illumina
Clinical and biological predictors of response to standardised paediatric colitis therapy (PROTECT): a multicentre inception cohort study
Prof Jeffrey S Hyams, MD; Prof Sonia Davis Thomas, DrPH; Nathan Gotman, MS; Yael Haberman, MD; Rebekah Karns, PhD; Melanie Schirmer et al.;
Background
Lack of evidence-based outcomes data leads to uncertainty in developing treatment regimens in children who are newly diagnosed with ulcerative colitis. We hypothesised that pretreatment clinical, transcriptomic, and microbial factors predict disease course.
Methods
In this inception cohort study, we recruited paediatric patients aged 4–17 years with newly diagnosed ulcerative colitis from 29 centres in the USA and Canada. Patients initially received standardised mesalazine or corticosteroids, with pre-established criteria for escalation to immunomodulators (ie, thiopurines) or anti-tumor necrosis factor-α (TNFα) therapy. We used RNA sequencing to define rectal gene expression before treatment, and 16S sequencing to characterise rectal and faecal microbiota. The primary outcome was week 52 corticosteroid-free remission with no therapy beyond mesalazine. We assessed factors associated with the primary outcome using logistic regression models of the per-protocol population. This study is registered with ClinicalTrials.gov, number NCT01536535.
Findings
Between July 10, 2012, and April 21, 2015, of 467 patients recruited, 428 started medical therapy, of whom 400 (93%) were evaluable at 52 weeks and 386 (90%) completed the study period with no protocol violations. 150 (38%) of 400 participants achieved week 52 corticosteroid-free remission, of whom 147 (98%) were taking mesalazine and three (2%) were taking no medication. 74 (19%) of 400 were escalated to immunomodulators alone, 123 (31%) anti-TNFα therapy, and 25 (6%) colectomy. Low baseline clinical severity, high baseline haemoglobin, and week 4 clinical remission were associated with achieving week 52 corticosteroid-free remission (n=386, logistic model area under the curve [AUC] 0·70, 95% CI 0·65–0·75; specificity 77%, 95% CI 71–82). Baseline severity and remission by week 4 were validated in an independent cohort of 274 paediatric patients with newly diagnosed ulcerative colitis. After adjusting for clinical predictors, an antimicrobial peptide gene signature (odds ratio [OR] 0·57, 95% CI 0·39–0·81; p=0·002) and abundance of Ruminococcaceae (OR 1·43, 1·02–2·00; p=0·04), and Sutterella (OR 0·81, 0·65–1·00; p=0·05) were independently associated with week 52 corticosteroid-free remission.
Interpretation
Our findings support the utility of initial clinical activity and treatment response by 4 weeks to predict week 52 corticosteroid-free remission with mesalazine alone in children who are newly diagnosed with ulcerative colitis. The development of personalised clinical and biological signatures holds the promise of informing ulcerative colitis therapeutic decisions.
Funding
US National Institutes of Health.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina and Mix2 RNA-Seq Data Analysis Software
Dyslipidemia and the role of adipose tissue in early pregnancy in the BPH/5 mouse model for preeclampsia
Dorien Reijnders, Kelsey N. Olson, Chin-Chi Liu, Kalie F. Beckers, Sujoy Ghosh, Leanne M. Redman, and Jenny L. Sones
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
A systems biology approach uncovers cell-specific gene regulatory effects of genetic associations in multiple sclerosis
Lohith Madireddy, Nikolaos A. Patsopoulos, Chris Cotsapas, Steffan D. Bos, Ashley Beecham, Jacob McCauley, Kicheol Kim, Xiaoming Jia, Adam Santaniello, Stacy J. Caillier, Till F. M. Andlauer, Lisa F. Barcellos, Tone Berge, Luisa Bernardinelli, Filippo Martinelli-Boneschi, David R. Booth, Farren Briggs, Elisabeth G. Celius, Manuel Comabella, Giancarlo Comi, Bruce A. C. Cree, Sandra D’Alfonso, Katrina Dedham, Pierre Duquette, Efthimios Dardiotis, Federica Esposito, Bertrand Fontaine, Christiane Gasperi, An Goris, Bénédicte Dubois, Pierre-Antoine Gourraud, Georgios Hadjigeorgiou, Jonathan Haines, Clive Hawkins, Bernhard Hemmer, Rogier Hintzen, Dana Horakova, Noriko Isobe, Seema Kalra, Jun-ichi Kira, Michael Khalil, Ingrid Kockum, Christina M. Lill, Matthew R. Lincoln, Felix Luessi, Roland Martin, Annette Oturai, Aarno Palotie, Margaret A. Pericak-Vance, Roland Henry, Janna Saarela, Adrian Ivinson, Tomas Olsson, Bruce V. Taylor, Graeme J. Stewart, Hanne F. Harbo, Alastair Compston, Stephen L. Hauser, David A. Hafler, Frauke Zipp, Philip De Jager, Stephen Sawcer, Jorge R. Oksenberg & Sergio E. Baranzini
Genome-wide association studies (GWAS) have identified more than 50,000 unique associations with common human traits. While this represents a substantial step forward, establishing the biology underlying these associations has proven extremely difficult. Even determining which cell types and which particular gene(s) are relevant continues to be a challenge. Here, we conduct a cell-specific pathway analysis of the latest GWAS in multiple sclerosis (MS), which had analyzed a total of 47,351 cases and 68,284 healthy controls and found more than 200 non-MHC genome-wide associations. Our analysis identifies pan immune cell as well as cell-specific susceptibility genes in T cells, B cells and monocytes. Finally, genotype-level data from 2,370 patients and 412 controls is used to compute intra-individual and cell-specific susceptibility pathways that offer a biological interpretation of the individual genetic risk to MS. This approach could be adopted in any other complex trait for which genome-wide data is available.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Single-Cell RNA-Sequencing-Based CRISPRi Screening Resolves Molecular Drivers of Early Human Endoderm Development
Ryan M.J. Genga, Eric M. Kernfeld, Krishna M. Parsi, Teagan J. Parsons, Michael J. Ziller, René Maehr
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Aberrant oligodendroglial–vascular interactions disrupt the blood–brain barrier, triggering CNS inflammation
Jianqin Niu, Hui-Hsin Tsai, Kimberly K. Hoi, Nanxin Huang, Guangdan Yu, Kicheol Kim, Sergio E. Baranzini, Lan Xiao, Jonah R. Chan & Stephen P. J. Fancy
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
C/EBPβ regulates the M2 transcriptome in β-adrenergic-stimulated macrophages
Donald M. Lamkin, Shreyesi Srivastava, Karen P. Bradshaw, Jenna E. Betz, Kevin B. Muy, Anna M. Wiese, Shelby K. Yee, Rebecca M. Waggoner, Jesusa M. G. Arevalo, Alexander J. Yoon, Kym F. Faull, Erica K. Sloan, Steve W. Cole
Brain, Behavior, and Immunity, doi:10.1016/j.bbi.2019.05.034
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Regeneration associated transcriptional signature of retinal microglia and macrophages
Diana M. Mitchell, Chi Sun, Samuel S. Hunter, Daniel D. New & Deborah L. Stenkamp
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
The lung-enriched p53 mutants V157F and R158L/P regulate a gain of function transcriptome in lung cancer
Julie A Barta, Kristen Pauley, Andrew V Kossenkov, Steven B McMahon
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Differential gene regulatory pathways and co-expression networks associated with fire blight infection in apple (Malus × domestica)
Katchen Julliany Pereira Silva, Jugpreet Singh, Ryland Bednarek, Zhangjun Fei & Awais Khan
Apple cultivars with durable resistance are needed for sustainable management of fire blight, the most destructive bacterial disease of apples. Although studies have identified genetic resistance to fire blight in both wild species and cultivated apples, more research is needed to understand the molecular mechanisms underlying host–pathogen interaction and differential genotypic responses to fire blight infection. We have analyzed phenotypic and transcriptional responses of ‘Empire’ and ‘Gala’ apple cultivars to fire blight by infecting them with a highly aggressive E. amylovora strain. Disease progress, based on the percentage of visual shoot necrosis, started showing significant (p < 0.001) differences between ‘Empire’ and ‘Gala’ 4 days after infection (dai). ‘Empire’ seems to slow down bacterial progress more rapidly after this point. We further compared transcriptome profiles of ‘Empire’ and ‘Gala’ at three different time points after fire blight infection. More genes showed differential expression in ‘Gala’ at earlier stages, but the number of differentially expressed genes increased in ‘Empire’ at 3 dai. Functional classes related to defense, cell cycle, response to stress, and biotic stress were identified and a few co-expression gene networks showed particular enrichment for plant defense and abiotic stress response genes. Several of these genes also co-localized in previously identified quantitative trait locus regions for fire blight resistance on linkage groups 7 and 12, and can serve as functional candidates for future research. These results highlight different molecular mechanisms for pathogen perception and control in two apple cultivars and will contribute toward better understanding of E. amylovora-Malus pathosystem.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Improved annotation of the domestic pig genome through integration of Iso-Seq and RNA-seq data
H. Beiki, H. Liu, J. Huang, N. Manchanda, D. Nonneman, T. P. L. Smith, J. M. Reecy and C. K. Tuggle
Background
Our understanding of the pig transcriptome is limited. RNA transcript diversity among nine tissues was assessed using poly(A) selected single-molecule long-read isoform sequencing (Iso-seq) and Illumina RNA sequencing (RNA-seq) from a single White cross-bred pig.
Results
Across tissues, a total of 67,746 unique transcripts were observed, including 60.5% predicted protein-coding, 36.2% long non-coding RNA and 3.3% nonsense-mediated decay transcripts. On average, 90% of the splice junctions were supported by RNA-seq within tissue. A large proportion (80%) represented novel transcripts, mostly produced by known protein-coding genes (70%), while 17% corresponded to novel genes. On average, four transcripts per known gene (tpg) were identified; an increase over current EBI (1.9 tpg) and NCBI (2.9 tpg) annotations and closer to the number reported in human genome (4.2 tpg). Our new pig genome annotation extended more than 6000 known gene borders (5′ end extension, 3′ end extension, or both) compared to EBI or NCBI annotations. We validated a large proportion of these extensions by independent pig poly(A) selected 3′-RNA-seq data, or human FANTOM5 Cap Analysis of Gene Expression data. Further, we detected 10,465 novel genes (81% non-coding) not reported in current pig genome annotations. More than 80% of these novel genes had transcripts detected in > 1 tissue. In addition, more than 80% of novel intergenic genes with at least one transcript detected in liver tissue had H3K4me3 or H3K36me3 peaks mapping to their promoter and gene body, respectively, in independent liver chromatin immunoprecipitation data.
Conclusions
These validated results show significant improvement over current pig genome annotations.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Development and characterisation of acquired radioresistant breast cancer cell lines
Mark Gray, Arran K. Turnbull, Carol Ward, James Meehan, Carlos Martínez-Pérez, Maria Bonello, Lisa Y. Pang, Simon P. Langdon, Ian H. Kunkler, Alan Murray and David Argyle
Background
Radiotherapy plays an important role in the multimodal treatment of breast cancer. The response of a breast tumour to radiation depends not only on its innate radiosensitivity but also on tumour repopulation by cells that have developed radioresistance. Development of effective cancer treatments will require further molecular dissection of the processes that contribute to resistance.
Methods
Radioresistant cell lines were established by exposing MDA-MB-231, MCF-7 and ZR-751 parental cells to increasing weekly doses of radiation. The development of radioresistance was evaluated through proliferation and colony formation assays. Phenotypic characterisation included migration and invasion assays and immunohistochemistry. Transcriptomic data were also generated for preliminary hypothesis generation involving pathway-focused analyses.
Results
Proliferation and colony formation assays confirmed radioresistance. Radioresistant cells exhibited enhanced migration and invasion, with evidence of epithelial-to-mesenchymal-transition. Significantly, acquisition of radioresistance in MCF-7 and ZR-751 cell lines resulted in a loss of expression of both ERα and PgR and an increase in EGFR expression; based on transcriptomic data they changed subtype classification from their parental luminal A to HER2-overexpressing (MCF-7 RR) and normal-like (ZR-751 RR) subtypes, indicating the extent of phenotypic changes and cellular plasticity involved in this process. Radioresistant cell lines derived from ER+ cells also showed a shift from ER to EGFR signalling pathways with increased MAPK and PI3K activity.
Conclusions
This is the first study to date that extensively describes the development and characterisation of three novel radioresistant breast cancer cell lines through both genetic and phenotypic analysis. More changes were identified between parental cells and their radioresistant derivatives in the ER+ (MCF-7 and ZR-751) compared with the ER- cell line (MDA-MB-231) model; however, multiple and likely interrelated mechanisms were identified that may contribute to the development of acquired resistance to radiotherapy.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
A Crispr Knockout Screen Identifies Foxf1 as a Suppressor of Colorectal Cancer Metastasis that Acts through Reduced mTOR Signalling
Lennard Lee, Connor Woolley, Thomas Starkey, Luke Freeman-Mills, Andrew Bassett, Fanny Franchini, Lai Mun Wang, Annabelle Lewis, Roland Arnold, Ian Tomlinson
Introduction
A greater understanding of molecular mechanisms underlying metastasis is necessary for development of new strategies to prevent and treat cancer.
Methods
We performed a genome-wide CRISPR/Cas9 knockout screen in MC38 colorectal cancer (CRC) cells transplanted orthotopically into mice to identify genes that promote metastasis. We undertook focussed molecular analyses to identify mechanisms underlying metastasis.
Results
The screen identified several gene knockouts over-represented in lung metastases, including Dptor (mTOR signalling) and Foxf1 (gastrointestinal tumour predisposition). We validate that loss of Foxf1 promotes metastasis, increased Foxf1 expression restrained cellular migration in-vitro and human CRC metastases express lower Foxf1 than paired primary tumours. Analysis of gene expression changes downstream of Foxf1 identified increased mTOR signalling as a possible mechanism of metastasis caused by Foxf1 loss, consistent with Dptor identification. We confirmed this mechanism demonstrating that mTOR inhibitor sirolimus reduced lung metastasis burden in xenografts.
Conclusion
Mesenchymal Foxf1 plays a major role in intestinal development. We have shown for the first time, through an unbiased genetic screen, that reduced epithelial Foxf1 results in raised mTOR signalling and metastasis.
Authorship statement
Lennard Lee-study concept and design, acquisition of data, analysis, interpretation of data, drafting of the manuscript, statistical analysis and obtained funding. Connor Woolley-acquisition of data, analysis and interpretation of data. Thomas Starkey-acquisition of data, analysis, interpretation of data, drafting of the manuscript. Luke Freeman-Mills-interpretation of data. Andrew Bassett-technical and material support. Fanny Fanchini-technical support. Lai Mun Wang-acquisition of data and study supervision. Annabelle Lewis-study supervision. Roland Arnold-analysis, interpretation of data, statistical analysis. Ian Tomlinson-study supervision and critical revision of the manuscript.
Conflict of Interest
The authors whose names are listed above declare that they have no conflict of interest.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
BRCA2 deficiency instigates cGAS-mediated inflammatory signaling and confers sensitivity to tumor necrosis factor-alpha-mediated cytotoxicity
Anne Margriet Heijink, Francien Talens, Lucas T. Jae, Stephanie E. van Gijn, Rudolf S. N. Fehrmann, Thijn R. Brummelkamp & Marcel A. T. M. van Vugt
Loss of BRCA2 affects genome stability and is deleterious for cellular survival. Using a genome-wide genetic screen in near-haploid KBM-7 cells, we show that tumor necrosis factor-alpha (TNFα) signaling is a determinant of cell survival upon BRCA2 inactivation. Specifically, inactivation of the TNF receptor (TNFR1) or its downstream effector SAM68 rescues cell death induced by BRCA2 inactivation. BRCA2 inactivation leads to pro-inflammatory cytokine production, including TNFα, and increases sensitivity to TNFα. Enhanced TNFα sensitivity is not restricted to BRCA2 inactivation, as BRCA1 or FANCD2 inactivation, or hydroxyurea treatment also sensitizes cells to TNFα. Mechanistically, BRCA2 inactivation leads to cGAS-positive micronuclei and results in a cell-intrinsic interferon response, as assessed by quantitative mass-spectrometry and gene expression profiling, and requires ASK1 and JNK signaling. Combined, our data reveals that micronuclei induced by loss of BRCA2 instigate a cGAS/STING-mediated interferon response, which encompasses re-wired TNFα signaling and enhances TNFα sensitivity.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
The repair of articular cartilage needs a sufficient number of chondrocytes to replace the defect tissue. Direct reprogramming of fibroblasts into chondrocytes can provide a sufficient number of chondrocytes because fibroblasts can be expanded efficiently. Herein, we demonstrate for the first time that electrical stimulation can drive direct reprogramming of human dermal fibroblasts (HDFs) into hyaline chondrogenic cells. Our results shows that electrical stimulation drives condensation of HDFs and then enhances expression levels of chondrogenic markers, such as type II collagen, aggrecan, and Sox9, and decreases type I collagen levels without the addition of exogenous growth factors or gene transduction. Electrical stimulation-directly reprogrammed chondrogenic cells showed the normal karyotype. It was also found that electrical stimulation increased the secretion levels of TGF-beta1, PDGF-AA, and IGFBP-2, 3. These findings may contribute to not only novel approach of direct reprogramming but also cell therapy for cartilage regeneration.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Differential gene expression profiles of human periodontal ligament cells preserved in Hank’s balanced salt solution and milk
Ok Hyung Nam, Tae Jun Oh, Jae‐Hyung Lee, Yu‐Shik Hwang, Sung Chul Choi
Background/Aim
Various types of storage media have been investigated to preserve avulsed teeth. However, the efficacies of storage media mainly focus on the aspect of cell viability. The aim of this study was to evaluate and compare the gene expression profiles of human periodontal ligament cells preserved in Hank’s balanced salt solution (HBSS) and milk over different storage durations.
Material and Methods
Human periodontal ligament cells were cultured and preserved in HBSS and milk for 3 and 6 hours. Next, total RNA was isolated. QuantSeq 3′ mRNA‐Sequencing was used to examine differences in gene expression in HBSS‐ and milk‐grown periodontal ligament cells. Bioinformatics analysis was also performed to predict the function of the differentially expressed genes.
Results
The number of differentially expressed genes shared among all groups was 101. In gene set enrichment analysis, the shared differentially expressed genes in HBSS and milk were associated with the TNF‐α signaling pathway (P = 1.07E−7). Seven hallmark gene sets were also identified in HBSS. Moreover, hallmark gene sets associated with hypoxia (P = 7.26E−5) and apoptosis (P = 4.06E−4) were identified in HBSS. In milk, 10 hallmark gene sets along with gene sets for inflammatory response (P = 6.87E−3) were identified.
Conclusions
Compared to those in milk, genes in HBSS were differentially expressed with increasing storage duration, suggesting that diverse and different gene expression may be involved in HBSS and milk. However, a more detailed functional analysis of these differentially expressed genes in storage solutions should be performed in the future.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
The Suv420h histone methyltransferases regulate PPAR-γ and energy expenditure in response to environmental stimuli
Simona Pedrotti, Roberta Caccia, Maria Victoria Neguembor, Jose Manuel Garcia-Manteiga, Giulia Ferri, Clara de Palma, Tamara Canu, Matteo Giovarelli, Paolo Marra, Amleto Fiocchi, Ivan Molineris, Michele Raso, Francesca Sanvito, Claudio Doglioni, Antonio Esposito, Emilio Clementi and Davide Gabellini
Obesity and its associated metabolic abnormalities have become a global emergency with considerable morbidity and mortality. Epidemiologic and animal model data suggest an epigenetic contribution to obesity. Nevertheless, the cellular and molecular mechanisms through which epigenetics contributes to the development of obesity remain to be elucidated. Suv420h1 and Suv420h2 are histone methyltransferases responsible for chromatin compaction and gene repression. Through in vivo, ex vivo, and in vitro studies, we found that Suv420h1 and Suv420h2 respond to environmental stimuli and regulate metabolism by down-regulating peroxisome proliferator–activated receptor gamma (PPAR-γ), a master transcriptional regulator of lipid storage and glucose metabolism. Accordingly, mice lacking Suv420h proteins activate PPAR-γ target genes in brown adipose tissue to increase mitochondria respiration, improve glucose tolerance, and reduce adipose tissue to fight obesity. We conclude that Suv420h proteins are key epigenetic regulators of PPAR-γ and the pathways controlling metabolism and weight balance in response to environmental stimuli.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Janus effect of glucocorticoids on differentiation of muscle fibro/adipogenic progenitors
Andrea Cerquone Perpetuini, Alessio Reggio, Mauro Cerretani, Giulio Giuliani, Marisabella Santoriello, Roberta Stefanelli, Alessandro Palma, Steven Harper, Luisa Castagnoli, Alberto Bresciani, Gianni Cesareni
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
The response to the DNA damaging agent methyl methanesulfonate in a fungal plant pathogen
Shira Milo-Cochavi, Manish Pareek, Gregory Delulio, Yael Almog, Gautam Anand, Li-Jun Ma, Shay Covo
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
The lncRNA TP73-AS1 is linked to aggressiveness in glioblastoma and promotes temozolomide resistance in glioblastoma cancer stem cells
Gal Mazor, Liron Levin, Daniel Picard, Ulvi Ahmadov, Helena Carén, Arndt Borkhardt, Guido Reifenberger, Gabriel Leprivier, Marc Remke & Barak Rotblat
Glioblastoma multiform (GBM) is the most common brain tumor characterized by a dismal prognosis. GBM cancer stem cells (gCSC) or tumor-initiating cells are the cell population within the tumor-driving therapy resistance and recurrence. While temozolomide (TMZ), an alkylating agent, constitutes the first-line chemotherapeutic significantly improving survival in GBM patients, resistance against this compound commonly leads to GBM recurrence and treatment failure. Although the roles of protein-coding transcripts, proteins and microRNA in gCSC, and therapy resistance have been comprehensively investigated, very little is known about the role of long noncoding RNAs (lncRNAs) in this context. Using nonoverlapping, independent RNA sequencing and gene expression profiling datasets, we reveal that TP73-AS1 constitutes a clinically relevant lncRNA in GBM. Specifically, we demonstrate significant overexpression of TP73-AS1 in primary GBM samples, which is particularly increased in the gCSC. More importantly, we demonstrate that TP73-AS1 comprises a prognostic biomarker in glioma and in GBM with high expression identifying patients with particularly poor prognosis. Using CRISPRi to downregulate our candidate lncRNA in gCSC, we demonstrate that TP73-AS1 promotes TMZ resistance in gCSC and is linked to regulation of the expression of metabolism- related genes and ALDH1A1, a protein known to be expressed in cancer stem cell markers and protects gCSC from TMZ treatment. Taken together, our results reveal that high TP73-AS1predicts poor prognosis in primary GBM cohorts and that this lncRNA promotes tumor aggressiveness and TMZ resistance in gCSC.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Multiple Auxin-Response Regulators Enable Stability and Variability in Leaf Development
Alon Israeli, Yossi Capua, Ido Shwartz, Lior Tal, Zohar Meir, Matan Levy, Maya Bar, Idan Efroni, Naomi Ori
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Emergence of a floral colour polymorphism by pollinator-mediated overdominance
Roman T. Kellenberger, Kelsey J. R. P. Byers, Rita M. De Brito Francisco, Yannick M. Staedler, Amy M. LaFountain, Jürg Schönenberger, Florian P. Schiestl & Philipp M. Schlüter
Maintenance of polymorphism by overdominance (heterozygote advantage) is a fundamental concept in evolutionary biology. In most examples known in nature, overdominance is a result of homozygotes suffering from deleterious effects. Here we show that overdominance maintains a non-deleterious polymorphism with black, red and white floral morphs in the Alpine orchid Gymnadenia rhellicani. Phenotypic, metabolomic and transcriptomic analyses reveal that the morphs differ solely in cyanidin pigments, which are linked to differential expression of an anthocyanidin synthase (ANS) gene. This expression difference is caused by a premature stop codon in an ANS-regulating R2R3-MYBtranscription factor, which is heterozygous in the red colour morph. Furthermore, field observations show that bee and fly pollinators have opposite colour preferences; this results in higher fitness (seed set) of the heterozygous morph without deleterious effects in either homozygous morph. Together, these findings demonstrate that genuine overdominance exists in nature.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Cardiac endothelial cell transcriptome in neonatal, adult, and remodeling hearts
Zarha Vermeulen, Ligia Mateiu, Lindsey Dugaucquier, Gilles W. De Keulenaer, and Vincent F. M. Segers
Physiological Genomics, doi:10.1152/physiolgenomics.00002.2019
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Bcor loss perturbs myeloid differentiation and promotes leukaemogenesis
Madison J. Kelly, Joan So, Amy J. Rogers, Gareth Gregory, Jason Li, Magnus Zethoven, Micah D. Gearhart, Vivian J. Bardwell, Ricky W. Johnstone, Stephin J. Vervoort & Lev M. Kats
The BCL6 Corepressor (BCOR) is a component of a variant Polycomb repressive complex 1 (PRC1) that is essential for normal development. Recurrent mutations in the BCOR gene have been identified in acute myeloid leukaemia and myelodysplastic syndrome among other cancers; however, its function remains poorly understood. Here we examine the role of BCOR in haematopoiesis in vivo using a conditional mouse model that mimics the mutations observed in haematological malignancies. Inactivation of Bcor in haematopoietic stem cells (HSCs) results in expansion of myeloid progenitors and co-operates with oncogenic KrasG12D in the initiation of an aggressive and fully transplantable acute leukaemia. Gene expression analysis and chromatin immunoprecipitation sequencing reveals differential regulation of a subset of PRC1-target genes including HSC-associated transcription factors such as Hoxa7/9. This study provides mechanistic understanding of how BCOR regulates cell fate decisions and how loss of function contributes to the development of leukaemia.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Alternative 3′ UTRs direct localization of functionally diverse protein isoforms in neuronal compartments
Camilla Ciolli Mattioli, Aviv Rom, Vedran Franke, Koshi Imami, Gerard Arrey, Mandy Terne, Andrew Woehler, Altuna Akalin, Igor Ulitsky, Marina Chekulaeva
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
BRCA2 deficiency instigates cGAS-mediated inflammatory signaling and confers sensitivity to tumor necrosis factor-alpha-mediated cytotoxicity
Anne Margriet Heijink, Francien Talens, Lucas T. Jae, Stephanie E. van Gijn, Rudolf S. N. Fehrmann, Thijn R. Brummelkamp & Marcel A. T. M. van Vugt
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Emergence of a floral colour polymorphism by pollinator-mediated overdominance
Roman T. Kellenberger, Kelsey J. R. P. Byers, Rita M. De Brito Francisco, Yannick M. Staedler, Amy M. LaFountain, Jürg Schönenberger, Florian P. Schiestl & Philipp M. Schlüter
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
A comparison between whole transcript and 3’ RNA sequencing methods using Kapa and Lexogen library preparation methods
Feiyang Ma, Brie K. Fuqua, Yehudit Hasin, Clara Yukhtman, Chris D. Vulpe, Aldons J. Lusis and Matteo Pellegrini
Background
3’ RNA sequencing provides an alternative to whole transcript analysis. However, we do not know a priori the relative advantage of each method. Thus, a comprehensive comparison between the whole transcript and the 3′ method is needed to determine their relative merits. To this end, we used two commercially available library preparation kits, the KAPA Stranded mRNA-Seq kit (traditional method) and the Lexogen QuantSeq 3’ mRNA-Seq kit (3′ method), to prepare libraries from mouse liver RNA. We then sequenced and analyzed the libraries to determine the advantages and disadvantages of these two approaches.
Results
We found that the traditional whole transcript method and the 3’ RNA-Seq method had similar levels of reproducibility. As expected, the whole transcript method assigned more reads to longer transcripts, while the 3′ method assigned roughly equal numbers of reads to transcripts regardless of their lengths. We found that the 3’ RNA-Seq method detected more short transcripts than the whole transcript method. With regard to differential expression analysis, we found that the whole transcript method detected more differentially expressed genes, regardless of the level of sequencing depth.
Conclusions
The 3’ RNA-Seq method was better able to detect short transcripts, while the whole transcript RNA-Seq was able to detect more differentially expressed genes. Thus, both approaches have relative advantages and should be selected based on the goals of the experiment.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Commensal-specific T cell plasticity promotes rapid tissue adaptation to injury
Oliver J. Harrison, Jonathan L. Linehan, Han-Yu Shih, Nicolas Bouladoux, Seong-Ji Han, Margery Smelkinson, Shurjo K. Sen, Allyson L. Byrd, Michel Enamorado, Chen Yao, Samira Tamoutounour, Francois Van Laethem, Charlotte Hurabielle1, Nicholas Collins, Andrea Paun, Rosalba Salcedo, John J. O’Shea, Yasmine Belkaid
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Elevated pro-inflammatory gene expression in the third trimester of pregnancy in mothers who experienced stressful life events
Kharah M. Ross, Steve W. Coleb, Judith E. Carroll, Christine Dunkel Schettera
Brain, Behavior, and Immunity, doi:10.1016/j.bbi.2018.11.009
Background
Stress exposure is associated with risk for adverse pregnancy outcomes, potentially in part through dysregulated immune and inflammatory activity. Evidence suggests that stress during pregnancy is associated with inflammation during pregnancy, consistent with risk for preterm birth. However, research has not tested whether complementary changes are reflected in immune cell gene expression, or upstream regulation of inflammation. The purpose of this study was to test associations between preconception and prenatal stress exposure and third trimester immune cell gene expression, focusing specifically on sets of genes previously linked to stress in non-pregnant samples: Pro-inflammatory genes, and antiviral and antibody genes.
Methods
A sample of 116 low-income, diverse women was recruited from 5 U.S. sites by the Community Child and Health Network at the birth of a child. This study is of the subgroup of women who became pregnant again over the two-year follow-up period, and provided information on stressful life events that occurred both preconception and during the third trimester of the subsequent pregnancy. Dried blood spots (DBS) were collected in the third trimester of pregnancy, and used for gene expression analysis.
Results
Women with more prenatal stressful life events had higher expression of pro-inflammatory genes when compared to those with fewer life events, and the effect was driven by increased activation of pro-inflammatory transcription factors, NF-κB and AP-1. Preconception stressful life event exposure was not associated with gene expression profiles. When entered into models simultaneously, only prenatal stressful life events were associated with up-regulation of pro-inflammatory genes. No differences between high or low stress groups emerged for antiviral or antibody genes.
Conclusions
Prenatal stress exposure was associated with up-regulated pro-inflammatory gene expression during pregnancy, and increased activity of NF-κB and AP-1. In contrast, stress occurring preconception was not associated with gene expression. These results are consistent with the hypothesis that stress-induced activation of pro-inflammatory transcriptional pathways in pregnancy, but not earlier, may increase risk for inflammation-driven adverse pregnancy outcomes.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Sasa quelpaertensis leaf extract mitigates fatigue and regulates the transcriptome profile in mice
Hana Song, Mi Gyeong Jang, Ju Yeop Lee, Hee Chul Ko, Sung-Pyo Hur, Se-Jae Kim
Introduction
It has been reported that various plant species may enhance the elimination of fatigue-related metabolites. However, relatively few studies have directly addressed the potential anti-fatigue effects.
Objective
The objective of this study was to investigate the anti-fatigue potential of a hot water extract of Sasa quelpaertensis Nakai leaf (SQH) in male ICR mice.
Methods
The animals were divided into three groups. The normal control (NC) group was administered saline without exercise every day for 7 days. The exercise control (EC) and exercise with SQH (ES) groups were administered saline and SQH (50 mg/kg of body weight), respectively, every day for 7 days and underwent swimming exercise. RNA sequencing technology was used to analyze the transcriptome profiles of muscle.
Results
Swimming times were prolonged in the ES group compared with the EC group. The ES group had higher blood glucose and lower blood lactate levels, and higher muscular glycogen and lower muscular lactate levels, compared with the EC group. The groups did not differ in histopathological parameters of the muscle and liver, but muscle cell sizes were smaller in the EC group than in the ES and NC groups. RNA sequencing analysis revealed that SQH administration regulated genes associated with energy-generating metabolic pathways in skeletal muscle.
Conclusion
These results suggest that SQH exerts anti-fatigue properties by balancing various biological systems and helping maintain the basic harmonious pattern of the body.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Dynamic molecular monitoring reveals that SWI–SNF mutations mediate resistance to ibrutinib plus venetoclax in mantle cell lymphoma
Rishu Agarwal, Yih-Chih Chan, Constantine S. Tam, Tane Hunter, Dane Vassiliadis, Charis E. Teh, Rachel Thijssen, Paul Yeh, Stephen Q. Wong, Sarah Ftouni, Enid Y. N. Lam, Mary Ann Anderson, Christiane Pott, Omer Gilan, Charles C. Bell, Kathy Knezevic, Piers Blombery, Kathleen Rayeroux, Adrian Zordan, Jason Li, David C. S. Huang, Meaghan Wall, John F. Seymour, Daniel H. D. Gray, Andrew W. Roberts, Mark A. Dawson & Sarah-Jane Dawson
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
OKN-007 Increases temozolomide (TMZ) Sensitivity and Suppresses TMZ-Resistant Glioblastoma (GBM) Tumor Growth
Rheal A.Towner, Nataliya Smith, Debra Saunders, Chase A.Brown, Xue Cai, Jadith Ziegler, Samantha Mallory, Mikhail G. Dozmorov, Patricia Coutinho De Souza, Graham Wiley, Kyeongsoon Kim, Shinwook Kang, Doo-Sik Kong, Young-TaeKim, Kar-MingFung, Jonathan D. Wren, James Battiste
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
TBX2 is a neuroblastoma core regulatory circuitry component enhancing MYCN/FOXM1 reactivation of DREAM targets
Bieke Decaesteker, Geertrui Denecker, Christophe Van Neste, Emmy M. Dolman, Wouter Van Loocke, Moritz Gartlgruber, Carolina Nunes, Fanny De Vloed, Pauline Depuydt, Karen Verboom, Dries Rombaut, Siebe Loontiens, Jolien De Wyn, Waleed M. Kholosy, Bianca Koopmans, Anke H. W. Essing, Carl Herrmann, Daniel Dreidax, Kaat Durinck, Dieter Deforce, Filip van Nieuwerburgh, Anton Henssen, Rogier Versteeg, Valentina Boeva, Gudrun Schleiermacher, Johan van Nes, Pieter Mestdagh, Suzanne Vanhauwaert, Johannes H. Schulte, Frank Westermann, Jan J. Molenaar, Katleen De Preter & Frank Speleman
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
The Trithorax protein Ash1L promotes myoblast fusion by activating Cdon expression
Ilaria Castiglioni, Roberta Caccia, Jose Manuel Garcia-Manteiga, Giulia Ferri, Giuseppina Caretti, Ivan Molineris, Kenichi Nishioka & Davide Gabellini
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Inflammatory pathways are upregulated in the nasal epithelium in patients with idiopathic pulmonary fibrosis
Marc A. Sala, Yalbi Itzel Balderas-Martínez, Ivette Buendía-Roldan, Hiam Abdala-Valencia, Kiwon Nam, Manu Jain, Sangeeta Bhorade, Ankit Bharat, Paul A. Reyfman, Karen M. Ridge, Annie Pardo, Jacob I. Sznajder, G. R. Scott Budinger, Alexander V. Misharin and Moises Selman
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Multidimensional Assessment of the Host Response in Mechanically Ventilated Patients with Suspected Pneumonia
James M Walter, Ziyou Ren, Tyrone Yacoub, Paul A Reyfman, Raj D. Shah, Hiam Abdala-Valencia, Kiwon Nam, Vince K Morgan, Kishore R Anekalla, Nikita Joshi, Alexandra C McQuattie-Pimentel, Ching-I Chen, Monica Chi, SeungHye Han, Francisco J Gonzalez-Gonzalez, Saul Soberanes, Raul P. Aillon, Satoshi Watanabe, Kinola J.N. Williams, Ziyan Lu, Joseph Paonessa, Peter Hountras, Madonna Breganio, Nicole Borkowski, Helen K Donnelly, Jonathan P. Allen, Luis A Amaral, Ankit Bharat, Alexander V Misharin, Neda Bagheri, Alan R. Hauser, G.R. Scott Budinger, Richard G Wunderink
American Journal of Respiratory and Critical Care Medicine, doi:10.1164/rccm.201804-0650OC
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
HEXIM1-Tat chimera inhibits HIV-1 replication
Marie Leoz, Petra Kukanja, Zeping Luo, Fang Huang, Daniele C. Cary, B. Matija Peterlin, Koh Fujinaga
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Investigation of the skeletal muscle transcriptome in lambs fed β adrenergic agonists and subjected to heat stress for 21 d
Rachel M Kubik, Shauna M Tietze, Ty B Schmidt, Dustin Tyler Yates, Jessica L Petersen
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Intestinal barrier tightening by a cell‐penetrating antibody to Bin1, a candidate target for immunotherapy of ulcerative colitis
Sunil Thomas, Kevther Hoxha, Walker Alexander, John Gilligan, Rima Dilbarova, Kelly Whittaker, Andrew Kossenkov, George C. Prendergast, James M. Mullin
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Transcriptome-wide effects of expansin gene manipulation in etiolated Arabidopsis seedling
Iqmal Asyraf Ilias, Kohei Negishi, Keito Yasue, Naohiro Jomura, Kengo Morohashi, Syarul Nataqain Baharum, Hoe-Han Goh
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
A stably self-renewing adult blood-derived induced neural stem cell exhibiting patternability and epigenetic rejuvenation
Chao Sheng, Johannes Jungverdorben, Hendrik Wiethoff, Qiong Lin, Lea J. Flitsch, Daniela Eckert, Matthias Hebisch, Julia Fischer, Jaideep Kesavan, Beatrice Weykopf, Linda Schneider, Dominik Holtkamp, Heinz Beck, Andreas Till, Ullrich Wüllner, Michael J. Ziller, Wolfgang Wagner, Michael Peitz & Oliver Brüstle
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Maternal immune activation alters brain microRNA expression in mouse offspring
Jun‐Sang Sunwoo, Daejong Jeon, Soon‐Tae Lee, Jangsup Moon, Jung‐Suk Yu, Dong‐Kyu Park, Ji‐Yeon Bae, Doo Young Lee, Sangwoo Kim, Keun‐Hwa Jung, Kyung‐Il Park, Ki‐Young Jung, Manho Kim, Sang Kun Lee, Kon Chu
Annals of Clinical and Translational Neurology, doi: 10.1002/acn3.652
Objective
Maternal immune activation (MIA) is associated with an increased risk of autism spectrum disorder (ASD) in offspring. Herein, we investigate the altered expression of microRNAs (miRNA), and that of their target genes, in the brains of MIA mouse offspring.
Methods
To generate MIA model mice, pregnant mice were injected with polyriboinosinic:polyribocytidylic acid on embryonic day 12.5. We performed miRNA microarray and mRNA sequencing in order to determine the differential expression of miRNA and mRNA between MIA mice and controls, at 3 weeks of age. We further identified predicted target genes of dysregulated miRNAs, and miRNA‐target interactions, based on the inverse correlation of their expression levels.
Results
Mice prenatally subjected to MIA exhibited behavioral abnormalities typical of ASD, such as a lack of preference for social novelty and reduced prepulse inhibition. We found 29 differentially expressed miRNAs (8 upregulated and 21 downregulated) and 758 differentially expressed mRNAs (542 upregulated and 216 downregulated) in MIA offspring compared to controls. Based on expression levels of the predicted target genes, 18 downregulated miRNAs (340 target genes) and three upregulated miRNAs (60 target genes) were found to be significantly enriched among the differentially expressed genes. miRNA and target gene interactions were most significant between mmu‐miR‐466i‐3p and Hfm1 (ATP‐dependent DNA helicase homolog), and between mmu‐miR‐877‐3p and Aqp6 (aquaporin 6).
Interpretation
Our results provide novel information regarding miRNA expression changes and their putative targets in the early postnatal period of brain development. Further studies will be needed to evaluate potential pathogenic roles of the dysregulated miRNAs.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Differential effects of partial and complete loss of TREM2 on microglial injury response and tauopathy
Faten A. Sayed, Maria Telpoukhovskaia, Lay Kodama, Yaqiao Li, Yungui Zhou, David Le, Axel Hauduc, Connor Ludwig, Fuying Gao, Claire Clelland, Lihong Zhan, Yonatan A. Cooper, Dimitrios Davalos, Katerina Akassoglou, Giovanni Coppola, and Li Gan
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Prospective Isolation and Characterization of Genetically and Functionally Distinct AML Subclones
Bauke de Boer, Janine Prick, Maurien G. Pruis, Peter Keane, Maria Rosaria Imperato, Jennifer Jaques, Annet Z. Brouwers-Vos, Shanna M. Hogeling, Carolien M. Woolthuis, Marije T. Nijk, Arjan Diepstra, Sebastian Wandinger, Matthias Versele, Ricardo M. Attar, Peter N. Cockerill, Gerwin Huls, Edo Vellenga, André B. Mulder, Constanze Bonifer, Jan Jacob Schuringa
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Transcriptional profiling of Zygosaccharomyces bailii early response to acetic acid or copper stress mediated by ZbHaa1
Miguel Antunes, Margarida Palma & Isabel Sá-Correia
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Transcriptome profiling in blood before and after hepatitis B vaccination shows significant differences in gene expression between responders and non-responders
Esther Bartholomeus, Nicolas De Neuter, Pieter Meysman, Arvid Suls, Nina Keersmaekers, George Elias, Hilde Jansens, Niel Hens, Evelien Smits, Viggo Van Tendeloo, Philippe Beutels, Pierre Van Damme, Benson Ogunjimi, Kris Laukens, Geert Mortier
As the hepatitis B virus is widely spread and responsible for considerable morbidity and mortality, WHO recommends vaccination from infancy to reduce acute infection and chronic carriers. However, current subunit vaccines are not 100% efficacious and leave 5–10% of recipients unprotected.
Methods
To evaluate immune responses after Engerix-B vaccination, we determined, using mRNA-sequencing, whole blood early gene expression signatures before, at day 3 and day 7 after the first dose and correlated this with the resulting antibody titer after two vaccine doses.
Results
Our results indicate that immune related genes are differentially expressed in responders mostly at day 3 and in non-responders mostly at day 7. The most remarkable difference between responders and non-responders were the differentially expressed genes before vaccination. The granulin precursor gene (GRN) was significantly downregulated in responders while upregulated in non-responders at day 0. Furthermore, absolute granulocytes numbers were significantly higher in non-responders at day 0.
Conclusion
The non-responders already showed an activated state of the immune system before vaccination. Furthermore, after vaccination, they exhibited a delayed and partial immune response in comparison to the responders. Our data may indicate that the baseline and untriggered immune system can influence the response upon hepatitis B vaccination.
Keywords
Differential gene expressionEngerix-B vaccineHepatitis BImmune response
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Lipocalin-2 is a pathogenic determinant and biomarker of neuropsychiatric lupus
Elise V. Mike, Hadijat M. Makinde, Maria Gulinello, Kamala Vanarsa, Leal Herlitz, Gaurav Gadhvi, Deborah R. Winter, Chandra Mohan, John G. Hanly, C.C. Mok, Carla M. Cuda, Chaim Puttermanah
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Self-Renewing Trophoblast Organoids Recapitulate the Developmental Program of the Early Human Placenta
Sandra Haider, Gudrun Meinhardt, Leila Saleh, Viktoria Kunihs, Magdalena Gamperl, Ulrich Kaindl, Adolf Ellinger, Thomas R. Burkard, Christian Fiala, Jürgen Pollheimer, Sasha Mendjan, Paulina A. Latos, Martin Knöfler
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
GRHL2-Dependent Enhancer Switching Maintains a Pluripotent Stem Cell Transcriptional Subnetwork after Exit from Naive Pluripotency
Amy F. Chen, Arthur J. Liu, Raga Krishnakumar, Jacob W. Freimer, Brian DeVeale, Robert Blelloch
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis
Matthias Muhar, Anja Ebert, Tobias Neumann, Christian Umkehrer, Julian Jude, Corinna Wieshofer, Philipp Rescheneder, Jesse J. Lipp, Veronika A. Herzog, Brian Reichholf, David A. Cisneros, Thomas Hoffmann, Moritz F. Schlapansky, Pooja Bhat, Arndt von Haeseler, Thomas Köcher, Anna C. Obenauf, Johannes Popow, Stefan L. Ameres, Johannes Zuber
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina and SLAMseq Metabolic RNA Labeling Kit for RNA-Seq
Dysregulation of expression correlates with rare-allele burden and fitness loss in maize
Karl A. G. Kremling, Shu-Yun Chen, Mei-Hsiu Su, Nicholas K. Lepak, M. Cinta Romay, Kelly L. Swarts, Fei Lu, Anne Lorant, Peter J. Bradbury & Edward S. Buckler
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Neurogenomic Profiling Reveals Distinct Gene Expression Profiles Between Brain Parts That Are Consistent in Ophthalmotilapia Cichlids
Sofie Derycke, Loic Kéver, Koen Herten, Koen Van den Berge, Maarten Van Steenberge, Jeroen Van Houdt, Lieven Clement, Pascal Poncin, Eric Parmentier and Erik Verheyen
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Combined Ectopic Expression of Homologous Recombination Factors Promotes Embryonic Stem Cell Differentiation
Eui-Hwan Choi, Seobin Yoon, Keun P. Kim
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Single Cell Molecular Alterations Reveal Pathogenesis and Targets of Concussive Brain Injury *REVIEW
Douglas Arneson, Yumei Zhuang, Hyae Ran Byun, In Sook Ahn, Zhe Ying, Guanglin Zhang, Fernando Gomez-Pinilla, Xia Yang
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Gene-level differential analysis at transcript-level resolution
Lynn Yi, Harold Pimentel, Nicolas L. Bray, Lior Pachter
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Combined CDK4/6 and PI3Kα Inhibition Is Synergistic and Immunogenic in Triple-Negative Breast Cancer
Zhi Ling Teo, Stephanie Versaci, Sathana Dushyanthen, Franco Caramia, Peter Savas, Chris P. Mintoff, Magnus Zethoven, Balaji Virassamy, Stephen J. Luen, Grant A. McArthur, Wayne A. Phillips, Phillip K. Darcy and Sherene Loi
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
A Cell-Permeable Stapled Peptide Inhibitor of the Estrogen Receptor/Coactivator Interaction
Thomas E. Speltz, Jeanne M. Danes, Joshua D. Stender, Jonna Frasor, and Terry W. Moore
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Development of zebrafish medulloblastoma-like PNET model by TALEN-mediated somatic gene inactivation
Jaegal Shim, Jung-Hwa Choi, Moon-Hak Park, Hyena Kim, Jong Hwan Kim, Seon-Young Kim, Dongwan Hong, Sunshin Kim, Ji Eun Lee, Cheol-Hee Kim, Jeong-Soo Lee and Young-Ki Bae
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Lin28B and miR-142-3p regulate neuronal differentiation by modulating Staufen1 expression
Younseo Oh, Jungyun Park, Jin-Il Kim, Mi-Yoon Chang, Sang-Hun Lee, Youl-Hee Cho & Jungwook Hwang
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
IL-1R8 is a checkpoint in NK cells regulating anti-tumour and anti-viral activity
Martina Molgora, Eduardo Bonavita, Andrea Ponzetta, Federica Riva, Marialuisa Barbagallo, Sébastien Jaillon, Branka Popović, Giovanni Bernardini, Elena Magrini, Francesca Gianni, Santiago Zelenay, Stipan Jonjić, Angela Santoni, Cecilia Garlanda & Alberto Mantovani
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
SLAM-ITseq: Sequencing cell type-specific transcriptomes without cell sorting
Wayo Matsushima, Veronika A Herzog, Tobias Neumann, Katharina Gapp, Johannes Zuber, Stefan L Ameres, Eric A Miska
Features SLAMseq Metabolic RNA Labeling Kit for RNA-Seq and QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Post-transcriptional 3´-UTR cleavage of mRNA transcripts generates thousands of stable uncapped autonomous RNA fragments
Yuval Malka, Avital Steiman-Shimony, Eran Rosenthal, Liron Argaman, Leonor Cohen-Daniel, Eliran Arbib, Hanah Margalit, Tommy Kaplan & Michael Berger
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Identification of USP7 as an essential component to maintain integrity and function of non-canonical PRC1.1 in leukemia
Henny Maat, Jennifer Jaques, Aida Rodriguez Lopez, Marcel P de Vries, Chantal Gravesteijn, Annet Brouwers-Vos, Gerwin Huls, Edo Vellenga, Vincent van den Boom, Jan Jacob Schuringa
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Non-classical Immunity Controls Microbiota Impact on Skin Immunity and Tissue Repair
Jonathan L. Linehan, Oliver J. Harrison, Seong-Ji Han, Allyson L. Byrd, Ivan Vujkovic-Cvijin, Alejandro V. Villarino, Shurjo K. Sen, Jahangheer Shaik, Margery Smelkinson, Samira Tamoutounour, Nicholas Collins, Nicolas Bouladoux, Amiran Dzutsev, Stephan P. Rosshart, Jesse H. Arbuckle, Chyung-Ru Wang, Thomas M. Kristie, Barbara Rehermann, Giorgio Trinchieri, Jason M. Brenchley, John J. O’Shea, Yasmine Belkaid
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Network-Guided Discovery of Extensive Epistasis between Transcription Factors Involved in Aliphatic Glucosinolate Biosynthesis
Baohua Li, Michelle Tang, Ayla Nelson, Hart Caligagan, Xue Zhou, Caitlin Clark-Wiest, Richard Ngo, Siobhan M. Brady, Daniel J. Kliebenstein
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
An ultra-effective method of generating extramultipotent cells from human fibroblasts by ultrasound
Yong Seung Lee, Hyejung Heo, Jonghwan Lee, Sung Ung Moon, Woon Yong Jung, Yong Keun Park, Min Geun Park, Seung-Hun Oh, Soonhag Kim
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
The Transcriptional Landscape of Radiation-Treated Human Prostate Cancer: Analysis of a Prospective Tissue Cohort
Simon P. Keam, PhD, Franco Caramia, BCompSci, MBioinf, Cristina Gamell, PhD, Piotr J. Paul, MD, Gisela Mir Arnau, PhD, Paul J. Neeson, PhD, Scott G. Williams, MBBS, MD, MD Scott G. WilliamsEmail the author MBBS, MD Scott G. Williams, Ygal Haupt, PhD
International journal of radiation oncology, biology, physics, doi:10.1016/j.ijrobp.2017.09.037
Purpose
The resistance of prostate cancer to radiation therapy (RT) is a significant clinical issue and still largely unable to be guided by patient-specific molecular characteristics. The present study describes the gene expression changes induced in response to RT in human prostate tissue obtained from a prospective tissue acquisition study designed for radiobiology research.
Methods and Materials
A prospective cohort of 5 men with intermediate-risk and clinically localized tumors were treated with high-dose-rate brachytherapy with 2 × 10-Gy fractions. Image-guided transperineal biopsy specimens were taken immediately before and 14 days after the first high-dose-rate brachytherapy fraction. Using genome-wide 3′ RNA sequencing on total RNA extracted from 10 biopsy specimens, we obtained quantitative expression data for a median of 13,244 genes. We computed the fold-change information for each gene and extracted high-confidence lists of transcripts with either increased or decreased expression (≥1.5-fold) after radiation in ≥4 of the 5 patients. Several gene ontology analyses were then used to identify functionally enriched pathways.
Results
The predominant change in response to RT was elevation of the transcript levels, including that of DNA damage binding protein 2 and p21, and collagens, laminins, and integrins. We observed strong upregulation of the p53 pathway, without observable dysregulation of p53 itself. Interstitial remodeling, extracellular matrix proteins, and focal adhesion pathways were also strongly upregulated, as was inflammation. Functional network analysis showed clustering of the changes inherent in apoptosis and programmed cell death, extracellular matrix organization, and immune regulation.
Conclusions
In the present prospective study of matched clinical tissues, we successfully recognized known radiation-sensitive transcriptional pathways and identified numerous other novel and significantly altered genes with no current association with RT. These data could be informative in the development of future personalized therapeutic agents.
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Cdon deficiency causes cardiac remodeling through hyperactivation of WNT/β-catenin signaling
Myong-Ho Jeong, Hyun-Ji Kim, Jung-Hoon Pyun, Kyu-Sil Choi, Dong I. Lee, Soroosh Solhjoo, Brian O’Rourke, Gordon F. Tomaselli, Dong Seop Jeong, Hana Cho and Jong-Sun Kang
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Abstract 2682: IL-6/STAT3 activation in hepatocytes drives the formation of a pro-metastatic niche in the liver during pancreatic tumorigenesis
Jae W. Lee, Paige M. Porrett, Chad A. Komar, Whitney L. Gladney and Gregory L. Beatty
Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Transcriptomic analysis of purified human cortical microglia reveals age-associated changes
Thais F Galatro, Inge R Holtman, Antonio M Lerario, Ilia D Vainchtein, Nieske Brouwer, Paula R Sola, Mariana M Veras, Tulio F Pereira, Renata E P Leite, Thomas Möller, Paul D Wes, Mari C Sogayar, Jon D Laman, Wilfred den Dunnen, Carlos A Pasqualucci, Sueli M Oba-Shinjo, Erik W G M Boddeke, Suely K N Marie & Bart J L Eggen