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

EMBO Reports, doi:10.15252/embr.201847592

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 (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

The British journal of radiology, doi:10.1259/bjr.20190373

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.

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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

Hepatology, doi:10.1002/hep.30881

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.

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  • 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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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

Life science alliance, doi:10.26508/lsa.201900437

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

Cell Reports, doi:10.1016/j.celrep.2019.06.034

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

Stem cell reports, doi:10.1016/j.stemcr.2019.05.005

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

Scientific Reports, doi:10.1038/s41598-019-47101-y

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

Cancer Cell, doi:10.1016/j.ccell.2019.05.013

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.

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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

bioRxiv, doi:10.1101/693168

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.

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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.

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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

Journal of Molecular Endocrinology, doi:10.1530/JME-19-0018

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

bioRxiv, doi:10.1101/680462

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.

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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

Immunology and Cell Biology, doi:10.1111/imcb.12281

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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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

Scientific Reports, doi:10.1038/s41598-019-45047-9

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+CD15CD14+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

JCI Insight, doi:10.1172/jci.insight.126124

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.

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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

Nature Communications, doi:10.1038/s41467-019-10652-9

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.

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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

Nature Communications, doi:10.1038/s41467-019-10523-3

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.

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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,

Developmental Cell, doi:10.1016/j.devcel.2019.05.023

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.

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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

Pigment Cell & Melanoma Research, doi:10.1111/pcmr.12802

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.

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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

Molecular Autism, doi:10.1186/s13229-019-0278-0

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.

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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

Cancers, doi:10.3390/cancers11060812

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.

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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.

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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

Nucleic Acids Research, doi:10.1093/nar/gkaa060

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

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.

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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

Science Signaling, doi:10.1126/scisignal.aaw9450

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.

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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.

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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.

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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

The Journal of Immunology

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.

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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

Cell Reports, doi:10.1016/j.celrep.2019.03.064

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.

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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

Molecular Cell, doi:10.1016/j.molcel.2019.01.027

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.

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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

Cancers, doi:10.3390/cancers12030608

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

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

Brain, doi:10.1093/brain/awaa045

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

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

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

Molecular Cell, doi:10.1016/j.molcel.2019.03.041

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

G3: GENES, GENOMES, GENETICS, doi:10.1534/g3.119.400330

The human pathogenic yeast Cryptococcus neoformans silences transposable elements using endo-siRNAs and an Argonaute, Ago1. Endo-siRNAs production requires the RNA-dependent RNA polymerase, Rdp1, and two partially redundant Dicer enzymes, Dcr1 and Dcr2, but is independent of histone H3 lysine 9 methylation. We describe here an insertional mutagenesis screen for factors required to suppress the mobilization of the C. neoformans HARBINGER family DNA transposon HAR1. Validation experiments uncovered five novel genes (RDE1-5) required for HAR1 suppression and global production of suppressive endo-siRNAs. The RDE genes do not impact transcript levels, suggesting the endo-siRNAs do not act by impacting target transcript synthesis or turnover. RDE3 encodes a non-Dicer RNase III related to S. cerevisiae Rnt1, RDE4 encodes a predicted terminal nucleotidyltransferase, while RDE5 has no strongly predicted encoded domains. Affinity purification-mass spectrometry studies suggest that Rde3 and Rde5 are physically associated. RDE1 encodes a G-patch protein homologous to the S. cerevisiae Sqs1/Pfa1, a nucleolar protein that directly activates the essential helicase Prp43 during rRNA biogenesis. Rde1 copurifies Rde2, another novel protein obtained in the screen, as well as Ago1, a homolog of Prp43, and numerous predicted nucleolar proteins. We also describe the isolation of conditional alleles of PRP43, which are defective in RNAi. This work reveals unanticipated requirements for a non-Dicer RNase III and presumptive nucleolar factors for endo-siRNA biogenesis and transposon mobilization suppression in C. neoformans.

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.;

The Lancet, doi:10.1016/S0140-6736(18)32592-3

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

The hypertensive pregnancy disorder preeclampsia (PE) is a leading cause of fetal and maternal morbidity/mortality. Obesity increases the risk to develop PE, presumably via the release of inflammatory mediators from the adipose tissue, but the exact etiology remains largely unknown. Using obese PE-like blood pressure high subline 5 (BPH/5) and lean gestational age-matched C57Bl6 mice, we aimed to obtain insight into differential reproductive white adipose tissue (rWAT) gene expression, circulating lipids and inflammation at the maternal-fetal interface during early pregnancy. In addition, we investigated the effect of 7 days 25% calorie restriction (CR) in early pregnancy on gene expression in rWAT and implantation sites. Compared with C57Bl6, female BPH/5 are dyslipidemic before pregnancy and show an amplification of rWAT mass, circulating cholesterol, free fatty acids, and triacylglycerol levels throughout pregnancy. RNA sequencing showed that pregnant BPH/5 mice have elevated gene enrichment in pathways related to inflammation and cholesterol biosynthesis at embryonic day (e7.5. Expression of cholesterol-related HMGCS1MVDCyp51a1, and DHCR was validated by quantitative reverse-transcription-polymerase chain reaction. CR during the first 7 days of pregnancy restored the relative mRNA expression of these genes to a level comparable to C57Bl6 pregnant females and reduced the expression of circulating leptin and proinflammatory prostaglandin synthase 2 in both rWAT and implantation sites in BPH/5 mice at e7.5. Our data suggest a possible role for rWAT in the dyslipidemic state and inflammatory uterine milieu that might underlie the pathogenesis of PE. Future studies should further address the physiological functioning of the adipose tissue in relation to PE-related pregnancy outcomes.

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

Nature Communications, doi:10.1038/s41467-019-09773-y

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

Studies in vertebrates have outlined conserved molecular control of definitive endoderm (END) development. However, recent work also shows that key molecular aspects of human END regulation differ even from rodents. Differentiation of human embryonic stem cells (ESCs) to END offers a tractable system to study the molecular basis of normal and defective human-specific END development. Here, we interrogated dynamics in chromatin accessibility during differentiation of ESCs to END, predicting DNA-binding proteins that may drive this cell fate transition. We then combined single-cell RNA-seq with parallel CRISPR perturbations to comprehensively define the loss-of-function phenotype of those factors in END development. Following a few candidates, we revealed distinct impairments in the differentiation trajectories for mediators of TGFβ signaling and expose a role for the FOXA2 transcription factor in priming human END competence for human foregut and hepatic END specification. Together, this single-cell functional genomics study provides high-resolution insight on human END development.

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

Nature Neuroscience, doi:10.1038/s41593-019-0369-4

Disruption of the blood–brain barrier (BBB) is critical to initiation and perpetuation of disease in multiple sclerosis (MS). We report an interaction between oligodendroglia and vasculature in MS that distinguishes human white matter injury from normal rodent demyelinating injury. We find perivascular clustering of oligodendrocyte precursor cells (OPCs) in certain active MS lesions, representing an inability to properly detach from vessels following perivascular migration. Perivascular OPCs can themselves disrupt the BBB, interfering with astrocyte endfeet and endothelial tight junction integrity, resulting in altered vascular permeability and an associated CNS inflammation. Aberrant Wnt tone in OPCs mediates their dysfunctional vascular detachment and also leads to OPC secretion of Wif1, which interferes with Wnt ligand function on endothelial tight junction integrity. Evidence for this defective oligodendroglial–vascular interaction in MS suggests that aberrant OPC perivascular migration not only impairs their lesion recruitment but can also act as a disease perpetuator via disruption of the BBB.

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

At the M2 terminal of the macrophage activation spectrum, expression of genes is regulated by transcription factors that include STAT6, CREB, and C/EBPβ. Signaling through β-adrenergic receptors drives M2 activation of macrophages, but little is known about the transcription factors involved. In the present study, we found that C/EBPβ regulates the signaling pathway between β-adrenergic stimulation and expression of Arg1 and several other specific genes in the greater M2 transcriptome. β-adrenergic signaling induced Cebpb gene expression relatively early with a peak at 1 h post-stimulation, followed by peak Arg1 gene expression at 8 h. C/EBPβ transcription factor activity was elevated at the enhancer region for Arg 1 at both 4 and 8 h after stimulation but not near the more proximal promoter region. Knockdown of Cebpb suppressed the β-adrenergic-induced peak in Cebpb gene expression as well as subsequent accumulation of C/EBPβ protein in the nucleus, which resulted in suppression of β-adrenergic-induced Arg1 gene expression. Analysis of genome-wide transcriptional profiles identified 20 additional M2 genes that followed the same pattern of regulation by β-adrenergic- and C/EBPβ-signaling. Promoter-based bioinformatic analysis confirmed enrichment of binding motifs for C/EBPβ transcription factor across these M2 genes. These findings pinpoint a mechanism that may be targeted to redirect the deleterious influence of β-adrenergic signaling on macrophage involvement in M2-related diseases such as cancer.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

Zebrafish have the remarkable capacity to regenerate retinal neurons following a variety of damage paradigms. Following initial tissue insult and a period of cell death, a proliferative phase ensues that generates neuronal progenitors, which ultimately regenerate damaged neurons. Recent work has revealed that Müller glia are the source of regenerated neurons in zebrafish. However, the roles of another important class of glia present in the retina, microglia, during this regenerative phase remain elusive. Here, we examine retinal tissue and perform QuantSeq. 3′mRNA sequencing/transcriptome analysis to reveal localization and putative functions, respectively, of mpeg1 expressing cells (microglia/macrophages) during Müller glia-mediated regeneration, corresponding to a time of progenitor proliferation and production of new neurons. Our results indicate that in this regenerative state, mpeg1-expressing cells are located in regions containing regenerative Müller glia and are likely engaged in active vesicle trafficking. Further, mpeg1+ cells congregate at and around the optic nerve head. Our transcriptome analysis reveals several novel genes not previously described in microglia. This dataset represents the first report, to our knowledge, to use RNA sequencing to probe the microglial transcriptome in such context, and therefore provides a resource towards understanding microglia/macrophage function during successful retinal (and central nervous tissue) regeneration.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

Lung cancer is the leading cause of cancer-related deaths in the USA, and alterations in the tumor suppressor gene TP53 are the most frequent somatic mutation among all histologic subtypes of lung cancer. Mutations in TP53 frequently result in a protein that exhibits not only loss of tumor suppressor capability but also oncogenic gain-of-function (GOF). The canonical p53 hotspot mutants R175H and R273H, for example, confer upon tumors a metastatic phenotype in murine models of mutant p53. To the best of our knowledge, GOF phenotypes of the less often studied V157, R158 and A159 mutants—which occur with higher frequency in lung cancer compared with other solid tumors—have not been defined. In this study, we aimed to define whether the lung mutants are simply equivalent to full loss of the p53 locus, or whether they additionally acquire the ability to drive new downstream effector pathways. Using a publicly available human lung cancer dataset, we characterized patients with V157, R158 and A159 p53 mutations. In addition, we show here that cell lines with mutant p53-V157F, p53-R158L and p53-R158P exhibit a loss of expression of canonical wild-type p53 target genes. Furthermore, these lung-enriched p53 mutants regulate genes not previously linked to p53 function including PLAU. Paradoxically, mutant p53 represses genes associated with increased cell viability, migration and invasion. These findings collectively represent the first demonstration that lung-enriched p53 mutations at V157 and R158 regulate a novel transcriptome in human lung cancer cells and may confer de novo function.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

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

BMC Genomics, doi:10.1186/s12864-019-5709-y

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

Radiation Oncology, doi:10.1186/s13014-019-1268-2

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

BioRxiv, doi:10.1101/613356

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

Nature Communications, doi:10.1038/s41467-018-07927-y

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

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

Science Advances, doi:10.1126/sciadv.aav1472

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

bioRxiv, doi:10.1101/581363

Muscle resident fibro-adipogenic progenitors (FAPs), support muscle regeneration by releasing cytokines that stimulate the differentiation of myogenic stem cells. However, in non-physiological contexts (myopathies, atrophy, aging) FAPs cause fibrotic and fat infiltrations that impair muscle function. We set out to perform a fluorescence microscopy-based screening to identify compounds that perturb the differentiation trajectories of these multipotent stem cells. From a primary screen of 1120 FDA/EMA approved drugs, we identified 34 compounds as potential inhibitors of adipogenic differentiation of FAPs isolated from the murine model (mdx) of Duchenne muscular dystrophy (DMD). The hit list from this screen was surprisingly enriched with compounds from the glucocorticoid (GCs) chemical class, drugs that are known to promote adipogenesis in vitro and in vivo. To shed light on these data, three GCs identified in our screening efforts were characterized by different approaches. We found that like dexamethasone, budesonide inhibits adipogenesis induced by insulin in subconfluent FAPs. However, both drugs have a proadipogenic impact when the adipogenic mix contains factors that increase the concentration of cAMP. Gene expression analysis demonstrated that treatment with glucocorticoids induces the transcription of Gilz/Tsc22d3, an inhibitor of the adipogenic master regulator PPARγ, only in anti-adipogenic conditions. Additionally, alongside their anti-adipogenic effect, GCs are shown to promote terminal differentiation of satellite cells. Both the anti-adipogenic and pro-myogenic effects are mediated by the glucocorticoid receptor and are not observed in the presence of receptor inhibitors. Steroid administration currently represents the standard treatment for DMD patients, the rationale being based on their anti-inflammatory effects. The findings presented here offer new insights on additional glucocorticoid effects on muscle stem cells that may affect muscle homeostasis and physiology.

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

Fungal Biology, doi:10.1016/j.funbio.2019.03.007

DNA damage can cause mutations that in fungal plant pathogens lead to hypervirulence and resistance to pesticides. Almost nothing is known about the response of these fungi to DNA damage. We performed transcriptomic and phosphoproteomic analyses of Fusarium oxysporum exposed to methyl methanesulfonate (MMS). At the RNA level we observe massive induction of DNA repair pathways including the global genome nucleotide excision. Cul3, Cul4, several Ubiquitin-like ligases and components of the proteasome are significantly induced. In agreement, we observed drug synergismbetween a proteasome inhibitor and MMS. While our data suggest that Yap1 and Xbp1 networks are similarly activated in response to damage in yeast and F. oxysporum we were able to observe modules that were MMS-responsive in F. oxysporum and not in yeast. These include transcription/splicing modules that are upregulated and respiration that is down-regulated. In agreement, MMS treated cells are much more sensitive to a respiration inhibitor. At the phosphoproteomic level, Adenylate cyclase, which generates cAMP, is phosphorylated in response to MMS and forms a network of phosphorylated proteins that include cell cycle regulators and several MAPKs. Our analysis provides a starting point in understanding how genomic changes in response to DNA damage occur in Fusarium species.

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

Cell Death & Disease, doi:10.1038/s41419-019-1477-5

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

Current Biology, doi:10.1016/j.cub.2019.04.047

Auxin-signal transduction is mediated by the antagonistic activity of transcriptional activators and repressors. Both activators and repressors belong to gene families, but the biological importance of this complexity is not clear. Here, we addressed this question using tomato leaf development as a model by generating and analyzing mutants in multiple auxin-response components. In developing compound tomato leaves, auxin promotes leaflet formation and blade growth, and in the intercalary regions between leaflets, auxin response is inhibited by the Aux/IAA protein ENTIRE (E). e mutants form simple leaves due to ectopic blade growth in the intercalary domain. Using this unique loss-of-function phenotype and genome editing of auxin-response factor (ARF) genes, encoding auxin-response activators, we identified the contribution of specific ARFs to the e phenotype. Mutations in the related ARFs SlMP, SlARF19A, and SlARF19B, but not SlARF7, reduced the leaf blade and suppressed the e phenotype in a dosage-dependent manner that correlated with their relative expression, leading to a continuum of shapes. While single e and slmp mutants affected blade growth in an opposite manner, leaves of e slmp double mutants were similar to those of the wild type. However, the leaf shape of e slmp was more variable than that of the wild type, and it showed increased sensitivity to auxin. Our findings demonstrate that the existence of multiple auxin-response repressors and activators stabilizes the developmental output of auxin and that tuning their activity enables shape variability. The increased complexity of the auxin response therefore balances stability and flexibility in leaf patterning.

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

Nature Communications, doi:10.1038/s41467-018-07936-x

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

Cardiac microvascular endothelial cells (CMVECs) are the most numerous cells in the myocardium and orchestrate cardiogenesis during development, regulate adult cardiac function, and modulate pathophysiology of heart failure. It has been shown that the transcriptome of CMVECs differs from other endothelial cell types, but transcriptomic changes in cardiac endothelial cells during cardiac maturation and cardiac remodeling have not been studied. CMVECs were isolated from rat hearts based on CD31 expression and were immediately processed for RNA sequencing. We compared gene expression levels from primary CMVECs of neonatal hearts, normal adult hearts, and infarcted hearts. Between neonatal and adult CMVECs, 6,838 genes were differentially expressed, indicating that CMVECs undergo a substantial transformation during postnatal cardiac growth. A large fraction of genes upregulated in neonatal CMVECs are part of mitosis pathways, whereas a large fraction of genes upregulated in adult CMVECs are part of cellular response, secretory, signaling, and cell adhesion pathways. Between CMVECs of normal adult hearts and infarcted hearts, 159 genes were differentially expressed. We found a limited degree of overlap (55 genes) between the differentially expressed genes in neonatal and infarcted-hearts. Of 46 significantly upregulated genes in the infarcted heart, 46% were also upregulated in neonatal hearts relative to sham. Of 113 significantly downregulated genes in the infarcted-hearts, 30% were also downregulated in neonatal hearts relative to sham. These data demonstrate that CMVECs undergo dramatic changes from neonatal to adult and more subtle changes between normal state and cardiac remodeling.

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

Nature Communications, doi:10.1038/s41467-019-09250-6

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

Nucleic Acids Research, doi:10.1093/nar/gky1270

The proper subcellular localization of RNAs and local translational regulation is crucial in highly compartmentalized cells, such as neurons. RNA localization is mediated by specific cis-regulatory elements usually found in mRNA 3′UTRs. Therefore, processes that generate alternative 3′UTRs—alternative splicing and polyadenylation—have the potential to diversify mRNA localization patterns in neurons. Here, we performed mapping of alternative 3′UTRs in neurites and soma isolated from mESC-derived neurons. Our analysis identified 593 genes with differentially localized 3′UTR isoforms. In particular, we have shown that two isoforms of Cdc42 gene with distinct functions in neuronal polarity are differentially localized between neurites and soma of mESC-derived and mouse primary cortical neurons, at both mRNA and protein level. Using reporter assays and 3′UTR swapping experiments, we have identified the role of alternative 3′UTRs and mRNA transport in differential localization of alternative CDC42 protein isoforms. Moreover, we used SILAC to identify isoform-specific Cdc42 3′UTR-bound proteome with potential role in Cdc42 localization and translation. Our analysis points to usage of alternative 3′UTR isoforms as a novel mechanism to provide for differential localization of functionally diverse alternative protein isoforms.

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

Nature Communicationsvolume 10, Article number: 100 (2019)

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

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

Nature Communicationsvolume 10, Article number: 63 (2019)

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

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

BMC Genomics2019, doi:10.1186/s12864-018-5393-3

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

Science, doi:10.1126/science.aat6280

Barrier tissues are primary targets of environmental stressors and home to the largest number of antigen-experienced lymphocytes in the body, including commensal-specific T cells. Here, we show that skin-resident commensal-specific T cells harbor a paradoxical program characterized by a type-17 program associated with a poised type-2 state. Thus, in the context of injury and exposure to inflammatory mediators such as IL-18, these cells rapidly release type-2 cytokines, thereby acquiring contextual functions. Notably, such acquisition of a type-2 effector program promotes tissue repair. Aberrant type-2 responses can also be unleashed in the context of local defects in immunoregulation. Thus, commensal-specific T cells co-opt tissue residency and cell-intrinsic flexibility as a means to promote both local immunity and tissue adaptation to injury.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

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

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

Nature Medicine, doi:10.1038/s41591-018-0243-z

Ibrutinib plus venetoclax is a highly effective combination in mantle cell lymphoma. However, strategies to enable the evaluation of therapeutic response are required. Our prospective analyses of patients within the AIM study revealed genomic profiles that clearly dichotomized responders and nonresponders. Mutations in ATM were present in most patients who achieved a complete response, while chromosome 9p21.1–p24.3 loss and/or mutations in components of the SWI–SNF chromatin-remodeling complex were present in all patients with primary resistance and two-thirds of patients with relapsed disease. Circulating tumor DNA analysis revealed that these alterations could be dynamically monitored, providing concurrent information on treatment response and tumor evolution. Functional modeling demonstrated that compromise of the SWI–SNF complex facilitated transcriptional upregulation of BCL2L1 (Bcl-xL) providing a selective advantage against ibrutinib plus venetoclax. Together these data highlight important insights into the molecular basis of therapeutic response and provide a model for real-time assessment of innovative targeted therapies.

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

Translational Oncology, doi:10.1016/j.tranon.2018.10.002

Treatment of glioblastoma (GBM) remains a challenge using conventional chemotherapy, such as temozolomide (TMZ), and is often ineffective as a result of drug resistance. We have assessed a novel nitrone-based agent, OKN-007, and found it to be effective in decreasing tumor volumes and increasing survival in orthotopic GBM xenografts by decreasing cell proliferation and angiogenesis and increasing apoptosis. In this study, we assessed combining OKN-007 with TMZ in vivo in a human G55 GBM orthotopic xenograft model and in vitro in TMZ-resistant and TMZ-sensitive human GBM cell lines. For the in vivo studies, magnetic resonance imaging was used to assess tumor growth and vascular alterations. Percent animal survival was also determined. For the in vitro studies, cell growth, IC50 values, RNA-seq, RT-PCR, and ELISA were used to assess growth inhibition, possible mechanism-of actions (MOAs) associated with combined OKN-007 + TMZ versus TMZ alone, and gene and protein expression levels, respectively. Microarray analysis of OKN-007–treated rat F98 glioma tumors was also carried out to determine possible MOAs of OKN-007 in glioma-bearing animals either treated or not treated with OKN-007. OKN-007 seems to elicit its effect on GBM tumors via inhibition of tumorigenic TGF-β1, which affects the extracellular matrix. When combined with TMZ, OKN-007 significantly increases percent survival, decreases tumor volumes, and normalizes tumor blood vasculature in vivocompared to untreated tumors and seems to affect TMZ-resistant GBM cells possibly via IDO-1SUMO2, and PFN1 in vitro. Combined OKN-007 + TMZ may be a potentially potent treatment strategy for GBM patients.

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

Nature Communications, doi:10.1038/s41467-018-06699-9

Chromosome 17q gains are almost invariably present in high-risk neuroblastoma cases. Here, we perform an integrative epigenomics search for dosage-sensitive transcription factors on 17q marked by H3K27ac defined super-enhancers and identify TBX2 as top candidate gene. We show that TBX2 is a constituent of the recently established core regulatory circuitry in neuroblastoma with features of a cell identity transcription factor, driving proliferation through activation of p21-DREAM repressed FOXM1 target genes. Combined MYCN/TBX2 knockdown enforces cell growth arrest suggesting that TBX2 enhances MYCN sustained activation of FOXM1 targets. Targeting transcriptional addiction by combined CDK7 and BET bromodomain inhibition shows synergistic effects on cell viability with strong repressive effects on CRC gene expression and p53 pathway response as well as several genes implicated in transcriptional regulation. In conclusion, we provide insight into the role of the TBX2 CRC gene in transcriptional dependency of neuroblastoma cells warranting clinical trials using BET and CDK7 inhibitors.

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

Nature Communications, doi:10.1038/s41467-018-07313-8

Myoblast fusion (MF) is required for muscle growth and repair, and its alteration contributes to muscle diseases. The mechanisms governing this process are incompletely understood, and no epigenetic regulator has been previously described. Ash1L is an epigenetic activator belonging to the Trithorax group of proteins and is involved in FSHD muscular dystrophy, autism and cancer. Its physiological role in skeletal muscle is unknown. Here we report that Ash1L expression is positively correlated with MF and reduced in Duchenne muscular dystrophy. In vivo, ex vivo and in vitro experiments support a selective and evolutionary conserved requirement for Ash1L in MF. RNA- and ChIP-sequencing indicate that Ash1L is required to counteract Polycomb repressive activity to allow activation of selected myogenesis genes, in particular the key MF gene Cdon. Our results promote Ash1L as an important epigenetic regulator of MF and suggest that its activity could be targeted to improve cell therapy for muscle diseases.

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

Respiratory Research, doi:10.1186/s12931-018-0932-7

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive scarring of the lung parenchyma, leading to respiratory failure and death. High resolution computed tomography of the chest is often diagnostic for IPF, but its cost and the risk of radiation exposure limit its use as a screening tool even in patients at high risk for the disease. In patients with lung cancer, investigators have detected transcriptional signatures of disease in airway and nasal epithelial cells distal to the site of disease that are clinically useful as screening tools. Here we assessed the feasibility of distinguishing patients with IPF from age-matched controls through transcriptomic profiling of nasal epithelial curettage samples, which can be safely and repeatedly sampled over the course of a patient’s illness. We recruited 10 patients with IPF and 23 age-matched healthy control subjects. Using 3′ messenger RNA sequencing (mRNA-seq), we identified 224 differentially expressed genes, most of which were upregulated in patients with IPF compared with controls. Pathway enrichment analysis revealed upregulation of pathways related to immune response and inflammatory signaling in IPF patients compared with controls. These findings support the concept that fibrosis is associated with upregulation of inflammatory pathways across the respiratory epithelium with possible implications for disease detection and pathobiology.

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

Rationale: The identification of informative elements of the host response to infection may improve the diagnosis and management of bacterial pneumonia. Objective: To determine whether the absence of alveolar neutrophilia can exclude bacterial pneumonia in critically ill patients with suspected infection and to test whether signatures of bacterial pneumonia can be identified in the alveolar macrophage transcriptome. Methods: We determined the test characteristics of alveolar neutrophilia for the diagnosis of bacterial pneumonia in 3 cohorts of mechanically ventilated patients. In one cohort, we also isolated macrophages from alveolar lavage fluid and used the transcriptome to identify signatures of bacterial pneumonia. Finally, we developed a humanized mouse model of Pseudomonas aeruginosa pneumonia to determine if pathogen-specific signatures can be identified in human alveolar macrophages. Measurements and Main Results: An alveolar neutrophil percentage < 50% had a negative predictive value of > 90% for bacterial pneumonia in both the retrospective (N = 851) and validation cohorts (N = 76 and N = 79). A transcriptional signature of bacterial pneumonia was present in both resident and recruited macrophages. Gene signatures from both cell types identified patients with bacterial pneumonia with test characteristics similar to alveolar neutrophilia. Conclusions: The absence of alveolar neutrophilia has a high negative predictive value for bacterial pneumonia in critically ill patients with suspected infection. Macrophages can be isolated from alveolar lavage fluid obtained during routine care and used for RNA-Seq analysis. This novel approach may facilitate a longitudinal and multidimensional assessment of the host response to bacterial pneumonia.

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

PLoS Pathogens, doi:10.1371/journal.ppat.1007402

Transcription of HIV provirus is a key step of the viral cycle, and depends on the recruitment of the cellular positive transcription elongation factor (P-TEFb) to the HIV promoter. The viral transactivator Tat can displace P-TEFb from the 7SK small nuclear ribonucleoprotein, where it is bound and inactivated by HEXIM1, and bring it to TAR, which allows the stalled RNA polymerase II to transition to successful transcription elongation. In this study, we designed a chimeric inhibitor of HIV transcription by combining functional domains from HEXIM1 and Tat. The chimera (HT1) potently inhibited gene expression from the HIV promoter, by competing with Tat for TAR and P-TEFb binding, while keeping the latter inactive. HT1 inhibited spreading infection as well as viral reactivation in lymphocyte T cell line models of HIV latency, with little effect on cellular transcription and metabolism. This proof-of-concept study validates an innovative approach to interfering with HIV transcription via peptide mimicry and competition for RNA-protein interactions. HT1 represents a new candidate for HIV therapy, or HIV cure via the proposed block and lock strategy.

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Maize (Zea mays) is one of the most important staple crops worldwide. Every year, significant proportion of the commercial maize production is lost to insect herbivores and phytopathogens despite extensive control measures. This global issue can be alleviated by harnessing the innate biochemical defense mechanisms of maize, which may have been sacrificed over the course of crop domestication for higher yield. In this thesis, I use Fusarium graminearum, a widespread fungal pathogen of maize, as a model pathosystem, to study the genetic and physiological control of maize specialized metabolism and biochemical defense. By integrating untargeted metabolomics and transcriptomics data into quantitative genetics framework, I am able to discover novel regulatory genes and mechanisms of specific metabolites, as well as to establish a metabolome-scale resource of metabolite-genetic loci associations at high resolution. Through comparative metabolomics analyses, I identify two F. graminearum-resistancerelated acetylated diferuloylsucrose compounds, smiglaside C and smilaside A, which have not been confirmed in maize previously. In an in vitro fungal growth inhibition assay, only the diacetylated smilaside A demonstrates significant bioactivity, whereas the tri-acetylated smiglaside C does not. Genetic mapping of these two compounds, alongside with mutant analyses and physiological experiments, show that ethylene signaling can regulate the metabolism of these two compounds. While ethylene production is required for the accumulation of both compounds in planta, their relative abundance is fine-tuned by ethylene sensitivity. Interestingly, the relative abundance, rather than the absolute amount of these two compounds appears to have a more significant influence on maize resistance against F. graminearum infection. In the same genetic mapping population, genetic mapping and metabolite-transcript correlation analyses suggest that a putative vesicular transport protein is a negative regulator of accumulation of benzaoxazinoids, the most abundant class of specialized metabolites in maize seedlings. This hypothesis is partially supported by genetic mutant analyses and pharmacological disruption of the vesicular transport system in planta. However, further experimental evidence is required to establish a role for the vesicular transport system in benzoxazinoid metabolism. Finally, the chemical genetics approach is extended to a much more diverse maize genome-wide association mapping diversity panel. Multivariate statistical analyses of the large untargeted metabolomics dataset reveal that different classes of specialized metabolites are selectively differentiated between developmental stages and genetic subpopulations. Using liquid chromatography retention time as a proxy of metabolite structure relatedness, it is shown that structurally similar metabolites tend to be co-regulated by shared genetic loci. To demonstrate the utility of the thousands of metabolite-genetic loci association, I experimentally validate that different alleles of a maize citrate synthase gene is responsible for the different structural isomers of hydroxycinnamic acid-hydroxycitric acid conjugates accumulated in the tropical versus temperate maize inbred lines. In summary, work presented in this thesis demonstrates the power of integrating multiomics dataset to dissect specialized metabolism in maize. It provides examples of metabolic and regulatory gene discovery using a forward genetics approach, and set up a platform for future validation of candidate genetic loci associated with potential metabolites of interest.

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Dietary β-adrenergic agonists (β-AA) are used in livestock to increase muscle protein accretion and decrease adipose deposition during the last 20 to 40 d of the finishing period (Johnson et al., 2014). These β-AA act through specific seven transmembrane receptors and are classified by the receptor isoform to which they primarily bind (Mersmann, 1998). Two β-AA are approved for use in beef cattle in the United States: ractopamine HCl (RAC, β1 agonist) and zilpaterol HCl (ZH, β2 agonist) (Johnson et al., 2014). Supplementation of β-AA increases efficiency of the animal and results in a leaner carcass (Elam et al., 2009). However, the skeletal muscle’s genomic response to these treatments is not well understood. Heat stress (HS) has long been a major concern in the livestock industry. HS occurs when an animal’s body temperature rises above its thermoneutral zone, at which point the heat load exceeds the animal’s capacity for heat dissipation (Bernabucci et al., 2010), resulting in decreased feed intake and poor performance (Marai et al., 2007). Therefore, growth and production decrease during HS, affecting economically important carcass and reproductive traits. As a result, millions of dollars are lost each year due to HS (Renaudeau et al., 2012). Individually, HS and β-AA supplementation have antagonistic effects on muscle growth. However, there is a gap in understanding of the genomic mechanisms through which animals respond to these factors individually and in concert. The purpose of this study is to investigate the effects of β-AA, HS, and their interaction in skeletal muscle using transcriptomic analyses.

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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

Journal of Cellular Biochemistry, doi:10.1002/jcb.27716

Patients afflicted with ulcerative colitis (UC) are at increased risk of colorectal cancer. While its causes are not fully understood, UC is associated with defects in colonic epithelial barriers that sustain inflammation of the colon mucosa caused by recruitment of lymphocytes and neutrophils into the lamina propria. Based on genetic evidence that attenuation of the bridging integrator 1 (Bin1) gene can limit UC pathogenicity in animals, we have explored Bin1 targeting as a therapeutic option. Early feasibility studies in the dextran sodium sulfate mouse model of experimental colitis showed that administration of a cell‐penetrating Bin1 monoclonal antibody (Bin1 mAb 99D) could prevent lesion formation in the colon mucosa in part by preventing rupture of lymphoid follicles. In vivo administration of Bin1 mAb altered tight junction protein expression and cecal barrier function. Strikingly, electrophysiology studies in organ cultures showed that Bin1 mAb could elevate resistance and lower 14C‐mannitol leakage across the cecal mucosa, consistent with a direct strengthening of colonic barrier function. Transcriptomic analyses of colitis tissues highlighted altered expression of genes involved in circadian rhythm, lipid metabolism, and inflammation, with a correction of the alterations by Bin1 mAb treatment to patterns characteristic of normal tissues. Overall, our results suggest that Bin1 mAb protects against UC by directly improving colonic epithelial barrier function to limit gene expression and cytokine programs associated with colonic inflammation.

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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

Journal of Plant Research, doi:10.1007/s10265-018-1067-0

Expansin is a non-enzymatic protein which plays a pivotal role in cell wall loosening by inducing stress relaxation and extension in the plant cell wall. Previous studies on Arabidopsis, Petunia × hybrida, and tomato demonstrated that the suppression of expansin gene expression reduced plant growth but expansin overexpression does not necessarily promotes growth. In this study, both expansin gene suppression and overexpression in dark-grown transgenic Arabidopsis seedlings resulted in reduced hypocotyl length at late growth stages with a more pronounced effect for the overexpression. This defect in hypocotyl elongation raises questions about the molecular effect of expansin gene manipulation. RNA-seq analysis of the transcriptomic changes between day 3 and day 5 seedlings for both transgenic lines found numerous differentially expressed genes (DEGs) including transcription factors and hormone-related genes involved in different aspects of cell wall development. These DEGs imply that the observed hypocotyl growth retardation is a consequence of the concerted effect of regulatory factors and multiple cell-wall related genes, which are important for cell wall remodelling during rapid hypocotyl elongation. This is further supported by co-expression analysis through network-centric approach of differential network cluster analysis. This first transcriptome-wide study of expansin manipulation explains why the effect of expansin overexpression is greater than suppression and provides insights into the dynamic nature of molecular regulation during etiolation.

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

Nature Communications, doi:10.1038/s41467-018-06398-5

Recent reports suggest that induced neurons (iNs), but not induced pluripotent stem cell (iPSC)-derived neurons, largely preserve age-associated traits. Here, we report on the extent of preserved epigenetic and transcriptional aging signatures in directly converted induced neural stem cells (iNSCs). Employing restricted and integration-free expression of SOX2 and c-MYC, we generated a fully functional, bona fide NSC population from adult blood cells that remains highly responsive to regional patterning cues. Upon conversion, low passage iNSCs display a profound loss of age-related DNA methylation signatures, which further erode across extended passaging, thereby approximating the DNA methylation age of isogenic iPSC-derived neural precursors. This epigenetic rejuvenation is accompanied by a lack of age-associated transcriptional signatures and absence of cellular aging hallmarks. We find iNSCs to be competent for modeling pathological protein aggregation and for neurotransplantation, depicting blood-to-NSC conversion as a rapid alternative route for both disease modeling and neuroregeneration.

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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.

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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

PNAS, doi: 10.1073/pnas.1811411115

Alzheimer’s disease (AD), the most common form of dementia, is characterized by the abnormal accumulation of amyloid plaques and hyperphosphorylated tau aggregates, as well as microgliosis. Hemizygous missense variants in Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) are associated with elevated risk for developing late-onset AD. These variants are hypothesized to result in loss of function, mimicking TREM2 haploinsufficiency. However, the consequences of TREM2 haploinsufficiency on tau pathology and microglial function remain unknown. We report the effects of partial and complete loss of TREM2 on microglial function and tau-associated deficits. In vivo imaging revealed that microglia from aged TREM2-haploinsufficient mice show a greater impairment in their injury response compared with microglia from aged TREM2-KO mice. In transgenic mice expressing mutant human tau, TREM2 haploinsufficiency, but not complete loss of TREM2, increased tau pathology. In addition, whereas complete TREM2 deficiency protected against tau-mediated microglial activation and atrophy, TREM2 haploinsufficiency elevated expression of proinflammatory markers and exacerbated atrophy at a late stage of disease. The differential effects of partial and complete loss of TREM2 on microglial function and tau pathology provide important insights into the critical role of TREM2 in AD pathogenesis.

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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

Cancer Cell, doi: 10.1016/j.ccell.2018.08.014

Intra-tumor heterogeneity caused by clonal evolution is a major problem in cancer treatment. To address this problem, we performed label-free quantitative proteomics on primary acute myeloid leukemia (AML) samples. We identified 50 leukemia-enriched plasma membrane proteins enabling the prospective isolation of genetically distinct subclones from individual AML patients. Subclones differed in their regulatory phenotype, drug sensitivity, growth, and engraftment behavior, as determined by RNA sequencing, DNase I hypersensitive site mapping, transcription factor occupancy analysis, in vitro culture, and xenograft transplantation. Finally, we show that these markers can be used to identify and longitudinally track distinct leukemic clones in patients in routine diagnostics. Our study describes a strategy for a major improvement in stratifying cancer diagnosis and treatment.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

The non-conventional yeast species Zygosaccharomyces bailii is remarkably tolerant to acetic acid, a highly important microbial inhibitory compound in Food Industry and Biotechnology. ZbHaa1 is the functional homologue of S. cerevisiae Haa1 and a bifunctional transcription factor able to modulate Z. bailii adaptive response to acetic acid and copper stress. In this study, RNA-Seq was used to investigate genomic transcription changes in Z. bailii during early response to sublethal concentrations of acetic acid (140 mM, pH 4.0) or copper (0.08 mM) and uncover the regulatory network activated by these stresses under ZbHaa1 control. Differentially expressed genes in response to acetic acid exposure (297) are mainly related with the tricarboxylic acid cycle, protein folding and stabilization and modulation of plasma membrane composition and cell wall architecture, 17 of which, directly or indirectly, ZbHaa1-dependent. Copper stress induced the differential expression of 190 genes mainly involved in the response to oxidative stress, 15 ZbHaa1-dependent. This study provides valuable mechanistic insights regarding Z. bailiiadaptation to acetic acid or copper stress, as well as useful information on transcription regulatory networks in pre-whole genome duplication (WGD) (Z. bailii) and post-WGD (S. cerevisiae) yeast species, contributing to the understanding of transcriptional networks’ evolution in yeasts.

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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

Vaccine, doi: 10.1016/j.vaccine.2018.09.001

Introduction
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

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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

Journal of Autoimmunity, 10.1016/j.jaut.2018.08.005

Neuropsychiatric manifestations in lupus (NPSLE) affect ∼20–40% of patients. In the central nervous system, lipocalin-2 (LCN2) can promote injury through mechanisms directly linked to NPSLE, including brain barrier disruption, neurotoxicity, and glial activation. Since LCN2 is elevated in lupus and has been implicated in neuroinflammation, we investigated whether LCN2 is required for the pathogenesis of NPSLE. Here, we investigated the effects of LCN2 deficiency on the development of neurobehavioral deficits in the B6.Sle1.Sle3 (Sle1,3) mouse lupus model. Sle1,3 mice exhibited depression-like behavior and impaired spatial and recognition memory, and these deficits were attenuated in Sle1,3-LCN2KO mice. Whole-brain flow cytometry showed a significant increase in brain infiltrating leukocytes in Sle1,3 mice that was not reduced by LCN2 deficiency. RNA sequencing on sorted microglia revealed that several genes differentially expressed between B6 and Sle1,3 mice were regulated by LCN2, and that these genes are key mediators of the neuroinflammatory cascade. Importantly, LCN2 is upregulated in the cerebrospinal fluid of NPSLE patients across 2 different ethnicities. Our findings establish the Sle1,3 strain as an NPSLE model, demonstrate that LCN2 is a major regulator of the detrimental neuroimmune response in NPSLE, and identify CSF LCN2 as a novel biomarker for NPSLE.

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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

Stem Cell Reports, 10.1016/j.stemcr.2018.07.004

Defective placentation is the underlying cause of various pregnancy complications, such as severe intrauterine growth restriction and preeclampsia. However, studies on human placental development are hampered by the lack of a self-renewing in vitro model that would recapitulate formation of trophoblast progenitors and differentiated subtypes, syncytiotrophoblast (STB) and invasive extravillous trophoblast (EVT), in a 3D orientation. Hence, we established long-term expanding organoid cultures from purified first-trimester cytotrophoblasts (CTBs). Molecular analyses revealed that the CTB organoid cultures (CTB-ORGs) express markers of trophoblast stemness and proliferation and are highly similar to primary CTBs at the level of global gene expression. Whereas CTB-ORGs spontaneously generated STBs, withdrawal of factors for self-renewal induced trophoblast outgrowth, expressing the EVT progenitor marker NOTCH1, and provoked formation of adjacent, distally located HLA-G+ EVTs. In summary, we established human CTB-ORGs that grow and differentiate under defined culture conditions, allowing future human placental disease modeling.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

The enhancer landscape of pluripotent stem cells undergoes extensive reorganization during early mammalian development. The functions and mechanisms behind such reorganization, however, are unclear. Here, we show that the transcription factor GRHL2 is necessary and sufficient to activate an epithelial subset of enhancers as naive embryonic stem cells (ESCs) transition into formative epiblast-like cells (EpiLCs). Surprisingly, many GRHL2 target genes do not change in expression during the ESC-EpiLC transition. Instead, enhancers regulating these genes in ESCs diminish in activity in EpiLCs while GRHL2-dependent alternative enhancers become activated to maintain transcription. GRHL2 therefore assumes control over a subset of the naive network via enhancer switching to maintain expression of epithelial genes upon exit from naive pluripotency. These data evoke a model where the naive pluripotency network becomes partitioned into smaller, independent networks regulated by EpiLC-specific transcription factors, thereby priming cells for lineage specification.

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

Science, doi:10.1126/science.aao2793

Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. Here we combine SLAM-seq, a method for direct quantification of newly synthesized mRNAs, with pharmacological and chemical-genetic perturbation to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETi). We find that BRD4 acts as general co-activator of RNA polymerase II (Pol2)-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETi deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de-novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway.

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

Nature, doi:10.1038/nature25966

Here we report a multi-tissue gene expression resource that represents the genotypic and phenotypic diversity of modern inbred maize, and includes transcriptomes in an average of 255 lines in seven tissues. We mapped expression quantitative trait loci and characterized the contribution of rare genetic variants to extremes in gene expression. Some of the new mutations that arise in the maize genome can be deleterious; although selection acts to keep deleterious variants rare, their complete removal is impeded by genetic linkage to favourable loci and by finite population size. Modern maize breeders have systematically reduced the effects of this constant mutational pressure through artificial selection and self-fertilization, which have exposed rare recessive variants in elite inbred lines. However, the ongoing effect of these rare alleles on modern inbred maize is unknown. By analysing this gene expression resource and exploiting the extreme diversity and rapid linkage disequilibrium decay of maize, we characterize the effect of rare alleles and evolutionary history on the regulation of expression. Rare alleles are associated with the dysregulation of expression, and we correlate this dysregulation to seed-weight fitness. We find enrichment of ancestral rare variants among expression quantitative trait loci mapped in modern inbred lines, which suggests that historic bottlenecks have shaped regulation. Our results suggest that one path for further genetic improvement in agricultural species lies in purging the rare deleterious variants that have been associated with crop fitness.

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

Frontiers in Neuroscience, doi:10.3389/fnins.2018.00136

The detection of external and internal cues alters gene expression in the brain which in turn may affect neural networks that underly behavioral responses. Previous studies have shown that gene expression profiles differ between major brain regions within individuals and between species with different morphologies, cognitive abilities and/or behaviors. A detailed description of gene expression in all macroanatomical brain regions and in species with similar morphologies and behaviors is however lacking. Here, we dissected the brain of two cichlid species into six macroanatomical regions. Ophthalmotilapia nasuta and O. ventralis have similar morphology and behavior and occasionally hybridize in the wild. We use 3′ mRNA sequencing and a stage-wise statistical testing procedure to identify differential gene expression between females that were kept in a social setting with other females. Our results show that gene expression differs substantially between all six brain parts within species: out of 11,577 assessed genes, 8,748 are differentially expressed (DE) in at least one brain part compared to the average expression of the other brain parts. At most 16% of these DE genes have |log2FC| significantly higher than two. Functional differences between brain parts were consistent between species. The majority (61–79%) of genes that are DE in a particular brain part were shared between both species. Only 32 genes show significant differences in fold change across brain parts between species. These genes are mainly linked to transport, transmembrane transport, transcription (and its regulation) and signal transduction. Moreover, statistical equivalence testing reveals that within each comparison, on average 89% of the genes show an equivalent fold change between both species. The pronounced differences in gene expression between brain parts and the conserved patterns between closely related species with similar morphologies and behavior suggest that unraveling the interactions between genes and behavior will benefit from neurogenomic profiling of distinct brain regions.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

Homologous recombination (HR), which ensures accurate DNA replication and strand-break repair, is necessary to preserve embryonic stem cell (ESC) self-renewal. However, little is known about how HR factors modulate ESC differentiation and replication stress-associated DNA breaks caused by unique cell-cycle progression. Here, we report that ESCs utilize Rad51-dependent HR to enhance viability and induce rapid proliferation through a replication-coupled pathway. In addition, ESC differentiation was shown to be enhanced by ectopic expression of a subset of recombinases. Abundant expression of HR proteins throughout the ESC cycle, but not during differentiation, facilitated immediate HR-mediated repair of single-stranded DNA (ssDNA) gaps incurred during S-phase, via a mechanism that does not perturb cellular progression. Intriguingly, combined ectopic expression of two recombinases, Rad51 and Rad52, resulted in efficient ESC differentiation and diminished cell death, indicating that HR factors promote cellular differentiation by repairing global DNA breaks induced by chromatin remodeling signals. Collectively, these findings provide insight into the role of key HR factors in rapid DNA break repair following chromosome duplication during self-renewal and differentiation of ESCs.

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

bioRxiv, doi:10.1101/250381

The complex neuropathology of traumatic brain injury (TBI) is difficult to dissect in the hippocampus considering the convoluted hippocampal cytoarchitecture. As a major casualty of TBI, hippocampal dysfunction results in cognitive decline that may escalate to other neurological disorders, and the molecular basis is hidden in the genomic programs of individual hippocampal cells. Using the unbiased single cell sequencing method Drop-seq, we uncovered the hippocampal cell types most sensitive to concussive mild TBI (mTBI) as well as the vulnerable genes, pathways and cell-cell interactions predictive of disease pathogenesis in a cell-type specific manner, revealing hidden pathogenic mechanisms and potential targets. Targeting Ttr, encoding the thyroid hormone T4 transporter transthyretin, mitigated the genomic and behavioral abnormalities associated with mTBI. Single cell genomics provides unique evidence about altered circuits and pathogenic pathways, and pinpoints new targets amenable to therapeutics in mTBI and related disorders.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

Gene-level differential expression analysis based on RNA-Seq is more robust, powerful and biologically actionable than transcript-level differential analysis. However aggregation of transcript counts prior to analysis results can mask transcript-level dynamics. We demonstrate that aggregating the results of transcript-level analysis allow for gene-level analysis with transcript-level resolution. We also show that p-value aggregation methods, typically used for meta-analyses, greatly increase the sensitivity of gene-level differential analyses. Furthermore, such aggregation can be applied directly to transcript compatibility counts obtained during pseudoalignment, thereby allowing for rapid and accurate model-free differential testing. The methods are general, allowing for testing not only of genes but also of any groups of transcripts, and we showcase an example where we apply them to perturbation analysis of gene ontologies.

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

Cancer Research, doi:10.1158/0008-5472.CAN-17-2210

New treatments for triple-negative breast cancer (TNBC) are urgently needed. Despite there being little evidence of clinical activity as single-agent therapies, we show that dual blockade of PI3Kα and CDK4/6 is synergistically effective against multiple RB1-wild-type TNBC models. Combined PI3Kα and CDK4/6 inhibition significantly increased apoptosis, cell-cycle arrest, and tumor immunogenicity and generated immunogenic cell death in human TNBC cell lines. Combination treatment also significantly improved disease control in human xenograft models compared with either monotherapy. Combined PI3Kα and CDK4/6 inhibition significantly increased tumor-infiltrating T-cell activation and cytotoxicity and decreased the frequency of immunosuppressive myeloid-derived suppressor cells in a syngeneic TNBC mouse model. Notably, combined PI3Kα and CDK4/6 inhibition, along with inhibition of immune checkpoints PD-1 and CTLA-4, induced complete and durable regressions (>1 year) of established TNBC tumors in vivo. Overall, our results illustrate convergent mechanisms of PI3Kα and CDK4/6 blockade on cell-cycle progression, DNA damage response, and immune-modulation and may provide a novel therapeutic approach for TNBC.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

We and others have proposed that coactivator binding inhibitors, which block the interaction of estrogen receptor and steroid receptor coactivators, may represent a potential class of new breast cancer therapeutics. The development of coactivator binding inhibitors has been limited, however, because many of the current molecules which are active in in vitro and biochemical assays are not active in cell-based assays. Our goal in this work was to prepare a coactivator binding inhibitor active in cellular models of breast cancer. To accomplish this, we used molecular dynamics simulations to convert a high-affinity stapled peptide with poor cell permeability into R4K1, a cell-penetrating stapled peptide. R4K1 displays high binding affinity for estrogen receptor α, inhibits the formation of estrogen receptor/coactivator complexes, and distributes throughout the cell with a high percentage of nuclear localization. R4K1 represses native gene transcription mediated by estrogen receptor α and inhibits proliferation of estradiol-stimulated MCF-7 cells. Using RNA-Seq, we demonstrate that almost all of the effects of R4K1 on global gene transcription are estrogen-receptor-associated. This chemical probe provides a significant proof-of-concept for preparing cell-permeable stapled peptide inhibitors of the estrogen receptor/coactivator interaction.

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

Oncotarget, doi:10.18632/oncotarget.19424

Genetically engineered animal tumor models have traditionally been generated by the gain of single or multiple oncogenes or the loss of tumor suppressor genes; however, the development of live animal models has been difficult given that cancer phenotypes are generally induced by somatic mutation rather than by germline genetic inactivation. In this study, we developed somatically mutated tumor models using TALEN-mediated somatic gene inactivation of cdkn2a/b or rb1 tumor suppressor genes in zebrafish. One-cell stage injection of cdkn2a/b-TALEN mRNA resulted in malignant peripheral nerve sheath tumors with high frequency (about 39%) and early onset (about 35 weeks of age) in F0 tp53e7/e7 mutant zebrafish. Injection of rb1-TALEN mRNA also led to the formation of brain tumors at high frequency (58%, 31 weeks of age) in F0 tp53e7/e7 mutant zebrafish. Analysis of each tumor induced by somatic inactivation showed that the targeted genes had bi-allelic mutations. Tumors induced by rb1 somatic inactivation were characterized as medulloblastoma-like primitive neuroectodermal tumors based on incidence location, histopathological features, and immunohistochemical tests. In addition, 3′ mRNA Quanti-Seq analysis showed differential activation of genes involved in cell cycle, DNA replication, and protein synthesis; especially, genes involved in neuronal development were up-regulated.

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

Cell Death and Differentiation, doi:10.1038/cdd.2017.182

Staufen1 (STAU1) and Lin28B are RNA-binding proteins that are involved in neuronal differentiation as a function of post-transcriptional regulation. STAU1 triggers post-transcriptional regulation, including mRNA export, mRNA relocation, translation and mRNA decay. Lin28B also has multiple functions in miRNA biogenesis and the regulation of translation. Here, we examined the connection between STAU1 and Lin28B and found that Lin28B regulates the abundance of STAU1 mRNA via miRNA maturation. Decreases in the expression of both STAU1 and Lin28B were observed during neuronal differentiation. Depletion of STAU1 or Lin28B inhibited neuronal differentiation, and overexpression of STAU1 or Lin28B enhanced neuronal differentiation. Interestingly, the stability of STAU1 mRNA was modulated by miR-142-3p, whose maturation was regulated by Lin28B. Thus, miR-142-3p expression increased as Lin28B expression decreased during differentiation, leading to the reduction of STAU1 expression. The transcriptome from Staufen-mediated mRNA decay (SMD) targets during differentiation was analyzed, confirming that STAU1 was a key factor in neuronal differentiation. In support of this finding, regulation of STAU1 expression in mouse neural precursor cells had the same effects on neuronal differentiation as it did in human neuroblastoma cells. These results revealed the collaboration of two RNA-binding proteins, STAU1 and Lin28B, as a regulatory mechanism in neuronal differentiation.

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

Nature, doi:10.1038/nature24293

Interleukin-1 receptor 8 (IL-1R8, also known as single immunoglobulin IL-1R-related receptor, SIGIRR, or TIR8) is a member of the IL-1 receptor (ILR) family with distinct structural and functional characteristics, acting as a negative regulator of ILR and Toll-like receptor (TLR) downstream signalling pathways and inflammation. Natural killer (NK) cells are innate lymphoid cells which mediate resistance against pathogens and contribute to the activation and orientation of adaptive immune responses. NK cells mediate resistance against haematopoietic neoplasms but are generally considered to play a minor role in solid tumour carcinogenesis. Here we report that IL-1R8 serves as a checkpoint for NK cell maturation and effector function. Its genetic blockade unleashes NK-cell-mediated resistance to hepatic carcinogenesis, haematogenous liver and lung metastasis, and cytomegalovirus infection.

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

Development, doi:10.1242/dev.164640

Cell type-specific transcriptome analysis is an essential tool in understanding biological processes in which diverse types of cells are involved. Although cell isolation methods such as fluorescence-activated cell sorting (FACS) in combination with transcriptome analysis have widely been used so far, their time-consuming and harsh procedures limit their applications. Here, we report a novel in vivo metabolic RNA sequencing method, SLAM-ITseq, which metabolically labels RNA with 4-thiouracil in a specific cell type in vivo followed by detection through an RNA-seq-based method that specifically distinguishes the thiolated uridine by base conversion. This method has successfully identified the cell type-specific transcriptome in three different tissues: endothelial cells in brain, epithelial cells in intestine, and adipocytes in white adipose tissue. Since this method does not require isolation of cells or RNA prior to the transcriptomic analysis, SLAM-ITseq provides an easy yet accurate snapshot of the transcriptional state in vivo.

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

Nature Communicationsvolume, doi:10.1038/s41467-017-02099-7

The majority of mammalian genes contain one or more alternative polyadenylation sites. Choice of polyadenylation sites was suggested as one of the underlying mechanisms for generating longer/shorter transcript isoforms. Here, we demonstrate that mature mRNA transcripts can undergo additional cleavage and polyadenylation at a proximal internal site in the 3′-UTR, resulting in two stable, autonomous, RNA fragments: a coding sequence with a shorter 3′-UTR (body) and an uncapped 3′-UTR sequence downstream of the cleavage point (tail). Analyses of the human transcriptome has revealed thousands of such cleavage positions, suggesting a widespread post-transcriptional phenomenon producing thousands of stable 3′-UTR RNA tails that exist alongside their transcripts of origin. By analyzing the impact of microRNAs, we observed a significantly stronger effect for microRNA regulation at the body compared to the tail fragments. Our findings open a variety of future research prospects and call for a new perspective on 3′-UTR-dependent gene regulation.

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

BioRxiv, doi:10.1101/221093

Polycomb proteins are essential epigenetic regulators of gene transcription. KDM2B, the chromatin-binding moiety of non-canonical PRC1.1, is critically important for human leukemias. Here, we investigated the complete interactome of KDM2B in human leukemic cells and identified that the deubiquitinase USP7 is an essential component of PRC1.1 and required for its stability and function. USP7 inhibition results in disassembly of the PRC1.1 complex and consequently loss of binding to its target loci. PRC1.1 can be associated with active loci and loss of PRC1.1 binding coincided with loss of H2AK119ub, reduced H3K27ac levels and reduced gene transcription, whereas H3K4me3 levels remained unaffected. Survival was reduced in (primary) acute myeloid leukemia cells in both cycling as well as quiescent populations upon USP7 inhibition, also independent of the USP7-MDM2-p53 axis. Finally, we evaluated the efficacy of USP7 inhibition in vivo and find that progression of MLL-AF9-induced leukemia is delayed, although in a niche-dependent manner.

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

Cell, doi:10.1016/j.cell.2017.12.033

Mammalian barrier surfaces are constitutively colonized by numerous microorganisms. We explored how the microbiota was sensed by the immune system and the defining properties of such responses. Here, we show that a skin commensal can induce T cell responses in a manner that is restricted to non-classical MHC class I molecules. These responses are uncoupled from inflammation and highly distinct from pathogen-induced cells. Commensal-specific T cells express a defined gene signature that is characterized by expression of effector genes together with immunoregulatory and tissue-repair signatures. As such, non-classical MHCI-restricted commensal-specific immune responses not only promoted protection to pathogens, but also accelerated skin wound closure. Thus, the microbiota can induce a highly physiological and pleiotropic form of adaptive immunity that couples antimicrobial function with tissue repair. Our work also reveals that non-classical MHC class I molecules, an evolutionarily ancient arm of the immune system, can promote homeostatic immunity to the microbiota.

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

The Plant cell, doi:10.1105/tpc.17.00805

Plants use diverse mechanisms influenced by vast regulatory networks of indefinite scale to adapt to their environment. These regulatory networks have an unknown potential for epistasis between genes within and across networks. To test for epistasis within an adaptive trait genetic network, we generated and tested 47 Arabidopsis thaliana double mutant combinations for 20 transcription factors, which all influence the accumulation of aliphatic glucosinolates, the defense metabolites that control fitness. The epistatic combinations were used to test if there is more or less epistasis depending on gene membership within the same or different phenotypic subnetworks. Extensive epistasis was observed between the transcription factors, regardless of subnetwork membership. Metabolite accumulation displayed antagonistic epistasis, suggesting the presence of a buffering mechanism. Epistasis affecting enzymatic estimated activity was highly conditional on the tissue and environment and shifted between both antagonistic and synergistic forms. Transcriptional analysis showed that epistasis shifts depend on how the trait is measured. Because the 47 combinations described here represent a small sampling of the potential epistatic combinations in this genetic network, there is potential for significantly more epistasis. Additionally, the main effect of the individual gene was not predictive of the epistatic effects, suggesting that there is a need for further studies.

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

Biomaterials, doi:10.1016/j.biomaterials.2017.07.033

Multipotent cells have similar basic features of all stem cells but limitation in ability of self-renewal and differentiation compared with pluripotent cells. Here, we have developed an ultra effective, gene- and chemical-free method of generating extra multipotent (xpotent) cells which have differentiation potential more than limited cell types, by the mechanism of ultrasound-directed permeation of environmental transition-guided cellular reprogramming (Entr). Ultrasound stimulus generated a massive number of Entr-mediated xpotent (x/Entr) spheroids from human dermal fibroblasts (HDFs) 6 days after treatment. The emergence of x/Entr was first initiated by the introduction of human embryonic stem cell (ESC) environments into the HDFs to start fast cellular reprogramming including activation of stress-related kinase signaling pathways, subsequent chromatin remodeling, and expression of pluripotent-related genes via transient membrane damage caused by ultrasound-induced cavitation. And then, pluripotent markers were transported into their adjacent HDFs via direct cell-to-cell connections in order to generate xpotent clusters. The features of x/Entr cells were intermediate between pluripotency and multipotency in terms of pluripotency with three germ layer markers, multi-lineage differentiation potential, and no teratoma formation. This physical stimulus-mediated reprogramming strategy was cost-effective, simple, quick, produced significant yields, and was safe, and can therefore provide a new paradigm for clinical application.

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

Proceedings of the National Academy of Sciences of the United States of America, doi:10.1073/pnas.1615105114

On pathological stress, Wnt signaling is reactivated and induces genes associated with cardiac remodeling and fibrosis. We have previously shown that a cell surface receptor Cdon (cell-adhesion associated, oncogene regulated) suppresses Wnt signaling to promote neuronal differentiation however its role in heart is unknown. Here, we demonstrate a critical role of Cdon in cardiac function and remodeling. Cdon is expressed and predominantly localized at intercalated disk in both mouse and human hearts. Cdon-deficient mice develop cardiac dysfunction including reduced ejection fraction and ECG abnormalities. Cdon−/− hearts exhibit increased fibrosis and up-regulation of genes associated with cardiac remodeling and fibrosis. Electrical remodeling was demonstrated by up-regulation and mislocalization of the gap junction protein, Connexin 43 (Cx43) in Cdon−/− hearts. In agreement with altered Cx43 expression, functional analysis both using Cdon−/− cardiomyocytes and shRNA-mediated knockdown in rat cardiomyocytes shows aberrant gap junction activities. Analysis of the underlying mechanism reveals that Cdon−/− hearts exhibit hyperactive Wnt signaling as evident by β-catenin accumulation and Axin2 up-regulation. On the other hand, the treatment of rat cardiomyocytes with a Wnt activator TWS119 reduces Cdon levels and aberrant Cx43 activities, similarly to Cdon-deficient cardiomyocytes, suggesting a negative feedback between Cdon and Wnt signaling. Finally, inhibition of Wnt/β-catenin signaling by XAV939, IWP2 or dickkopf (DKK)1 prevented Cdon depletion-induced up-regulation of collagen 1a and Cx43. Taken together, these results demonstrate that Cdon deficiency causes hyperactive Wnt signaling leading to aberrant intercellular coupling and cardiac fibrosis. Cdon exhibits great potential as a target for the treatment of cardiac fibrosis and cardiomyopathy.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

Pancreatic ductal adenocarcinoma (PDAC) is the fourth-leading cause of cancer-related deaths in the United States with metastasis to the liver as the major cause of mortality. While the propensity of PDAC to spread to the liver may reflect mechanical trapping of tumor cells that enter the portal circulation, primary tumor cells have also been suggested to secrete factors that may promote recruitment of myeloid cells to establish a pro-metastatic niche. In this study, we used the LSL-KrasG12D/+;LSL-Trp53R127H/+;Pdx-1-Cre (KPC) mouse model of PDAC to investigate the impact of PDAC development on the formation of a pro-metastatic niche in the liver. We found that KPC mice (compared to age- and gender-matched control mice) demonstrated an increased susceptibility to tumor seeding in the liver even prior to development of invasive PDAC. Examination of the liver of KPC mice revealed diffuse activation of Signal Transducer and Activator of Transcription 3 (STAT3) signaling, particularly in hepatocytes. Although hepatocytes are recognized as important regulators of inflammation, their role in establishing a pro-metastatic niche is unknown. To define changes in the liver associated with development of a pro-metastatic niche, we performed QuantSeq analysis on RNA isolated from the liver of KPC versus control PC mice. Our results showed increased transcriptional levels of myeloid chemoattractants, particularly serum amyloid A proteins that are predominantly produced by hepatocytes. Consistent with this finding, we observed an accumulation of F4/80+ and Ly6G+ myeloid cells in the liver of KPC mice by immunofluorescence microscopy. We next determined the role of tumor cells in driving cellular activation seen in the liver by establishing intraperitoneal and orthotopic models of PDAC. Using these models, we found that implantation of pancreatic tumor cells induced STAT3 activation in hepatocytes and stimulated F4/80+ and Ly6G+ myeloid cell recruitment to the liver. To determine whether cellular activation in the liver was associated with systemic release of soluble factors, we performed parabiotic joining of tumor-implanted mice and control wild type mice, and we found evidence of STAT3 activation and myeloid recruitment to the liver in parabiotic pairs. As interleukin-6 (IL-6) is a key inflammatory cytokine that can activate STAT3 signaling, we hypothesized a role for IL-6 directed STAT3 activation in hepatocytes for development of a pro-metastatic niche in the liver. Consistent with this hypothesis, we found that IL-6 receptor blocking antibodies administered after tumor implantation reduced STAT3 activation in hepatocytes and decreased transcriptional levels of hepatocyte-derived chemoattractants. Together, our findings support a role for IL-6/STAT3 signaling in hepatocytes in driving a pro-metastatic niche in the liver during PDAC development.

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

Nature Neuroscience, doi:10.1038/nn.4597