Ancestral dietary change alters the development of Drosophila larvae through MAPK signalling.

Studies in a broad range of animal species have revealed phenotypes that are caused by ancestral life experiences, including stress and diet. Ancestral dietary macronutrient composition and quantity (over- and under-nutrition) have been shown to alter descendent growth, metabolism and behaviour. Molecules have been identified in gametes that are changed by ancestral diet and are required for transgenerational effects. However, there is less understanding of the developmental pathways altered by inherited molecules during the period between fertilization and adulthood. To investigate this non-genetic inheritance, we exposed great grand-parental and grand-parental generations to defined protein to carbohydrate (P:C) dietary ratios. Descendent developmental timing was consistently faster in the period between the embryonic and pupal stages when ancestors had a higher P:C ratio diet. Transcriptional analysis revealed extensive and long-lasting changes to the MAPK signalling pathway, which controls growth rate through the regulation of ribosomal RNA transcription. Pharmacological inhibition of both MAPK and rRNA pathways recapitulated the ancestral diet-induced developmental changes. This work provides insight into non-genetic inheritance between fertilization and adulthood.

Transcriptomic insights into the zinc homeostasis of MCF-7 breast cancer cells via next-generation RNA sequencing.

A significant gap in the knowledge of zinc homeostasis exists for breast cancer cells. In this study, we investigated the transcriptomic response of the luminal breast cancer cells (MCF-7) to the exposure of extracellular zinc using next-generation RNA sequencing. The dataset was collected for three time points (T0, T30, and T120) in the time course of zinc treatment, which revealed the dramatic increase, up to 869-fold, of the gene expression for metallothioneins (MT1B, MT1F, MT1X, and MT2A) and the zinc exporter ZnT1 (SLC30A1) at T30, continuingly through to T120. The similar dynamic expression pattern was found for the autophagy-related gene (VMP1) and numerous genes for zinc finger proteins (e.g. RNF165, ZNF365, ZBTB2, SNAI1, ZNF442, ZNF547, ZNF563, and ZNF296). These findings point to the all-hands-on-deck strategy adopted by the cancer cells for maintaining zinc homeostasis. The stress responsive genes encoding heat shock proteins (HSPA1A, HSPA1B, HSPA1L, HSPA4L, HSPA6, HSPA8, HSPH1, HSP90AA1, and HSP90AB1) and the MTF-1 biomarker genes (AKR1C2, CLU, ATF3, GDF15, HMOX1, MAP1A, MAFG, SESN2, and UBC) were also differentially up-regulated at T120, suggesting a role of heat shock proteins and the MTF-1 related stress proteins in dealing with zinc exposure. It is for the first time that the gene encoding Polo-like kinase 2 (PLK2) was found to be involved in zinc-related response. The top differentially expressed genes were validated by qRT-PCR and further extended to the basal type breast cancer cells (MDA-MB-231). It was found that the expression level of SLC30A1 in MDA-MB-231 was higher than MCF-7 in response to zinc exposure. Taken together, the findings contribute to our knowledge and understanding of zinc homeostasis in breast cancer cells.

QuantSeq. 3' Sequencing combined with Salmon provides a fast, reliable approach for high throughput RNA expression analysis.

RNA-Seq is increasingly used for the diagnosis of patients, targeting of therapies and for single cell transcriptomics. These applications require cost effective, fast and reliable ways of capturing and analyzing gene expression data. Here we compared Lexogen's QuantSeq which captures only the 3' end of RNA transcripts and Illumina's TruSeq, using both Tophat2 and Salmon for gene quantification. We also compared these results to microarray. This analysis was performed on peripheral blood mononuclear cells stimulated with Poly (I:C), a viral mimic that induces innate antiviral responses. This provides a well-established model to determine if RNA-Seq and QuantSeq identify the same biological signatures. Gene expression levels in QuantSeq and RNA-Seq were strongly correlated (Spearman's rho ~0.8), Salmon and Tophat2 (Spearman's rho > 0.9). There was high consistency in protein coding genes, non-concordant genes had a high proportion of shorter, non-coding features. RNA-Seq identified more differentially expressed genes than QuantSeq, both methods outperformed microarray. The same key biological signals emerged in each of these approaches. We conclude that QuantSeq, coupled with a fast quantification method such as Salmon, should provide a viable alternative to traditional RNA-Seq in many applications and may be of particular value in the study of the 3'UTR region of mRNA.

Plau and Tgfbr3 are YAP-regulated genes that promote keratinocyte proliferation.

Yes-associated protein (YAP) is a mechanosensor protein and a downstream effector of the Hippo kinase pathway, which controls organ growth, cell proliferation, survival, maintenance and regeneration. Unphosphorylated YAP translocates to the nucleus where it acts as a cofactor of primarily the TEAD transcription factors to activate target gene transcription and cell proliferation. Perturbed YAP activation results in tumorigenesis. The pathways downstream of activated YAP that drive cell proliferation remain relatively unexplored. In this study, we employed YAP2-5SA-∆C transgenic mice, which overexpress a mildly activated YAP mutant protein in basal keratinocytes leading to increased proliferation of the epidermal stem/progenitor cell populations. We performed massively-parallel sequencing of skin biopsy mRNA (RNA-Seq) and found dysregulation of 1491 genes in YAP2-5SA-∆C skin, including many with roles in cell activation and proliferation. Furthermore, we found that 150 of these dysregulated genes harbored YAP/TEAD binding motifs in the 3' UTR, suggesting that these may be direct YAP/TEAD target genes in the control of epidermal regeneration. Further validation and functional characterization assays identified Plau and Tgfbr3 as prime candidate genes that may be activated by epidermal YAP activity in the mouse skin in vivo to promote keratinocyte proliferation. This study provides novel insights into the mechanisms regulated by YAP that control tissue homeostasis, and in particular in conditions where YAP is aberrantly activated such as in neoplastic and regenerative skin disease.

Nuclear Receptors and Neuroinflammation in Schizophrenia.

INTRODUCTION: Several nuclear receptor family members have been associated with schizophrenia and inflammation. Vitamins A and D exert anti-inflammatory actions, but their receptors (mainly nuclear receptors) have not been extensively studied in either schizophrenia brains or in association with neuroinflammation. We examined the expression of vitamin A (RARs and RXRs) and vitamin D and protein disulphide-isomerase A3 (PDIA3) receptors, as well as nuclear orphan receptors (NR4As), in the context of elevated cytokine expression in the dorsolateral prefrontal cortex (DLPFC). METHODS: mRNA levels of nuclear receptors were measured in DLPFC tissues via RT-qPCR. ANCOVAs comparing high inflammation schizophrenia, low inflammation schizophrenia and low inflammation control groups were performed. RESULTS: RARG, RXRB, NR4A1 and NR4A3 transcripts showed significant differential expression across the three groups (ANCOVA p = 0.02-0.001). Post hoc testing revealed significant reductions in RARG expression in schizophrenia with low inflammation compared to schizophrenia with high inflammation and to controls, and RXRB mRNA was significantly reduced in schizophrenia with low inflammation compared to controls. NR4A1 and NR4A3 mRNAs were decreased in schizophrenia with high inflammation compared to schizophrenia with low inflammation, with NR4A1 also significantly different to controls. CONCLUSION: In schizophrenia, changes in nuclear receptor mRNA levels involved with mediating actions of vitamin A derivatives vary according to the inflammatory state of brains.

RNA-Seq analysis and comparison of corneal epithelium in keratoconus and myopia patients.

Keratoconus is a common degenerative corneal disease that can lead to significant visual morbidity, and both genetic and environmental factors have been implicated in its pathogenesis. We compared the transcriptome of keratoconus and control epithelium using RNA-Seq. Epithelial tissues were obtained prior to surgery from keratoconus and myopia control patients, undergoing collagen cross-linking and photorefractive keratectomy, respectively. We identified major differences in keratoconus linked to cell-cell communication, cell signalling and cellular metabolism. The genes associated with the Hedgehog, Wnt and Notch1 signaling pathways were down-regulated in keratoconus. We also identified plasmolipin and Notch1 as being significantly reduced in keratoconus for both gene and protein expression (p < 0.05). Plasmolipin is a novel protein identified in human corneal epithelium, and has been demonstrated to have a key role in epithelial cell differentiation in other tissues. This study shows altered gene and protein expression of these three proteins in keratoconus, and further studies are clearly warranted to confirm the functional role of these proteins in the pathogenesis of keratoconus.

Differentially expressed genes from RNA-Seq and functional enrichment results are affected by the choice of single-end versus paired-end reads and stranded versus non-stranded protocols.

BACKGROUND: RNA-Seq is now widely used as a research tool. Choices must be made whether to use paired-end (PE) or single-end (SE) sequencing, and whether to use strand-specific or non-specific (NS) library preparation kits. To date there has been no analysis of the effect of these choices on identifying differentially expressed genes (DEGs) between controls and treated samples and on downstream functional analysis. RESULTS: We undertook four mammalian transcriptomics experiments to compare the effect of SE and PE protocols on read mapping, feature counting, identification of DEGs and functional analysis. For three of these experiments we also compared a non-stranded (NS) and a strand-specific approach to mapping the paired-end data. SE mapping resulted in a reduced number of reads mapped to features, in all four experiments, and lower read count per gene. Up to 4.3% of genes in the SE data and up to 12.3% of genes in the NS data had read counts which were significantly different compared to the PE data. Comparison of DEGs showed the presence of false positives (average 5%, using voom) and false negatives (average 5%, using voom) using the SE reads. These increased further, by one or two percentage points, with the NS data. Gene ontology functional enrichment (GO) of the DEGs arising from SE or NS approaches, revealed striking differences in the top 20 GO terms, with as little as 40% concordance with PE results. Caution is therefore advised in the interpretation of such results. By comparison, there was overall consistency in gene set enrichment analysis results. CONCLUSIONS: A strand-specific protocol should be used in library preparation to generate the most reliable and accurate profile of expression. Ideally PE reads are also recommended particularly for transcriptome assembly. Whilst SE reads produce a DEG list with around 5% of false positives and false negatives, this method can substantially reduce sequencing cost and this saving could be used to increase the number of biological replicates thereby increasing the power of the experiment. As SE reads, when used in association with gene set enrichment, can generate accurate biological results, this may be a desirable trade-off.

Towards an open, collaborative, reusable framework for sharing hands-on bioinformatics training workshops.

There is a clear demand for hands-on bioinformatics training. The development of bioinformatics workshop content is both time-consuming and expensive. Therefore, enabling trainers to develop bioinformatics workshops in a way that facilitates reuse is becoming increasingly important. The most widespread practice for sharing workshop content is through making PDF, PowerPoint and Word documents available online. While this effort is to be commended, such content is usually not so easy to reuse or repurpose and does not capture all the information required for a third party to rerun a workshop. We present an open, collaborative framework for developing and maintaining, reusable and shareable hands-on training workshop content.

Transcriptomic Analysis Shows Decreased Cortical Expression of NR4A1, NR4A2 and RXRB in Schizophrenia and Provides Evidence for Nuclear Receptor Dysregulation.

Many genes are differentially expressed in the cortex of people with schizophrenia, implicating factors that control transcription more generally. Hormone nuclear receptors dimerize to coordinate context-dependent changes in gene expression. We hypothesized that members of two families of nuclear receptors (NR4As), and retinoid receptors (RARs and RXRs), are altered in the dorsal lateral prefrontal cortex (DLPFC) of people with schizophrenia. We used next generation sequencing and then qPCR analysis to test for changes in mRNA levels for transcripts encoding nuclear receptors: orphan nuclear receptors (3 in the NR4A, 3 in the RAR, 3 in the RXR families and KLF4) in total RNA extracted from the DLPFC from people with schizophrenia compared to controls (n = 74). We also correlated mRNA levels with demographic factors and with estimates of antipsychotic drug exposure (schizophrenia group only). We tested for correlations between levels of transcription factor family members and levels of genes putatively regulated by these transcription factors. We found significantly down regulated expression of NR4A1 (Nurr 77) and KLF4 mRNAs in people with schizophrenia compared to controls, by both NGS and qPCR (p = or <0.01). We also detected decreases in NR4A2 (Nurr1) and RXRB mRNAs by using qPCR in the larger cohort (p<0.05 and p<0.01, respectively). We detected decreased expression of RARG and NR4A2 mRNAs in females with schizophrenia (p<0.05). The mRNA levels of NR4A1, NR4A2 and NR4A3 were all negative correlated with lifetime estimates of antipsychotic exposure. These novel findings, which may be influenced by antipsychotic drug exposure, implicate the orphan and retinoid nuclear receptors in the cortical pathology found in schizophrenia. Genes down stream of these receptors can be dysregulated as well, but the direction of change is not immediately predictable based on the putative transcription factor changes.

RNA-Seq analysis of Gtf2ird1 knockout epidermal tissue provides potential insights into molecular mechanisms underpinning Williams-Beuren syndrome.

BACKGROUND: Williams-Beuren Syndrome (WBS) is a genetic disorder associated with multisystemic abnormalities, including craniofacial dysmorphology and cognitive defects. It is caused by a hemizygous microdeletion involving up to 28 genes in chromosome 7q11.23. Genotype/phenotype analysis of atypical microdeletions implicates two evolutionary-related transcription factors, GTF2I and GTF2IRD1, as prime candidates for the cause of the facial dysmorphology. RESULTS: Using a targeted Gtf2ird1 knockout mouse, we employed massively-parallel sequencing of mRNA (RNA-Seq) to understand changes in the transcriptional landscape associated with inactivation of Gtf2ird1 in lip tissue. We found widespread dysregulation of genes including differential expression of 78 transcription factors or coactivators, several involved in organ development including Hey1, Myf6, Myog, Dlx2, Gli1, Gli2, Lhx2, Pou3f3, Sox2, Foxp3. We also found that the absence of GTF2IRD1 is associated with increased expression of genes involved in cellular proliferation, including growth factors consistent with the observed phenotype of extreme thickening of the epidermis. At the same time, there was a decrease in the expression of genes involved in other signalling mechanisms, including the Wnt pathway, indicating dysregulation in the complex networks necessary for epidermal differentiation and facial skin patterning. Several of the differentially expressed genes have known roles in both tissue development and neurological function, such as the transcription factor Lhx2 which regulates several genes involved in both skin and brain development. CONCLUSIONS: Gtf2ird1 inactivation results in widespread gene dysregulation, some of which may be due to the secondary consequences of gene regulatory network disruptions involving several transcription factors and signalling molecules. Genes involved in growth factor signalling and cell cycle progression were identified as particularly important for explaining the skin dysmorphology observed in this mouse model. We have noted that a number of the dysregulated genes have known roles in brain development as well as epidermal differentiation and maintenance. Therefore, this study provides clues as to the underlying mechanisms that may be involved in the broader profile of WBS.

Cardiac and skeletal muscles show molecularly distinct responses to cancer cachexia.

Cancer cachexia is a systemic, paraneoplastic syndrome seen in patients with advanced cancer. There is growing interest in the altered muscle pathophysiology experienced by cachectic patients. This study reports the microarray analysis of gene expression in cardiac and skeletal muscle in the colon 26 (C26) carcinoma mouse model of cancer cachexia. A total of 268 genes were found to be differentially expressed in cardiac muscle tissue, compared with nontumor-bearing controls. This was fewer than the 1,533 genes that changed in cachectic skeletal muscle. In addition to different numbers of genes changing, different cellular functions were seen to change in each tissue. The cachectic heart showed signs of inflammation, similar to cachectic skeletal muscle, but did not show the upregulation of ubiquitin-dependent protein catabolic processes or downregulation of genes involved in cellular energetics and muscle regeneration that characterizes skeletal muscle cachexia. Quantitative PCR was used to investigate a subset of inflammatory genes in the cardiac and skeletal muscle of independent cachectic samples; this revealed that B4galt1, C1s, Serpina3n, and Vsig4 were significantly upregulated in cardiac tissue, whereas C1s and Serpina3n were significantly upregulated in skeletal tissue. Our skeletal muscle microarray results were also compared with those from three published microarray studies and found to be consistent in terms of the genes differentially expressed and the functional processes affected. Our study highlights that skeletal and cardiac muscles are affected differently in the C26 mouse model of cachexia and that therapeutic strategies cannot assume that both muscle types will show a similar response.

The major pathway by which polymeric formula reduces inflammation in intestinal epithelial cells: a microarray-based analysis.

Nutritional therapy is well established as a means to induce remission in active Crohn's disease (CD). Evidence indicates that exclusive enteral nutrition (EEN) therapy for CD both alters the intestinal microbiota and directly suppresses the inflammatory response in the intestinal mucosa. However, the pathway(s) through which EEN suppresses inflammation is still unknown. Therefore, the aim of the current study was to use microarray technology to investigate the major pathway by which polymeric formula (PF) alters inflammatory processes in epithelial cells in vitro. HT-29 cells were grown to confluence and then co-cultured with tumour necrosis factor (TNF)-α (100 ng/ml) for 5 h in the presence or absence of PF, as used for EEN. Following incubation, RNA was extracted and subjected to polymerase chain reaction (PCR) and microarray analysis. Enzyme-linked immunosorbent assays were employed to evaluate cytokine protein levels. Neither TNF-α nor PF had a toxic effect on cells over the experimental period. Microarray analysis showed that PF modulated the expression of genes specifically linked to nuclear factor (NF)-κB, resulting in downregulation of a number of genes in this pathway. These findings were further confirmed by real-time PCR of selected dysregulated genes as well as reduced expression of IL-6 and IL-8 proteins following PF treatment. The results arising from this study provide evidence that PF alters the inflammatory responses in intestinal epithelial cells through modulation of the NF-κB pathway.

Identification of the RGG box motif in Shadoo: RNA-binding and signaling roles?

Using comparative genomics and in-silico analyses, we previously identified a new member of the prion-protein (PrP) family, the gene SPRN, encoding the protein Shadoo (Sho), and suggested its functions might overlap with those of PrP. Extended bioinformatics and conceptual biology studies to elucidate Sho's functions now reveal Sho has a conserved RGG-box motif, a well-known RNA-binding motif characterized in proteins such as FragileX Mental Retardation Protein. We report a systematic comparative analysis of RGG-box containing proteins which highlights the motif's functional versatility and supports the suggestion that Sho plays a dual role in cell signaling and RNA binding in brain. These findings provide a further link to PrP, which has well-characterized RNA-binding properties.