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Facilitated by the Schistosoma mansoni genome project, multiple transcriptomic studies were performed over the last decade to elucidate gene expression patterns among different developmental stages of the complex schistosome life cycle. While these analyses enable the identification of candidate genes with key functions in schistosome biology, a diverse molecular tool set is needed that allows comprehensive functional characterization at the single gene level. This includes the availability of reliable reference genes to confirm changes in the transcription of genes of interest over different biological samples and experimental conditions. In particular, the investigation of one key aspect of schistosome biology, the pairing-dependent gene expression in females and males, requires knowledge on reference genes that are expressed independently of both pairing and of in vitro culture effects. Therefore, the present study focused on the identification of quantitative reverse transcription (qRT)-PCR reference genes suitable for the investigation of pairing-dependent gene expression in the S. mansoni male. The "pipeline" we present here is based on qRT-PCR analyses of high biological replication combined with three different statistical analysis tools, BestKeeper, geNorm, and NormFinder. Our approach resulted in a statistically robust ranking of 15 selected reference genes with respect to their transcription stability between pairing-unexperienced and -experienced males. We further tested the top seven candidate genes for their transcription stability during invitro culture of adult S. mansoni. Of these, the two most suitable reference genes were used to investigate the influence of the pairing contact on the transcription of genes of interest, comprising a tyrosine decarboxylase gene Smtdc1, an ebony ortholog Smebony, and the follistatin ortholog Smfst in S. mansoni males. Performing pairing, separation and re-pairing experiments with adult S. mansoni in vitro, our results indicate for the first time that pairing can act as a molecular on/off-switch of specific genes to strictly control their expression in schistosome males., (Copyright © 2019 Australian Society for Parasitology. All rights reserved.)

RNA-Seq has proven excellence in providing information about the regulation and transcript levels of genes. We used this method for profiling genes in the flatworm Schistosoma mansoni. This parasite causes schistosomiasis, an infectious disease of global importance for human and animals. The pathology of schistosomiasis is associated with the eggs, which are synthesized as a final consequence of male and female adults pairing. The male induces processes in the female that lead to the full development of its gonads as a prerequisite for egg production. Unpaired females remain sexually immature. Based on an organ-isolation method we obtained gonad tissue for RNA extraction from paired and unpaired schistosomes, with whole adults included as controls. From a total of 23 samples, we used high-throughput cDNA sequencing (RNA-Seq) on the Illumina platform to profile gene expression between genders and tissues, with and without pairing influence. The data obtained provide a wealth of information on the reproduction biology of schistosomes and a rich resource for exploitation through basic and applied research activities.

The flatworm Schistosoma mansoni is an important but neglected pathogen that causes the disease schistosomiasis in millions of people worldwide. The parasite has a complex life cycle, undergoing sexual reproduction in a mammalian host and asexual replication in a snail host. Understanding the molecular mechanisms that the parasite uses to transition between hosts and develop into dimorphic reproductively competent adults may reveal new strategies for control. We present the first comprehensive transcriptomic analysis of S. mansoni, from eggs to sexually naïve worms. Focusing on eight life stages spanning free-living water-borne and parasitic stages from both intermediate and definitive hosts, we have generated deep RNA-seq data for five replicates per group for a total of 75 data sets. The data were produced using a single approach to increase the accuracy of stage-to-stage comparisons and made accessible via a user-friendly tool to visualise and explore gene expression (https://lifecycle.schisto.xyz/). These data are valuable for understanding the biology and sex-specific development of schistosomes and the interpretation of complementary genomic and functional genetics studies. [ABSTRACT FROM AUTHOR]

Background: The consequences of the earth's daily rotation have led to 24-h biological rhythms in most organisms. Even some parasites are known to have daily rhythms, which, when in synchrony with host rhythms, can optimise their fitness. Understanding these rhythms may enable the development of control strategies that take advantage of rhythmic vulnerabilities. Recent work on protozoan parasites has revealed 24-h rhythms in gene expression, drug sensitivity and the presence of an intrinsic circadian clock; however, similar studies on metazoan parasites are lacking. To address this, we investigated if a metazoan parasite has daily molecular oscillations, whether they reveal how these longer-lived organisms can survive host daily cycles over a lifespan of many years and if animal circadian clock genes are present and rhythmic. We addressed these questions using the human blood fluke Schistosoma mansoni that lives in the vasculature for decades and causes the tropical disease schistosomiasis. Results: Using round-the-clock transcriptomics of male and female adult worms collected from experimentally infected mice, we discovered that ~ 2% of its genes followed a daily pattern of expression. Rhythmic processes included a stress response during the host's active phase and a 'peak in metabolic activity' during the host's resting phase. Transcriptional profiles in the female reproductive system were mirrored by daily patterns in egg laying (eggs are the main drivers of the host pathology). Genes cycling with the highest amplitudes include predicted drug targets and a vaccine candidate. These 24-h rhythms may be driven by host rhythms and/or generated by a circadian clock; however, orthologs of core clock genes are missing and secondary clock genes show no 24-h rhythmicity. Conclusions: There are daily rhythms in the transcriptomes of adult S. mansoni, but they appear less pronounced than in other organisms. The rhythms reveal temporally compartmentalised internal processes and host interactions relevant to within-host survival and between-host transmission. Our findings suggest that if these daily rhythms are generated by an intrinsic circadian clock then the oscillatory mechanism must be distinct from that in other animals. We have shown which transcripts oscillate at this temporal scale and this will benefit the development and delivery of treatments against schistosomiasis. [ABSTRACT FROM AUTHOR]

Understanding oxidative stress and HTRA1 locus in abnormal angiogenesis resulting in wet AMD pathology is an important step in developing a novel therapeutic approach. Using subretinal injection of oxLDL into C57BL/6 mice, we observed a lesion resembling the features of choroidal neovascularization (CNV), including macrophage infiltration, increased VEGF expression, and neovascularization. However, incubating ARPE-19 cells with oxLDL–a carrier of oxidized phospholipids–resulted in increased expression of inflammatory cytokines and chemoattractant proteins that recruited monocytes, but no substantial increase in expression of VEGF. Furthermore, incubation of ARPE-19 with oxLDL induced higher expression of HTRA1, which we showed to synergize with oxLDL in elevating the expression of inflammatory cytokines and chemoattractant factors. To investigate the role of macrophage infiltration on these expression changes, we treated cultured J774 macrophages with oxLDL and applied the conditioned medium onto ARPE-19 cells. This treatment was found to greatly enhance the expression of VEGF in ARPE-19, indicating the necessity of macrophage secretory products to induce increased expression of VEGF in retinal pigment epithelium. Gene expression analysis revealed that oxLDL induced the expression of Wnt3A in macrophages, a key activator of canonical Wnt signaling pathways. In addition, western blot analysis showed that the macrophage conditioned media further enhanced the reduction of phosphorylated β-catenin induced by oxLDL. Lastly, we investigated HTRA1 as a potential target for AMD therapeutics. We demonstrated the ability of anti-HTRA1 antibody in vitro to neutralize the protease activity of HTRA1 and reduce the inflammatory and angiogenic response to oxidative stress. Finally, we validated the neutralizing effect of anti-HTRA1 antibody in vivo by evaluating lesion size and protein expression in a laser-photocoagulation murine model of CNV. We found that the combination of oxLDL and HTRA1 enhanced CNV size, which was reversed by the addition of anti-HTRA1 antibody. This study not only provides preliminary evidence that HTRA1 may be a viable target for AMD therapeutics but also elucidates the biochemical mechanisms by which this therapeutic effect may be mediated. [ABSTRACT FROM AUTHOR]

Background/Aims: Opiates are potent analgesics but their clinical use is limited by sex-associated side effects, such as drug tolerance, opioid-induced hyperalgesia and withdrawal reaction. OPRM1, as the main receptor of opioids, plays an important role in the pharmacological process of opioids in rodents and human. We have previously investigated OPRM1, the μ opioid receptor gene, which have dozens of alternatively spliced variants probably correlating with opioid-induced effects in brain regions of four inbred mouse strains and demonstrated the strain-specific expressions of these splice variants. Also, within a strain, the regional expression patterns of some of the variants were similar while others were opposite. Thus, we are aiming to seek out the relationship between sex differences and these alternatively spliced variants. Methods: The present studies follow a SYBR green quantitative PCR (qPCR) which we had used before to examine the expression of OPRM1 splice variant mRNAs in selected brain regions of male and female C57BL/6 mice. Sex-associated differences in baseline latency, opioid-induced tolerance, analgesia and addiction were examined and determined by Tail-flick test, jumps and statistical analysis. Results: The mRNA levels of opioid receptor gene splice variants in male and female mice showed significant differences among the brain regions, implying region-specific alternative splicing of the OPRM1 gene, which was consistent with our previous study. More importantly, the complete mRNA expression profiles of the OPRM1 splice variants was also gender-specific, suggesting a sexual influence on OPRM1 alternative splicing. Conclusion: In brief, we put forward that the distinctions among baseline latency, opioid-induced tolerance, analgesia and physical dependence in male and female mice might correlate with sex associated differential expressions of OPRM1 gene. [ABSTRACT FROM AUTHOR]

The sigma1 receptor acts as a chaperone at the endoplasmic reticulum, associates with multiple proteins in various cellular systems, and involves in a number of diseases, such as addiction, pain, cancer and psychiatric disorders. The sigma1 receptor is encoded by the single copy SIGMAR1 gene. The current study identifies five alternatively spliced variants of the mouse sigma1 receptor gene using a polymerase chain reaction cloning approach. All the splice variants are generated by exon skipping or alternative 3’ or 5’ splicing, producing the truncated sigma1 receptor. Similar alternative splicing has been observed in the human SIGMAR1 gene based on the molecular cloning or genome sequence prediction, suggesting conservation of alternative splicing of SIGMAR1 gene. Using quantitative polymerase chain reactions, we demonstrate differential expression of several splice variants in mouse tissues and brain regions. When expressed in HEK293 cells, all the splice variants fail to bind sigma ligands, implicating that each truncated region in these splice variants is important for ligand binding. However, co-immunoprecipitation (Co-IP) study in HEK293 cells co-transfected with tagged constructs reveals that all the splice variants maintain their ability to physically associate with a mu opioid receptor (mMOR-1), providing useful information to correlate the motifs/sequences necessary for their physical association. Furthermore, a competition Co-IP study showed that all the variants can disrupt in a dose-dependent manner the dimerization of the original sigma1 receptor with mMOR-1, suggesting a potential dominant negative function and providing significant insights into their function. [ABSTRACT FROM AUTHOR]

Conventional strategies are not particularly successful in the treatment of leukaemia, and identification of signalling pathways crucial to the activity of leukaemia stem cells will provide targets for the development of new therapies. Here we report that certain receptors containing the immunoreceptor tyrosine-based inhibition motif (ITIM) are crucial for the development of acute myeloid leukaemia (AML). Inhibition of expression of the ITIM-containing receptor LAIR1 does not affect normal haematopoiesis but abolishes leukaemia development. LAIR1 induces activation of SHP-1, which acts as a phosphatase-independent signalling adaptor to recruit CAMK1 for activation of downstream CREB in AML cells. The LAIR1-SHP-1-CAMK1-CREB pathway sustains the survival and self-renewal of AML stem cells. Intervention in the signalling initiated by ITIM-containing receptors such as LAIR1 may result in successful treatment of AML. [ABSTRACT FROM AUTHOR]

The µ opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing in rodents and humans, with dozens of alternatively spliced variants of the OPRM1 gene. The present studies establish a SYBR green quantitative PCR (qPCR) assay to more accurately quantify mouse OPRM1 splice variant mRNAs. Using these qPCR assays, we examined the expression of OPRM1 splice variant mRNAs in selected brain regions of four inbred mouse strains displaying differences in µ opioid-induced tolerance and physical dependence: C56BL/6J, 129P3/J, SJL/J and SWR/J. The complete mRNA expression profiles of the OPRM1 splice variants reveal marked differences of the variant mRNA expression among the brain regions in each mouse strain, suggesting region-specific alternative splicing of the OPRM1 gene. The expression of many variants was also strain-specific, implying a genetic influence on OPRM1 alternative splicing. The expression levels of a number of the variant mRNAs in certain brain regions appear to correlate with strain sensitivities to morphine analgesia, tolerance and physical dependence in four mouse strains. [ABSTRACT FROM AUTHOR]

AZD6244, an ATP-uncompetitive inhibitor of mitogen-activated protein kinase 1/2 (MEK1/2), has shown activity in several malignant tumours. However, whether AZD6244 has a function in lymphoma cells is not known. We report that AZD6244 treatment represses the growth of Raji and MOLT4 cells by inducing apoptosis and G1-phase arrest. Using miRNAs array and quantitative RT-PCR, miR-92a was downregulated byAZD6244 treatment through the ERK1/2-AP1 signalling pathway. Overexpression of miR-92a abrogated AZD6244-induced apoptosis and G1-phase arrest, indicating that it is involved in the cytotoxicity of AZD6244 in lymphoma cells. A luciferase reporter assay showed that miR-92a directly targetsthe 3′-UTRs of Bim. Overexpression of miR-92a mimics downregulated Bim mRNA and protein expression level, indicating that miR-92a negatively regulates its expression at both levels. Silencing Bim decreases AZD6244-induced apoptosis and G1-phase arrest, suggesting that Bim contributes to the growth arrest. Thus, miR-92a mediates AZD6244-induced cytotoxicity of lymphoma cells by targeting Bim. Downregulation of miR-92a by AZD6244 is mediated by the ERK1/2-AP1 signalling pathway. [ABSTRACT FROM AUTHOR]

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by overexpression of a defined set of transcription factors requires epigenetic changes in pluripotency genes. Nuclear reprogramming is an inefficient process and the molecular mechanisms that reset the epigenetic state during iPSC generation are largely unknown. Here, we show that downregulation of the nucleosome remodeling and deacetylation (NuRD) complex is required for efficient reprogramming. Overexpression of Mbd3, a subunit of NuRD, inhibits induction of iPSCs by establishing heterochromatic features and silencing embryonic stem cell-specific marker genes, including Oct4 and Nanog. Depletion of Mbd3, on the other hand, improves reprogramming efficiency and facilitates the formation of pluripotent stem cells that are capable of generating viable chimeric mice, even in the absence of c- Myc or Sox2. The results establish Mbd3/NuRD as an important epigenetic regulator that restricts the expression of key pluripotency genes, suggesting that drug-induced downregulation of Mbd3/NuRD may be a powerful means to improve the efficiency and fidelity of reprogramming. S TEM C ells 2013;31:1278-1286 [ABSTRACT FROM AUTHOR]

Schistosome parasites cause schistosomiasis, one of the most prevalent parasitemias worldwide affecting humans and animals. Constant pairing of schistosomes is essential for female sexual maturation and egg production, which causes pathogenesis. Female maturation involves signaling pathways controlling mitosis and differentiation within the gonads. In vitro studies had shown before that a Src-specific inhibitor, Herbimycin A (Herb A), and a TGFβ receptor (TβR) inhibitor (TRIKI) have physiological effects such as suppressed mitoses and egg production in paired females. As one Herb A target, the gonad-specifically expressed Src kinase SmTK3 was identified. Here, we comparatively analyzed the transcriptome profiles of Herb A- and TRIKI-treated females identifying transcriptional targets of Src-kinase and TβRI pathways. After demonstrating that TRIKI inhibits the schistosome TGFβreceptor SmTβRI by kinase assays in Xenopus oocytes, couples were treated with Herb A, TRIKI, or both inhibitors simultaneously in vitro. RNA was isolated from females for microarray hybridizations and transcription analyses. The obtained data were evaluated by Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA), but also by manual classification and intersection analyses. Finally, extensive qPCR experiments were done to verify differential transcription of candidate genes under inhibitor influence but also to functionally reinforce specific physiological effects. A number of genes found to be differentially regulated are associated with mitosis and differentiation. Among these were calcium-associated genes and eggshell-forming genes. In situ hybridization confirmed transcription of genes coding for the calcium sensor hippocalcin, the calcium transporter ORAI-1, and the calcium-binding protein calmodulin-4 in the reproductive system pointing to a role of calcium in parasite reproduction. Functional qPCR results confirmed an inhibitor-influenced, varying dependence of the transcriptional activities of Smp14, Smp48, fs800, a predicted eggshell precursor protein and SmTYR1. The results show that eggshell-formation is regulated by at least two pathways cooperatively operating in a balanced manner to control egg production. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is the most common adult acute leukemia. Despite treatment, the majority of the AML patients relapse within 5 years. In silico analysis of several available databases of AML patients showed that the expression of adenylate cyclase 7 (ADCY7) significantly inversely correlates with the overall survival of AML patients. To determine whether ADCY7 supports AML development, we employed an shRNA-encoding lentivirus system to inhibit adcy7 expression in human AML cells including U937, MV4-11, and THP-1 cells. The ADCY7 deficiency resulted in decreased cell growth, elevated apoptosis, and lower c-Myc expression of these leukemia cells. This indicates that G protein-coupled receptor signaling contributes to AML pathogenesis. Our study suggests that inhibition of ADCY7 may be novel strategy for treating leukemia. [ABSTRACT FROM AUTHOR]

Immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by low platelet count and bleeding, and is usually triggered by viral infections. We previously reported that 14 viral microRNAs of megakaryocytes cultured with serum from patients with ITP, including ebv-miR-BART6, are up-regulated. Previous research has reported that ebv-miR-BART6 down-regulated the expression of miR-185-5p. We therefore predicted that the ABCG4 gene, which is highly expressed in megakaryocyte progenitor cells, is a direct target of miR-185-5p. We hypothesized that ebv-miR-BART6 may play a role in development and differentiation of megakaryocytes. First, we verified the negative regulation of ABCG4 by miR-185-5p through luciferase assay analysis. Second, after transfection of ebv-miR-BART6 into megakaryocytes developing from normal cord blood mononuclear cells (MNCs), we found that the level of miR-185-5p in the ebv-miR-BART6 group was reduced to almost a third of that in the control groups, accompanied by up-regulation of ABCG4 at both the mRNA and protein levels. Meanwhile, proliferation of megakaryocytes was significantly repressed in the ebv-miR-BART6 group compared with the blank and negative control groups (14.89% ± 3.13%, 34.15% ± 2.42% and 30.96% ± 4.37%, respectively; P < 0.001). Our results further revealed that ebv-miR-BART6 inhibited megakaryocyte colony unit formation, decreased CD41 expression and inhibited megakaryocyte polyploidization. These data suggest a new paradigm to explain the mechanisms underlying ITP, involving the regulation of megakaryocytopoiesis by viral microRNAs through the intronic hsa-microRNA. [ABSTRACT FROM AUTHOR]

No new therapy for acute myeloid leukemia (AML) has been approved for more than 30 years. To effectively treat AML, new molecular targets and therapeutic approaches must be identified. In silico analysis of several databases of AML patients demonstrated that the expression of alkaline ceramidase 3 (ACER3) significantly inversely correlates with the overall survival of AML patients. To determine whether ACER3 supports AML development, we employed an shRNA-encoding lentivirus system to inhibit acer3 expression in human AML cells including NB4, U937, and THP-1 cells. The ACER3 deficiency resulted in decreased cell growth and colony formation, elevated apoptosis, and lower AKT signaling of leukemia cells. Our study indicates that ACER3 contributes to AML pathogenesis, and suggests that alkaline ceramidase inhibition is an option to treat AML. [ABSTRACT FROM AUTHOR]

Highlights: [•] NuRD binds to the promoter of rDNA methyltransferase recruiter TIP5. [•] NuRD enables demethylation of rDNA promoters through repressing TIP5 expression. [•] NuRD deficiency in reprogrammed cells activates TIP5 expression. [•] Reprogramming results in the accumulation of silent state of rRNA genes. [Copyright &y& Elsevier]