SENSE mRNA-Seq for Ion Torrent Publications

Major depressive disorder (MDD) is a debilitating illness that affects twice as many women than men postpuberty. This female bias is thought to be caused by greater heritability of MDD in women and increased vulnerability induced by female sex hormones. We tested this hypothesis by removing the ovaries from prepubertal Wistar Kyoto (WKY) more immobile (WMI) females, a genetic animal model of depression, and its genetically close control, the WKY less immobile (WLI). In adulthood, prepubertally ovariectomized (PrePubOVX) animals and their Sham-operated controls were tested for depression- and anxiety-like behaviors, using the routinely employed forced swim and open field tests, respectively, and RNA-sequencing was performed on their hippocampal RNA. Our results confirmed that the behavioral and hippocampal expression changes that occur after prepubertal ovariectomy are the consequences of an interaction between genetic predisposition to depressive behavior and ovarian hormone-regulated processes. Lack of ovarian hormones during and after puberty in the WLIs led to increased depression-like behavior. In WMIs, both depression- and anxiety-like behaviors worsened by prepubertal ovariectomy. The unbiased exploration of the hippocampal transcriptome identified sets of differentially expressed genes (DEGs) between the strains and treatment groups. The relatively small number of hippocampal DEGs resulting from the genetic differences between the strains confirmed the genetic relatedness of these strains. Nevertheless, the differences in DEGs between the strains in response to prepubertal ovariectomy identified different molecular processes, including the importance of glucocorticoid receptor-mediated mechanisms, that may be causative of the increased depression-like behavior in the presence or absence of genetic predisposition. This study contributes to the understanding of hormonal maturation-induced changes in affective behaviors and the hippocampal transcriptome as it relates to genetic predisposition to depression.

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Jjj1 Is a Negative Regulator of Pdr1-Mediated Fluconazole Resistance in Candida glabrata

Sarah G. Whaley, Kelly E. Caudle, Lucia Simonicova, Qing Zhang, W. Scott Moye-Rowley, P. David Rogers

mSphere, doi:10.1128/mSphere.00466-17

The high prevalence of fluconazole resistance among clinical isolates of Candida glabrata has greatly hampered the utility of fluconazole for the treatment of invasive candidiasis. Fluconazole resistance in this yeast is almost exclusively due to activating mutations in the transcription factor Pdr1, which result in upregulation of the ABC transporter genes CDR1, PDH1, and SNQ2 and therefore increased fluconazole efflux. However, the regulation of Pdr1 is poorly understood. In order to identify genes that interact with the Pdr1 transcriptional pathway and influence the susceptibility of C. glabrata to fluconazole, we screened a collection of deletion mutants for those exhibiting increased resistance to fluconazole. Deletion of the gene coding for a protein homologous to the Saccharomyces cerevisiae J protein Jjj1 resulted in decreased fluconazole susceptibility. We used the SAT1 flipper method to generate independent deletion mutants for JJJ1 in an SDD clinical isolate. Expression of both CDR1 and PDR1 was increased in the absence of JJJ1. In the absence of CDR1 or PDR1, deletion of JJJ1 has only a modest effect on fluconazole susceptibility. Transcriptional profiling using transcriptome sequencing (RNA-seq) revealed upregulation of genes of the Pdr1 regulon in the absence of JJJ1. Jjj1 appears to be a negative regulator of fluconazole resistance in C. glabrata and acts primarily through upregulation of the ABC transporter gene CDR1 via activation of the Pdr1 transcriptional pathway.

Features SENSE mRNA‐Seq Library Prep Kit for Ion Torrent

Loss of C-5 sterol desaturase activity results in increased resistance to azole and echinocandin antifungals in a clinical isolate of Candida parapsilosis

Jeffrey M. Rybak, C. Michael Dickens, Josie E. Parker, Kelly Caudle, Kayihura Manigaba, Sarah G. Whaley, Andrew Nishimoto, Arturo Luna-Tapia, Sujoy Roy, Qing Zhang, Katherine S. Barker, Glen E. Palmer, Thomas R. Sutter, Ramin Homayouni, Nathan P. Wiederhold, Steven L. Kelly and P. David Rogers

Antimicrobial Agents and Chemotherapy, doi:10.1128/AAC.00651-17

Among emerging non-albicans Candida species, C. parapsilosis is of particular concern as a cause of nosocomial bloodstream infections in neonatal and intensive care unit patients. While fluconazole and echinocandins are considered effective treatment of such infections, recent reports of fluconazole and echinocandin resistance in C. parapsilosis indicate a growing problem. The present study describes a novel mechanism of antifungal resistance in this organism affecting the susceptibility of azole and echinocandin antifungals in a clinical isolate obtained from a patient with prosthetic valve endocarditis. Transcriptome analysis indicated differential expression of several genes in the resistant isolate including upregulation of ergosterol biosynthesis pathway genes ERG2, ERG5, ERG6, ERG11, ERG24, ERG25, and UPC2. Whole-genome sequencing revealed the resistant isolate possessed an ERG3 mutation resulting in a G111R amino acid substitution. Sterol profiles indicated a reduction in sterol desaturase activity as a result of this mutation. Replacement of both mutant alleles in the resistant isolate with the susceptible isolate’s allele restored wild-type susceptibility to all azoles and echinocandins tested. Disruption of ERG3 in the susceptible and resistant isolates resulted in a loss of sterol desaturase activity, high-level azole resistance, and an echinocandin-intermediate to -resistant phenotype. While disruption of ERG3 in C. albicans resulted in azole resistance, echinocandin MICs, while elevated, remained within the susceptible range. This work demonstrates that the G111R substitution in Erg3 is wholly responsible for the altered azole and echinocandin susceptibilities observed in this C. parapsilosis isolate and is the first report of an ERG3 mutation influencing susceptibility to the echinocandins.

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Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals

Pedro M. Quirós, Miguel A. Prado, Nicola Zamboni, Davide D’Amico, Robert W. Williams, Daniel Finley, Steven P. Gygi, Johan Auwerx

The Journal of Cell Biology, doi:10.1083/jcb.201702058

Mitochondrial stress activates a mitonuclear response to safeguard and repair mitochondrial function and to adapt cellular metabolism to stress. Using a multiomics approach in mammalian cells treated with four types of mitochondrial stressors, we identify activating transcription factor 4 (ATF4) as the main regulator of the stress response. Surprisingly, canonical mitochondrial unfolded protein response genes mediated by ATF5 are not activated. Instead, ATF4 activates the expression of cytoprotective genes, which reprogram cellular metabolism through activation of the integrated stress response (ISR). Mitochondrial stress promotes a local proteostatic response by reducing mitochondrial ribosomal proteins, inhibiting mitochondrial translation, and coupling the activation of the ISR with the attenuation of mitochondrial function. Through a trans–expression quantitative trait locus analysis, we provide genetic evidence supporting a role for Fh1 in the control of Atf4 expression in mammals. Using gene expression data from mice and humans with mitochondrial diseases, we show that the ATF4 pathway is activated in vivo upon mitochondrial stress. Our data illustrate the value of a multiomics approach to characterize complex cellular networks and provide a versatile resource to identify new regulators of mitochondrial-related diseases.

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Tristetraprolin binding site atlas in the macrophage transcriptome reveals a switch for inflammation resolution

Vitaly Sedlyarov, Jörg Fallmann, Florian Ebner, Jakob Huemer, Lucy Sneezum, Masa Ivin, Kristina Kreiner, Andrea Tanzer, Claus Vogl, Ivo Hofacker, Pavel Kovarik

Molecular Systems Biology (2016) 12, 868, doi: 10.15252/msb.20156628

Precise regulation of mRNA decay is fundamental for robust yet not exaggerated inflammatory responses to pathogens. However, a global model integrating regulation and functional consequences of inflammation‐associated mRNA decay remains to be established. Using time‐resolved high‐resolution RNA binding analysis of the mRNA‐destabilizing protein tristetraprolin (TTP), an inflammation‐limiting factor, we qualitatively and quantitatively characterize TTP binding positions in the transcriptome of immunostimulated macrophages. We identify pervasive destabilizing and non‐destabilizing TTP binding, including a robust intronic binding, showing that TTP binding is not sufficient for mRNA destabilization. A low degree of flanking RNA structuredness distinguishes occupied from silent binding motifs. By functionally relating TTP binding sites to mRNA stability and levels, we identify a TTP‐controlled switch for the transition from inflammatory into the resolution phase of the macrophage immune response. Mapping of binding positions of the mRNA‐stabilizing protein HuR reveals little target and functional overlap with TTP, implying a limited co‐regulation of inflammatory mRNA decay by these proteins. Our study establishes a functionally annotated and navigable transcriptome‐wide atlas ( of cis‐acting elements controlling mRNA decay in inflammation.

Features SENSE mRNA‐Seq Library Prep Kit for Ion Torrent
Features Mix2 RNA-Seq Data Analysis Software

A Murine Hypertrophic Cardiomyopathy Model: The DBA/2J Strain

Wenyuan Zhao, Tieqiang Zhao, Yuanjian Chen, Fengbo Zhao, Qingqing Gu, Robert W. Williams, Syamal K. Bhattacharya, Lu Lu , Yao Sun

PLoS One. 2015 Aug 4;10(8):e0133132. doi: 10.1371/journal.pone.0133132.

Familial hypertrophic cardiomyopathy (HCM) is attributed to mutations in genes that encode for the sarcomere proteins, especially Mybpc3 and Myh7. Genotype-phenotype correlation studies show significant variability in HCM phenotypes among affected individuals with identical causal mutations. Morphological changes and clinical expression of HCM are the result of interactions with modifier genes. With the exceptions of angiotensin converting enzyme, these modifiers have not been identified. Although mouse models have been used to investigate the genetics of many complex diseases, natural murine models for HCM are still lacking. In this study we show that the DBA/2J (D2) strain of mouse has sequence variants in Mybpc3 and Myh7, relative to widely used C57BL/6J (B6) reference strain and the key features of human HCM. Four-month-old of male D2 mice exhibit hallmarks of HCM including increased heart weight and cardiomyocyte size relative to B6 mice, as well as elevated markers for cardiac hypertrophy including β-myosin heavy chain (MHC), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and skeletal muscle alpha actin (α1-actin). Furthermore, cardiac interstitial fibrosis, another feature of HCM, is also evident in the D2 strain, and is accompanied by up-regulation of type I collagen and α-smooth muscle actin (SMA)-markers of fibrosis. Of great interest, blood pressure and cardiac function are within the normal range in the D2 strain, demonstrating that cardiac hypertrophy and fibrosis are not secondary to hypertension, myocardial infarction, or heart failure. Because D2 and B6 strains have been used to generate a large family of recombinant inbred strains, the BXD cohort, the D2 model can be effectively exploited for in-depth genetic analysis of HCM susceptibility and modifier screens.

Features SENSE mRNA-Seq Library Prep Kit for Ion Torrent


In clinical and basic research custom panels for transcript profiling are gaining importance because only project specific informative genes are interrogated. This approach reduces costs and complexity of data analysis and allows multiplexing of samples. Polymerase-chain-reaction (PCR) based TaqMan assays have high sensitivity but suffer from a limited dynamic range and sample throughput. Hence, there is a gap for a technology able to measure expression of large gene sets in multiple samples.


We have adapted a commercially available mRNA quantification assay (AmpliSeq-RNA) that measures mRNA abundance based on the frequency of PCR amplicons determined by high-throughput semiconductor sequencing. This approach allows for parallel, accurate quantification of about 1000 transcripts in multiple samples covering a dynamic range of five orders of magnitude. Using samples derived from a well-characterized stem cell differentiation model, we obtained a good correlation (r = 0.78) of transcript levels measured by AmpliSeq-RNA and DNA-microarrays. A significant portion of low abundant transcripts escapes detection by microarrays due to limited sensitivity. Standard quantitative RNA sequencing of the same samples confirms expression of low abundant genes with an overall correlation coefficient of r = 0.87. Based on digital AmpliSeq-RNA imaging we show switches of signaling cascades at four time points during differentiation of stem cells into cardiomyocytes.


The AmpliSeq-RNA technology adapted to high-throughput semiconductor sequencing allows robust transcript quantification based on amplicon frequency. Multiplexing of at least 900 parallel PCR reactions is feasible because sequencing-based quantification eliminates artefacts coming from off-target amplification. Using this approach, RNA quantification and detection of genetic variations can be performed in the same experiment.

Features SENSE mRNA-Seq Library Prep Kit for Ion Torrent