Additional sex comb-like1 (Asxl1) is known as a chromatin modulator that plays dual functions in transcriptional regulation depending on the cell type. Recent studies using Asxl1 knockout mice revealed that Asxl1 is important for the proliferation and differentiation of hematopoietic progenitor cells, and the development of organs. Although we previously reported Asxl1 as a Sox2 target gene, its function in embryonic stem cells (ESCs) remains largely unknown. For this purpose, we isolated ESCs from the blastocyst inner cell mass of Asxl1^−/−mice. Asxl1 deficiency in ESCs exhibited no effect on cell proliferation, expression of core pluripotent transcription factors, or alkaline phosphataseactivity, suggesting dispensability of Asxl1 for self-renewal of ESCs. By contrast, the differentiation of Asxl1^−/− ESCs was significantly affected as shown by size reductions of embryoid bodies accompanied with apoptosis, aberrant expression of differentiation genes, downregulation of bivalent neurogenesisgenes, and abnormal axon formation in neurons. Overall, our findings indicated that Asxl1 played a critical role in regulating genes associated with neural differentiation without affecting self-renewal of mouse ESCs.

Features SENSE mRNA-Seq Library Prep Kit

Background

Molecular analyses such as whole-genome sequencing have become routine and are expected to be transformational for future healthcare and lifestyle decisions. Population-wide implementation of such analyses is, however, not without challenges, and multiple studies are ongoing to identify what these are and explore how they can be addressed.

Methods

Defined as a research project, the Personal Genome Project UK (PGP-UK) is part of the global PGP network and focuses on open data sharing and citizen science to advance and accelerate personalized genomics and medicine.

Results

Here we report our findings on using an open consent recruitment protocol, active participant involvement, open access release of personal genome, methylome and transcriptome data and associated analyses, including 47 new variants predicted to affect gene function and innovative reports based on the analysis of genetic and epigenetic variants. For this pilot study, we recruited 10 participants willing to actively engage as citizen scientists with the project. In addition, we introduce Genome Donation as a novel mechanism for openly sharing previously restricted data and discuss the first three donations received. Lastly, we present GenoME, a free, open-source educational app suitable for the lay public to allow exploration of personal genomes.

Conclusions

Our findings demonstrate that citizen science-based approaches like PGP-UK have an important role to play in the public awareness, acceptance and implementation of genomics and personalized medicine.

Features SENSE mRNA-Seq Library Prep Kit

Sporangia of Phytophthora infestans from pure cultures on agar plates are typically used in lab studies, whereas sporangia from leaflet lesions drive natural infections and epidemics. Multiple assays were performed to determine if sporangia from these two sources are equivalent. Sporangia from plate cultures showed much lower rates of indirect germination and produced much less disease in field and moist-chamber tests. This difference in aggressiveness was observed whether the sporangia had been previously incubated at 4 C (to induce indirect germination) or at 21 C (to prevent indirect germination). Furthermore, lesions caused by sporangia from plates produced much less sporulation. RNA-Seq analysis revealed that thousands of the >17,000 P. infestans genes with an RPKM >1 (reads per kilobase of exon model per million mapped reads >1) were differentially expressed in sporangia obtained from plate cultures of two independent field isolates compared to sporangia of those isolates from leaflet lesions. Among the significant differentially expressed genes (DEGs), putative RxLR effectors were over-represented, with almost half of the 355 effectors with RPKM>1 being up- or down-regulated. DEGs of both isolates included nine flagellar-associated genes, and all were down-regulated in plate sporangia. Ten elicitin genes were also detected as DEGs in both isolates, and nine (including INF1) were up-regulated in plate sporangia. These results corroborate previous observations that sporangia produced from plates and leaflets sometimes yield different experimental results and suggest hypotheses for potential mechanisms. We caution that use of plate sporangia in assays may not always produce results reflective of natural infections and epidemics.

Features SENSE mRNA-Seq Library Prep Kit and SPLIT RNA Extraction Kit

Recursive splicing (RS) starts by defining an “RS-exon,” which is then spliced to the preceding exon, thus creating a recursive 5′ splice site (RS-5ss). Previous studies focused on cryptic RS-exons, and now we find that the exon junction complex (EJC) represses RS of hundreds of annotated, mainly constitutive RS-exons. The core EJC factors, and the peripheral factors PNN and RNPS1, maintain RS-exon inclusion by repressing spliceosomal assembly on RS-5ss. The EJC also blocks 5ss located near exon-exon junctions, thus repressing inclusion of cryptic microexons. The prevalence of annotated RS-exons is high in deuterostomes, while the cryptic RS-exons are more prevalent in Drosophila, where EJC appears less capable of repressing RS. Notably, incomplete repression of RS also contributes to physiological alternative splicing of several human RS-exons. Finally, haploinsufficiency of the EJC factor Magoh in mice is associated with skipping of RS-exons in the brain, with relevance to the microcephaly phenotype and human diseases.

Features SENSE mRNA‐Seq Library Prep Kit

Adamussium colbecki is one of the most common bivalve mollusks found in the coastal waters of the Antarctic continent. Its widespread distribution, easiness of collection and sensitivity to slight alterations of water temperature and presence of pollutants make this cold-adapted scallop a potentially interesting sentinel species for the biomonitoring of the impact of anthropic activities on the Antarctic benthic communities. However, while the availability of genetic and molecular data would represent a resource of the utmost importance to enable the use of this species as a model for environmental studies, this data is presently nearly non-existing. Here we report a high quality de novo assembled and annotated transcriptome for A. colbecki, discussing the long-debated phylogenetic position of this species within the order Pectinida through a Bayesian phylogenomics approach, based on the concatenated multiple sequence alignment of 978 universally conserved orthologous genes.

Features SENSE mRNA‐Seq Library Prep Kit

RNA binding proteins (RBPs) mediate posttranscriptional gene regulatory events throughout development. During neurogenesis, many RBPs are required for proper dendrite morphogenesis within Drosophila sensory neurons. Despite their fundamental role in neuronal morphogenesis, little is known about the molecular mechanisms in which most RBPs participate during neurogenesis. In Drosophila, alan shepard (shep) encodes a highly conserved RBP that regulates dendrite morphogenesis in sensory neurons. Moreover, the C. elegans ortholog sup-26 has also been implicated in sensory neuron dendrite morphogenesis. Nonetheless, the molecular mechanism by which Shep/SUP-26 regulate dendrite development is not understood. Here we show that Shep interacts with the RBPs Trailer Hitch (Tral), Ypsilon schachtel (Yps), Belle (Bel), and Poly(A)-Binding Protein(PABP), to direct dendrite morphogenesis in Drosophila sensory neurons. Moreover, we identify a conserved set of Shep/SUP-26 target RNAs that include regulators of cell signaling, posttranscriptional gene regulators, and known regulators of dendrite development.

Features SENSE mRNA‐Seq Library Prep Kit

Chronic T cell activation is a hallmark of pulmonary tuberculosis (PTB). The mechanisms underpinning this important phenomenon are however, poorly elucidated, though known to rely on control of T effector cells (Teff) by regulatory T cells (Treg). Our studies show that circulating natural Treg cells in adults with PTB preserve their suppressive potential but Teff cells from such subjects are resistant to Treg-mediated suppression. We found this to be due to expansion of an activated Teff subset identified by Human Leukocyte Antigen (HLA)-DR expression. Sensitivity to suppression was restored to control levels by depletion of this subset. Comparative transcriptome analysis of Teff cells that contain HLA-DR⁺ cells versus the fraction depleted of this population identified putative resistance mechanisms linked to IFNG, IL17A, IL22, PD-L1 and β-chemokines CCL3L3, CCL4 expression. Antibody blocking experiments confirmed HLA-DR⁺ Teff cells, but not the fraction depleted of HLA-DR⁺ effectors, to be resistant to Treg suppression mediated via CCR5 and PD-L1 associated pathways. In the presence of HLA-DR⁺ Teff cells, activation of NFκB downstream of CCR5 and PD-L1 was perturbed. In addition, HLA-DR⁺ Teff cells expressed significantly higher levels of Th1/Th17 cytokines that may regulate Treg function through a reciprocal counter-balancing relationship. Taken together, our study provides novel insight on how activated HLA-DR⁺CD4⁺ T cells may contribute to disease associated inflammation by compromising Treg-mediated suppression in PTB.

Features SENSE mRNA‐Seq Library Prep Kit

Under stress conditions, the coactivator Multiprotein bridging factor 1 (Mbf1) translocates from the cytoplasm into the nucleus to induce stress-response genes. However, its role in the cytoplasm, where it is mainly located, has remained elusive. Here, we show that Drosophila Mbf1 associates with E(z)mRNA and protects it from degradation by the exoribonuclease Pacman (Pcm), thereby ensuring Polycomb silencing. In genetic studies, loss of mbf1 function enhanced a Polycomb phenotype in Polycomb group mutants, and was accompanied by a significant reduction in E(z) mRNA expression. Furthermore, a pcm mutation suppressed the Polycomb phenotype and restored the expression level of E(z) mRNA, while pcm overexpression exhibited the Polycomb phenotype in the mbf1 mutant but not in the wild-type background. In vitro, Mbf1 protected E(z) RNA from Pcm activity. Our results suggest that Mbf1 buffers fluctuations in Pcm activity to maintain an E(z) mRNA expression level sufficient for Polycomb silencing.

Features SENSE mRNA‐Seq Library Prep Kit

Glioblastoma multiforme is the most lethal malignant brain tumor. Despite many intensive studies, the prognosis of glioblastoma multiforme is currently very poor, with a median overall survival duration of 14 months and 2-year survival rates of less than 10%. Although viral infections have been emphasized as potential cofactors, their influences on pathways that support glioblastoma progression are not known. Some previous studies indicated that human Kaposi’s sarcoma-associated herpesvirus (KSHV) was detected in healthy brains, and its microRNA was also detected in glioblastoma patients’ plasma. However, a direct link between KSHV infection and glioblastoma is currently not known. In this study, we infected glioblastoma cells and glioma stem-like cells (GSCs) with KSHV to establish an in vitro cell model for KSHV-infected glioblastoma cells and glioma stem-like cells in order to identify virologic outcomes that overlap with markers of aggressive disease. Latently KSHV-infected glioblastoma cells and GSCs were successfully established. Additionally, using these cell models, we found that KSHV infection modulates the proliferation of glioma stem-like cells.

Features SENSE mRNA‐Seq Library Prep Kit

Animals have a carefully orchestrated relationship with oxygen. When exposed to low environmental oxygen concentrations, and during periods of increased energy expenditure, animals maintain cellular oxygen homeostasis by enhancing internal oxygen delivery, and by enabling the anaerobic production of ATP. These low-oxygen responses are thought to be controlled universally across animals by the hypoxia-inducible factor (HIF). We find, however, that sponge and ctenophore genomes lack key components of the HIF pathway. Since sponges and ctenophores are likely sister to all remaining animal phyla, the last common ancestor of extant animals likely lacked the HIF pathway as well. Laboratory experiments show that the marine sponge Tethya wilhelma maintains normal transcription under oxygen levels down to 0.25% of modern atmospheric saturation, the lowest levels we investigated, consistent with the predicted absence of HIF or any other HIF-like pathway. Thus, the last common ancestor of all living animals could have metabolized aerobically under very low environmental oxygen concentrations.

Features SENSE mRNA‐Seq Library Prep Kit

We performed a multiple ‘omics study by integrating data on epigenomic, transcriptomic, and proteomic perturbations associated with mitochondrial dysfunction in primary human hepatocytes caused by the liver toxicant valproic acid (VPA), to deeper understand downstream events following epigenetic alterations in the mitochondrial genome. Furthermore, we investigated persistence of cross-omics changes after terminating drug treatment. Upon transient methylation changes of mitochondrial genes during VPA-treatment, increasing complexities of gene-interaction networks across time were demonstrated, which normalized during washout. Furthermore, co-expression between genes and their corresponding proteins increased across time. Additionally, in relation to persistently decreased ATP production, we observed decreased expression of mitochondrial complex I and III–V genes. Persistent transcripts and proteins were related to citric acid cycle and β-oxidation. In particular, we identified a potential novel mitochondrial-nuclear signaling axis, MT-CO2–FN1–MYC–CPT1. In summary, this cross-omics study revealed dynamic responses of the mitochondrial epigenome to an impulse toxicant challenge resulting in persistent mitochondrial dysfunctioning. Moreover, this approach allowed for discriminating between the toxic effect of VPA and adaptation.

Features SENSE mRNA‐Seq Library Prep Kit

A single nucleotide substitution in the third intron of insulin-like growth factor 2 (IGF2) is associated with increased muscle mass and reduced subcutaneous fat in domestic pigs. This mutation disrupts the binding of the ZBED6 transcription factor and leads to a threefold up-regulation of IGF2 expression in pig skeletal muscle. Here, we investigated the biological significance of ZBED6–IGF2 interaction in the growth of placental mammals using two mouse models, ZBED6 knock-out (Zbed6^−/−) and Igf2knock-in mice that carry the pig IGF2 mutation. These transgenic mice exhibit markedly higher serum IGF2 concentrations, higher growth rate, increased lean mass, and larger heart, kidney, and liver; no significant changes were observed for white adipose tissues. The changes in body and lean mass were most pronounced in female mice. The phenotypic changes were concomitant with a remarkable up-regulation of Igf2expression in adult tissues. Transcriptome analysis of skeletal muscle identified differential expression of genes belonging to the extracellular region category. Expression analysis using fetal muscles indicated a minor role of ZBED6 in regulating Igf2 expression prenatally. Furthermore, transcriptome analysis of the adult skeletal muscle revealed that this elevated expression of Igf2 was derived from the P1 and P2 promoters. The results revealed very similar phenotypic effects in the Zbed6 knock-out mouse and in the Igf2 knock-in mouse, showing that the effect of ZBED6 on growth of muscle and internal organs is mediated through the binding site in the Igf2 gene. The results explain why this ZBED6 binding site is extremely well conserved among placental mammals.

Features SENSE mRNA‐Seq Library Prep Kit

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.

Features SENSE mRNA‐Seq Library Prep Kit

Plant genomes reduce in size following a whole genome duplication event, and one gene in a duplicate gene pair can lose function in absence of selective pressure to maintain duplicate gene copies. Maize and its sister genus, Tripsacum, share a genome duplication event that occurred 5 to 26 million years ago. Because few genomic resources for Tripsacum exist, it is unknown whether Tripsacum grasses and maize have maintained a similar set of genes under purifying selection. Here we present high quality de novo transcriptome assemblies for two species: Tripsacum dactyloides and Tripsacum floridanum. Genes with experimental protein evidence in maize were good candidates for genes under purifying selection in both genera because pseudogenes by definition do not produce protein. We tested whether genes with protein evidence are resisting gene loss in maize and whether their homologs are also resisting gene loss in Tripsacum. Protein-encoding maize transcripts and their Tripsacum homologs have higher GC content, higher gene expression levels, and more conserved expression levels than putatively untranslated maize transcripts and their Tripsacum homologs. These results indicate that gene loss is occurring in a similar fashion in both genera after a shared ancient polyploidy event. The Tripsacum transcriptome assemblies provide a high quality genomic resource that can provide insight into the evolution of maize, an highly valuable crop worldwide.

Features SENSE mRNA‐Seq Library Prep Kit

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 PDR1was 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

The ALAN is drawing the attention of researchers and environmentalists for its ever-increasing evidence on its capacity of “desynchronization” of organismal physiology. Photoperiod and circadian cycles are critical parameters to influence the biology of reproduction in several animals, including fish. The present study is the first proof of the development of an ovarian tumour with the effect of light in zebrafish (Danio rerio), an excellent model for circadian-related studies. Results of three experimental conditions, continuous light for one week, LLW, one month, LLM, and for one year, LLY revealed a clear desynchronization of clock associated genes (Clock1a, Bmal1a, Per2, and Cry2a). Interestingly, loss of rhythmicity and low concentration of melatonin found in these conditions in whole brain, retina, ovary, and serum through ELISA. RNA-Seq data of ovarian samples revealed the upregulation of Mid2, Tfg, Irak1, Pim2, Tradd, Tmem101, Nfkbib genes and ultimately increase the expression of NF-κB, a cellular transformer for tumourigenesis, confirmed by the western blot. The appearance of TNFα, inflammatory cytokines and activator of NF-κB also increased. Histology approved the formation of thecoma and granulosa cell tumour in the one year exposed ovarian sample. The whole transcriptome data analysis revealed 1791 significantly upregulated genes in an ovarian tumour. Among these genes, DAVID functional annotation tool identified 438 genes, directly linked to other physiological disorders. This study evidenced of an ovarian tumour induced by ALAN in zebrafish.

Features SENSE mRNA‐Seq Library Prep Kit

High-grade glioma (HGG) is highly angiogenic, but antiangiogenic therapy has transient clinical benefit in only a fraction of patients. Vascular regulators of these heterogeneous responses remain undetermined. We found up-regulation of Sox7 and down-regulation of Sox17 in tumor endothelial cells (tECs) in mouse HGG. Sox7 deletion suppressed VEGFR2 expression, vascular abnormality, hypoxia-driven invasion, regulatory T cell infiltration, and tumor growth. Conversely, Sox17 deletion exacerbated these phenotypes by up-regulating Sox7 in tECs. Anti-VEGFR2 antibody treatment delayed tumor growth by normalizing Sox17-deficient abnormal vessels with high Sox7 levels but promoted it by regressing Sox7-deficient vessels, recapitulating variable therapeutic responses to antiangiogenic therapy in HGG patients. Our findings establish that Sox7 promotes tumor growth via vessel abnormalization, and its level determines the therapeutic outcome of VEGFR2 inhibition in HGG. In 189 HGG patients, Sox7 expression was heterogeneous in tumor vessels, and high Sox7 levels correlated with poor survival, early recurrence, and impaired vascular function, emphasizing the clinical relevance of Sox7 in HGG.

Features SENSE mRNA‐Seq Library Prep Kit

Tripartite motif (TRIM) proteins have been shown to play important roles in cancer development and progression by modulating cell proliferation or resistance from cell death during non-homeostatic stress conditions found in tumor micro-environments. In this study, we set out to investigate the importance for cellular fitness of the virtually uncharacterized family member TRIM52.

The human TRIM52 gene has arisen recently in evolution, making it unlikely that TRIM52 is required for basic cellular functions in normal cells. However, a recent genome-wide ablation screening study has suggested that TRIM52 may be essential for optimal proliferation or survival in certain genetic cancer backgrounds. Identifying genes which fit this concept of genetic context-dependent fitness in cancer cells is of interest as they are promising targets for tumor-specific therapy.

We report here that TRIM52 ablation significantly diminished the proliferation of specific glioblastoma cell lines in cell culture and mouse xenografts by compromising their cell cycle progression in a p53-dependent manner. Together, our findings point to a non-redundant TRIM52 function that is required for optimal proliferation.

Features SENSE mRNA‐Seq Library Prep Kit

Chitinase-3-like-1 (Chi3l1) is known to play a significant role in the pathogenesis of Type 2 inflammation and cancer. However, the function of Chi3l1 in T cell and its clinical implications are largely unknown. Here we show that Chi3l1 expression was increased in activated T cells, especially in Th2 cells. In addition, Chi3l1-deficient T cells are hyper-responsive to TcR stimulation and are prone to differentiating into Th1 cells. Chi3l1-deficient Th1 cells show increased expression of anti-tumor immunity genes and decreased Th1 negative regulators. Deletion of Chi3l1 in T cells in mice show reduced melanoma lung metastasis with increased IFNγ and TNFα-producing T cells in the lung. Furthermore, silencing of Chi3l1 expression in the lung using peptide-siRNA complex (dNP2-siChi3l1) efficiently inhibit lung metastasis with enhanced Th1 and CTL responses. Collectively, this study demonstrates Chi3l1 is a regulator of Th1 and CTL which could be a therapeutic target to enhance anti-tumor immunity.

Features SENSE mRNA‐Seq Library Prep Kit

Long noncoding RNAs (lncRNAs) are emerging as key players in multiple cellular pathways, but their modes of action and how those are dictated by sequence remain unclear. lncRNAs tend to be enriched in the nuclear fraction, whereas most mRNAs are overtly cytoplasmic, although several studies have found that hundreds of mRNAs in various cell types are retained in the nucleus. It is thus conceivable that some mechanisms that promote nuclear enrichment are shared between lncRNAs and mRNAs. In order to identify elements that can force nuclear localization in lncRNAs and mRNAs we screened libraries of short fragments tiled across nuclear RNAs, which were cloned into the untranslated regions of an efficiently exported mRNA. The screen identified a short sequence derived from Alu elements and bound by HNRNPK that increases nuclear accumulation. We report that HNRNPK binding to C-rich motifs outside Alu elements is also associated with nuclear enrichment in both lncRNAs and mRNAs, and this mechanism is conserved across species. Our results thus detail a novel pathway for regulation of RNA accumulation and subcellular localization that has been co-opted to regulate the fate of transcripts that have integrated Alu elements.

Features SENSE mRNA‐Seq Library Prep Kit

Primary therapy resistance is a major problem in acute myeloid leukemia treatment. We set out to develop a powerful and robust predictor for therapy resistance for intensively treated adult patients. We used two large gene expression data sets (n=856) to develop a predictor of therapy resistance, which was validated in an independent cohort analyzed by RNA sequencing (n=250). In addition to gene expression markers, standard clinical and laboratory variables as well as the mutation status of 68 genes were considered during construction of the model. The final predictor (PS29MRC) consisted of 29 gene expression markers and a cytogenetic risk classification. A continuous predictor is calculated as a weighted linear sum of the individual variables. Additionally, a cut off was defined to divide patients into a high-risk and a low-risk group for resistant disease. PS29MRC was highly significant in the validation set, both as a continuous score (OR=2.39; p=8.63×10-9, AUC=0.76) and as a dichotomous classifier (OR=8.03; p=4.29×10-9). The accuracy was 77%. In multivariable models, only TP53 mutation, age and PS29MRC (continuous: OR=1.75; p=0.0011; dichotomous: OR=4.44, p=0.00021) were left as significant variables. PS29MRC dominated all models when compared with currently used predictors and also predicted overall survival independently of established markers. When integrated in the European LeukemiaNet 2017 genetic risk stratification, four groups (median survival [months] of 8, 18, 41, not reached) could be defined (p=4.01·10-10). PS29MRC will make it possible to design trials which stratify induction treatment according to the probability of response and refines the ELN 2017 classification.

Features SENSE mRNA‐Seq Library Prep Kit

Throughout life, the human eye is continuously exposed to sunlight and artificial lighting. Ambient light exposure can lead to visual impairment and transient or permanent blindness. To mimic benign light stress conditions, Mus musculus eyes were exposed to low-energy UVB radiation, ensuring no severe morphological changes in the retinal structure post-exposure. We performed RNA-seq analysis to reveal the early transcriptional changes and key molecular pathways involved before the activation of the canonical cell death pathway. RNA-seq analysis identified 537 genes that were differentially modulated, out of which 126 were clearly up regulated (>2-fold, P < .01) and 51 were significantly down regulated (<2-fold, P < .01) in response to UVB irradiation in the mouse retina. Gene ontology analysis revealed that UVB exposure affected pathways for cellular stress and signaling (eg, Creb3, Ddrgk1, Grin1, Map7, Uqcc2, Uqcrb), regulation of chromatin and gene expression (eg, Chd5, Jarid2, Kat6a, Smarcc2, Sumo1, Zfp84), transcription factors (eg, Asxl2, Atf7, Per1, Phox2a, Rxra), RNA processing, and neuronal genes (eg, B4gal2, Drd1, Grm5, Rnf40, Rnps1, Usp39, Wbp4). The differentially expressed genes from the RNA-seq analysis were validated by quantitative PCR. Both analyses yielded similar gene expression patterns. The genes and pathways identified here improve the understanding of early transcriptional responses to UVB irradiation. They may also help in elucidating the genes responsible for the inherent susceptibility of humans to UVB-induced retinal diseases.

Features SENSE mRNA‐Seq Library Prep Kit

Ethnopharmacological relevance

Gangjihwan (DF) which is composed of Ephedra intermedia, Lithospermum erythrorhizon, and Rheum palmatum has been used for the treatment of obesity in traditional medical clinics in Korea.

Aim of the study

This study was conducted to standardize DF and elucidate its mechanism of action for inhibiting fat accumulation in adipocytes and adipose tissues.

Materials and Methods

The herbal ingredients of DF were extracted in water, 30% ethanol or 70% ethanol and freeze-dried followed by HPLC analyses. 3T3-L1 adipocytes and high-fat diet-induced obese mice were treated with each of the three DF preparations. Messenger RNA and protein expression levels were measured by real-time qPCR and Western blotting. RNA-Seq analyses were conducted to examine the effects of DF treatment on whole transcriptome of adipocyte.

Results

(-)-Ephedrine and (+)-pseudoephedrine from E. intermedia, aloe-emodin and chrysophanol from R. palmatum and shikonin from L. erythrorhizon were identified as phytochemical components of DF. DF caused dose-dependent inhibition of fat accumulation in 3T3-L1 adipocytes. It also significantly reduced adipose tissue mass and adipocyte size in high-fat diet-induced obese mice. DF was found to down-regulate the expressions of the lipogenic transcription factors such as sterol regulatory element binding protein 1 C (SREBP1C), peroxisome proliferator activated receptor gamma (PPARγ), and CCAAT/enhancer binding protein alpha (C/EBPα). Among the three preparations of DF, the 70% ethanol extract was the most effective. RNA-Seq analyses showed that DF treatment decreased the expression levels of up-regulators and increased those of down-regulators of lipogenic transcription factors.

Conclusions

DF preparations, among which 70% ethanol extract was the most effective, reduced fat accumulation in 3T3-L1 adipocytes and high-fat diet-induced obese mice through the down-regulation of lipogenic transcription factors SREBP1C, PPARγ and C/EBPα.

Features SENSE mRNA‐Seq Library Prep Kit

Features SENSE mRNA‐Seq Library Prep Kit

Features SENSE mRNA‐Seq Library Prep Kit

Mycosporine-like amino acids (MAAs) have been highlighted as pharmacologically active secondary compounds to protect cells from harmful UV-radiation by absorbing its energy. Previous studies have mostly focused on characterizing their physiological properties such as antioxidant activity and osmotic regulation. However, molecular mechanisms underlying their UV-protective capability have not yet been revealed. In the present study, we investigated the expression profiling of porphyra-334-modulated genes or microRNA (miRNAs) in response to UV-exposure and their functional networks, using cDNA and miRNAs microarray. Based on our data, we showed that porphyra-334-regulated genes play essential roles in UV-affected biological processes such as Wnt (Wingless/integrase-1) and Notch pathways which exhibit antagonistic relationship in various biological processes; the UV-repressed genes were in the Wnt signaling pathway, while the activated genes were in the Notch signaling. In addition, porphyra-334-regulated miRNAs can target many genes related with UV-mediated biological processes such as apoptosis, cell proliferation and translational elongation. Notably, we observed that functional roles of the target genes for up-regulated miRNAs are inversely correlated with those for down-regulated miRNAs; the former genes promote apoptosis and translational elongation, whereas the latter function as inhibitors in these processes. Taken together, these data suggest that porphyra-334 protects cells from harmful UV radiation through the comprehensive modulation of expression patterns of genes involved in UV-mediated biological processes, and that provide a new insight to understand its functional molecular networks.

Features SENSE mRNA‐Seq Library Prep Kit

The bromodomain protein Brd4 is an epigenetic reader and plays a critical role in the development and maintenance of leukemia. Brd4 binds to acetylated histone tails and activates transcription by recruiting the positive elongation factor P-TEFb. Small molecule inhibitor JQ1 competitively binds the bromodomains of Brd4 and displaces the protein from acetylated histones. However, it remains unclear whether genes targeted by JQ1 are mainly regulated by Brd4 or by other bromodomain proteins such as Brd2 and Brd3. Here, we describe anti-proliferative dominant-negative Brd4 mutants that compete with the function of distinct Brd4 domains. We used these Brd4 mutants to compare the Brd4-specific transcriptome with the transcriptome of JQ1-treated cells. We found that most JQ1-regulated genes are also regulated by dominant-negative Brd4 mutants, including the mutant that competes with the P-TEFb recruitment function of Brd4. Importantly, JQ1 and dominant-negative Brd4 mutants regulated the same set of target genes of c-Myc, a key regulator of the JQ1 response in leukemia cells. Our results suggest that Brd4 mediates most of the anti-cancer effects of JQ1 and that the major function of Brd4 in this process is the recruitment of P-TEFb. In summary, our studies define the molecular targets of JQ1 in more detail.

Features SENSE mRNA‐Seq Library Prep Kit

Both p150 and p110 isoforms of ADAR1 convert adenosine to inosine in double-stranded RNA (dsRNA). ADAR1p150 suppresses the dsRNA-sensing mechanism that activates MDA5–MAVS–IFN signaling in the cytoplasm. In contrast, the biological function of the ADAR1p110 isoform, which is usually located in the nucleus, is largely unknown. Here, we show that stress-activated phosphorylation of ADAR1p110 by MKK6–p38–MSK MAP kinases promotes its binding to Exportin-5 and its export from the nucleus. After translocating to the cytoplasm, ADAR1p110 suppresses apoptosis in stressed cells by protecting many antiapoptotic gene transcripts that contain 3′-untranslated-region dsRNA structures primarily comprising inverted Alu repeats. ADAR1p110 competitively inhibits binding of Staufen1 to the 3′-untranslated-region dsRNAs and antagonizes Staufen1-mediated mRNA decay. Our study reveals a new stress-response mechanism in which human ADAR1p110 and Staufen1 regulate surveillance of a set of mRNAs required for survival of stressed cells.

Features SENSE mRNA‐Seq Library Prep Kit

Objective
Next-generation sequencing was performed to evaluate the effects of short-term application of dexamethasone on human gingiva-derived mesenchymal stem cells.

Methods
Human gingiva-derived stem cells were treated with a final concentration of 10−7 M dexamethasone and the same concentration of vehicle control. This was followed by mRNA sequencing and data analysis, gene ontology and pathway analysis, quantitative real-time polymerase chain reaction of mRNA, and western blot analysis of RUNX2 and β-catenin.

Results
In total, 26,364 mRNAs were differentially expressed. Comparison of the results of dexamethasone versus control at 2 hours revealed that 7 mRNAs were upregulated and 25 mRNAs were downregulated. The application of dexamethasone reduced the expression of RUNX2 and β-catenin in human gingiva-derived mesenchymal stem cells.

Conclusion
The effects of dexamethasone on stem cells were evaluated with mRNA sequencing, and validation of the expression was performed with qualitative real-time polymerase chain reaction and western blot analysis. The results of this study can provide new insights into the role of mRNA sequencing in maxillofacial areas.

Features SENSE mRNA‐Seq Library Prep Kit

Background/Aim: Ninjurin1 is a 17-kDa membrane protein that is highly expressed in circulating tumor cells (CTCs) obtained from locally-advanced prostate cancer patients. As CTCs are implicated in the initiation of distant metastasis, we examined the potential contribution of Ninjurin1 to the motility of prostate cancer cells.

Materials and Methods: Ninjurin1 expression was evaluated in CTCs harvested from seven locally advanced patients with no metastatic hallmarks using real-time polymerase chain reaction (PCR). The role of Ninjurin1 in cell motility was investigated using small interfering RNA (siRNA), neutralizing antibodies against Ninjurin1 and Ninjurin1-overexpressing adenoviruses.

Results: Ninjurin1 was ranked as the most significantly up-regulated adhesion protein identified by RNA-Seq analysis. Both Ninjurin1 down-regulation by siRNA and neutralizing antibodies blocking Ninjurin1 homophilic interactions effectively inhibited cell motility. In contrast, cell motility was enhanced in prostate cancer cells infected with adenovirus enabling Ninjurin1 overexpression.

Conclusion: Ninjurin1-neutralizing antibodies or Ninjurin1-targeting siRNA merit further development for patients with metastatic potential.

Features SENSE mRNA‐Seq Library Prep Kit

Autism spectrum disorders (ASD) are a group of genetic disorders often overlapping with other neurological conditions. We previously described abnormalities in the branched-chain amino acid (BCAA) catabolic pathway as a cause of ASD. Here, we show that the solute carrier transporter 7a5 (SLC7A5), a large neutral amino acid transporter localized at the blood brain barrier (BBB), has an essential role in maintaining normal levels of brain BCAAs. In mice, deletion of Slc7a5 from the endothelial cells of the BBB leads to atypical brain amino acid profile, abnormal mRNA translation, and severe neurological abnormalities. Furthermore, we identified several patients with autistic traits and motor delay carrying deleterious homozygous mutations in the SLC7A5 gene. Finally, we demonstrate that BCAA intracerebroventricular administration ameliorates abnormal behaviors in adult mutant mice. Our data elucidate a neurological syndrome defined by SLC7A5 mutations and support an essential role for the BCAA in human brain function.

Features SENSE mRNA‐Seq Library Prep Kit

Analysis of fusion transcripts has become increasingly important due to their link with cancer development. Since high-throughput sequencing approaches survey fusion events exhaustively, several computational methods for the detection of gene fusions from RNA-seq data have been developed. This kind of analysis, however, is complicated by native trans-splicing events, the splicing-induced complexity of the transcriptome and biases and artefacts introduced in experiments and data analysis. There are a number of tools available for the detection of fusions from RNA-seq data; however, certain differences in specificity and sensitivity between commonly used approaches have been found. The ability to detect gene fusions of different types, including isoform fusions and fusions involving non-coding regions, has not been thoroughly studied yet. Here, we propose a novel computational toolkit called InFusion for fusion gene detection from RNA-seq data. InFusion introduces several unique features, such as discovery of fusions involving intergenic regions, and detection of anti-sense transcription in chimeric RNAs based on strand-specificity. Our approach demonstrates superior detection accuracy on simulated data and several public RNA-seq datasets. This improved performance was also evident when evaluating data from RNA deep-sequencing of two well-established prostate cancer cell lines. InFusion identified 26 novel fusion events that were validated in vitro, including alternatively spliced gene fusion isoforms and chimeric transcripts that include intergenic regions. The toolkit is freely available to download from http:/bitbucket.org/kokonech/infusion.

Features SENSE mRNA‐Seq Library Prep Kit
Features SPLIT RNA Extraction Kit

Glucagon and thyroid hormone (T₃) exhibit therapeutic potential for metabolic disease but also exhibit undesired effects. We achieved synergistic effects of these two hormones and mitigation of their adverse effects by engineering chemical conjugates enabling delivery of both activities within one precisely targeted molecule. Coordinated glucagon and T₃ actions synergize to correct hyperlipidemia, steatohepatitis, atherosclerosis, glucose intolerance, and obesity in metabolically compromised mice. We demonstrate that each hormonal constituent mutually enriches cellular processes in hepatocytes and adipocytes via enhanced hepatic cholesterol metabolism and white fat browning. Synchronized signaling driven by glucagon and T₃ reciprocally minimizes the inherent harmful effects of each hormone. Liver-directed T₃ action offsets the diabetogenic liability of glucagon, and glucagon-mediated delivery spares the cardiovascular system from adverse T₃ action. Our findings support the therapeutic utility of integrating these hormones into a single molecular entity that offers unique potential for treatment of obesity, type 2 diabetes, and cardiovascular disease.

Features SENSE mRNA‐Seq Library Prep Kit

In analyzing grapevine clones infected with grapevine red blotch associated virus, we identified a small number of isometric particles of approximately 30 nm in diameter from an enriched fraction of leaf extract. A dominant protein of 25 kDa was isolated from this fraction using SDS-PAGE and was identified by mass spectrometry as belonging to grapevine asteroid mosaic associated virus (GAMaV). Using a combination of three methods RNA-Seq, sRNA-Seq, and Sanger sequencing of RT- and RACE-PCR products, we obtained a full-length genome sequence consisting of 6719 nucleotides without the poly(A) tail. The virus possesses all of the typical conserved functional domains concordant with the genus Marafivirus and lies evolutionarily between citrus sudden death associated virus and oat blue dwarf virus. A large shift in RNA-Seq coverage coincided with the predicted location of the subgenomic RNA involved in coat protein (CP) expression. Genus wide sequence alignments confirmed the cleavage motif LxG(G/A) to be dominant between the helicase and RNA dependent RNA polymerase (RdRp), and the RdRp and CP domains. A putative overlapping protein (OP) ORF lacking a canonical translational start codon was identified with a reading frame context more consistent with the putative OPs of tymoviruses and fig fleck associated virus than with those of marafiviruses. BLAST analysis of the predicted GAMaV OP showed a unique relatedness to the OPs of members of the genus Tymovirus.

Features SENSE mRNA‐Seq Library Prep Kit

The maternal-to-zygotic transition (MZT) is a process that occurs in animal embryos at the earliest developmental stages, during which maternally deposited mRNAs and other molecules are degraded and replaced by products of the zygotic genome. The zygotic genome is not activated immediately upon fertilization, and in the pre-MZT embryo post-transcriptional control by RNA-binding proteins (RBPs) orchestrates the first steps of development. To identify relevant Drosophila RBPs organism-wide, we refined the RNA interactome capture method for comparative analysis of the pre- and post-MZT embryos. We determine 523 proteins as high-confidence RBPs, half of which were not previously reported to bind RNA. Comparison of the RNA interactomes of pre- and post-MZT embryos reveals high dynamicity of the RNA-bound proteome during early development, and suggests active regulation of RNA binding of some RBPs. This resource provides unprecedented insight into the system of RBPs that govern the earliest steps of Drosophila development.

Features SENSE mRNA‐Seq Library Prep Kit

Circulating tumor cells serve as useful biomarkers with which to identify disease status associated with survival, metastasis and drug sensitivity. Here, we established a novel application for detecting PSA/PSMA-positive prostate cancer cells circulating in peripheral blood employing an adenovirus called Ad5/35E1aPSESE4. Ad5/35E1aPSESE4 utilized PSES, a chimeric enhancer derived from PSA/PSMA promoters that is highly active with and without androgen. A fluorescence signal mediated by GFP expression upon Ad5/35E1aPSESE4 infection was selectively amplified in PSA/PSMA-positive prostate cancer cells in vitro and ex vivo. Furthermore, for the in vivo model, blood drawn from TRAMP was tested for CTCs with Ad5/35E1aPSESE4 infection and was positive for CTCs at week 16. Validation was performed on patient blood at various clinical stages and found out 1–100 CTCs expressing GFP upon Ad5/35E1aPSESE4 infection. Interestingly, CTC from one patient was confirmed to be sensitive to docetaxel chemotherapeutic reagent and to abundantly express metastasis-related genes like MMP9, Cofilin1, and FCER1G through RNA-seq. Our study established that the usage of Ad5/35E1aPSESE4 is effective in marking PSA/PSMA-positive prostate cancer cells in patient blood to improve the efficacy of utilizing CTCs as a biomarker.

Features SENSE mRNA-Seq Library Prep Kit

The formation of RNA-DNA hybrids, referred to as R-loops, can promote genome instability and cancer development. Yet the mechanisms by which R-loops compromise genome instability are poorly understood. Here, we establish roles for the evolutionarily conserved Nrl1 protein in pre-mRNA splicing regulation, R-loop suppression and in maintaining genome stability. nrl1Δ mutants exhibit endogenous DNA damage, are sensitive to exogenous DNA damage, and have defects in homologous recombination (HR) repair. Concomitantly, nrl1Δ cells display significant changes in gene expression, similar to those induced by DNA damage in wild-type cells. Further, we find that nrl1Δ cells accumulate high levels of R-loops, which co-localize with HR repair factors and require Rad51 and Rad52 for their formation. Together, our findings support a model in which R-loop accumulation and subsequent DNA damage sequesters HR factors, thereby compromising HR repair at endogenously or exogenously induced DNA damage sites, leading to genome instability.

Features SENSE mRNA-Seq Library Prep Kit

Embryonic stem cells (ESCs) are unique in that they have the capacity to differentiate into all of the cell types in the body. We know a lot about the complex transcriptional control circuits that maintain the naive pluripotent state under self-renewing conditions but comparatively less about how cells exit from this state in response to differentiation stimuli. Here, we examined the role of Otx2 in this process in mouse ESCs and demonstrate that it plays a leading role in remodeling the gene regulatory networks as cells exit from ground state pluripotency. Otx2 drives enhancer activation through affecting chromatin marks and the activity of associated genes. Mechanistically, Oct4 is required for Otx2 expression, and reciprocally, Otx2 is required for efficient Oct4 recruitment to many enhancer regions. Therefore, the Oct4-Otx2 regulatory axis actively establishes a new regulatory chromatin landscape during the early events that accompany exit from ground state pluripotency.

Features SENSE mRNA-Seq Library Prep Kit

Background

A reduction of ADA2 activity due to autosomal recessive loss of function mutations in CECR1 results in a newly described vasculopathic phenotype reminiscent of polyarteritis nodosa, with manifestations ranging from fatal systemic vasculitis with multiple strokes in children to limited cutaneous disease in middle-aged individuals. Evidence indicates that ADA2 is essential for the endothelial integrity of small vessels. However, CECR1 is not expressed, nor is the ADA2 protein detectable, in cultured human endothelial cells, thus implicating additional cell types or circulating factors in disease pathogenesis.

Methods

Considering the phenotypic overlap of ADA2 deficiency with the type I interferonopathy Aicardi-Goutières syndrome due to mutations in SAMHD1, we looked for the presence of an interferon signature in the peripheral blood of two newly ascertained ADA2-deficient patients.

Results

We identified biallelic CECR1 mutations in two patients consistent with ADA2 deficiency. Both patients demonstrated an upregulation of interferon stimulated gene transcripts in peripheral blood. More strikingly however, genome-wide analysis revealed a marked overexpression of neutrophil-derived genes, suggesting that the vasculitis seen in ADA2 deficiency may be an indirect effect resulting from chronic and marked activity of neutrophils.

Conclusions

We hypothesise that ADA2 may act as a regulator of neutrophil activation, and that a reduction of ADA2 activity results in significant endothelial damage via a neutrophil-driven process.

Features SENSE mRNA-Seq Library Prep Kit