A new set of endogenous control genes for use in quantitative real-time PCR experiments show that formin Ldia2dex transcripts are enriched in the early embryo of the pond snail Lymnaea stagnalis (Panpulmonata)

© 2019 The Author(s). Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved. Although the pond snail Lymnaea stagnalis is an emerging model organism for molecular studies in a wide variety of fields, there are a limited number of verified endogenous control genes for use in quantitative real-Time PCR. As part of a larger study on snail chirality, or left-right asymmetry, we assayed gene expression in pond snail embryos. We evaluated six candidate control genes, by comparing their expression in three tissues (ovotestis, foot and embryo) and used three software programmes (geNorm, Normfinder and Bestkeeper) to do so. The specific utility of these control genes was then tested by investigating the relative expression of six experimental transcripts, including formin Ldia2, a gene that has been associated with chiral variation in L. stagnalis. All six control genes were found to be suitable for use in the three tissues tested. Of the six experimental genes, it was found that all were relatively depleted in the early embryo compared with other tissues, except the formin Ldia2 gene. Instead, transcripts of the wild-Type Ldia2dex were enriched in the embryo, whereas a nonfunctional frameshifted version, Ldia2sin, was severely depleted. These differences in Ldia2sin expression were less evident in the ovotestis and were not evident in the foot tissue, possibly because nonsense-mediated decay is obscured in actively transcribing tissues. Our work provides a set of control genes that may be useful to the wider community and illustrates how these genes may be used to assay differences in expression in a variety of tissues

Left-right asymmetry variation in the pond snail, Lymnaea stagnalis: exploring patterns of gene expression

The establishment of left-right (LR) asymmetry in animal development remains an unanswered, fundamental question in biology. Many mechanisms of symmetry-breaking have been proposed and supported, although as yet no universal mechanism has been verified across bilaterian animals. Snails provide an invaluable study organism for understanding LR asymmetry, due to the prevalence of chirally variable species. In the pond snail Lymnaea stagnalis LR asymmetry and resulting shell-coiling direction is a well described genetically tractable trait, inherited through a maternal effect. However, the ‘chirality gene’ is still unknown. In L. stagnalis, clockwise (dextral) coiling is the dominant genotype, therefore snails with homozygote genotype ‘DD’ or heterozygote ‘Dd’ both produce dextral offspring, whereas those with the homozygote recessive genotype ‘dd’ have anticlockwise (sinistral) coiling offspring. To further the Davison research group’s ongoing characterisation of the chirality gene in L. stagnalis, this project focussed on gene expression patterns exhibited between chiral genotypes. Differential gene expression was explored via a candidate gene approach, performing quantitative real-time PCR (qPCR) experiments on specific genes of interest, and also a transcriptomic sweep, utilising next generation sequencing. To enable accurate quantification of gene expression by relative qPCR, first, stable endogenous control genes had to be established. In light of general failings of the previously published control genes to meet the criteria for appropriate use of qPCR, five genes were verified for use as stable endogenous controls in L. stagnalis embryo, ovotestis and foot tissue, for the accurate comparison of gene expression between and within chiral genotypes. These endogenous control genes will enable other researchers of L. stagnalis to rapidly identify stable controls for relative qPCR experiments. qPCR experiments were performed to compare gene expression of 13 candidate genes between chiral genotypes in the single-cell embryo, ovotestis and foot tissues. Significant differential expression was observed between chiral genotypes only in the diaphanous related formin gene, Ldia2, and two actin-related protein genes, Larp2/3 1a and Larp2/3 3. A frameshift mutation in the sinistral copy of Ldia2, discovered by the Davison research group, has identified Ldia2 as the primary candidate for the causal gene in LR asymmetry determination in L. stagnalis. In support of this, Ldia2 mRNA was found to be dramatically underrepresented in the sinistral one cell embryo and significantly reduced in the sinistral ovotestis tissue, yet not in the somatic foot tissue. Ldia2 was also the only gene found to be overrepresented in the embryo tissue relative to the ovotestis and foot tissue, providing further support for the functional importance the gene in early development. The expression level of Ldia2 in the heterozygote genotype groups was calculated to be halfway between that of the homozygote groups, indicating equal expression dominance of the alleles at the chirality locus. The expression pattern observed in the actin-related proteins was less clear and will require further analysis to infer any true biological meaning. However due to the close interaction of actin-related proteins and formins the differential expression observed in the embryo tissue provides functional support for the role of Ldia2 in chiral dimorphism. Next generation transcriptome sequencing methods were employed to gain a transcriptome-wide scan of patterns of gene expression in the ovotestis tissue of snails of differing chiral genotype. A comparative analysis was initiated trialling a novel reduced-representation sequencing method, expression RAD sequencing (eRAD) and traditional RNA Seq. eRAD applies the method of restriction-site associated DNA Sequencing (RADSeq) to the transcriptome by utilising double-stranded complementary DNA (cDNA) in place of genomic DNA. Due to delays in sequencing, the RNA Seq data was not received in sufficient time to perform the comparative assessment within this thesis. Consequently, only the eRAD data is presented here. The eRAD data failed to identify reliable differences in gene expression between chiral genotypes, although did provide a transcriptomic resource of de novo assembled contigs, which has been verified through further analyses. Overall the lack of differential expression identified between chiral genotypes in both the qPCR and eRAD analyses has indicated that the sinistral morph of L. stagnalis does not exhibit a large-scale loss of gene function and pleiotropic effects on gene expression. Therefore, the negative consequences of chiral reversal in L. stagnalis, such as the low hatch rate observed in sinistral broods, may all result from the single chirality gene polymorphism

Left-right asymmetry variation in the pond snail, Lymnaea stagnalis: exploring patterns of gene expression

The establishment of left-right (LR) asymmetry in animal development remains an unanswered, fundamental question in biology. Many mechanisms of symmetry-breaking have been proposed and supported, although as yet no universal mechanism has been verified across bilaterian animals. Snails provide an invaluable study organism for understanding LR asymmetry, due to the prevalence of chirally variable species. In the pond snail Lymnaea stagnalis LR asymmetry and resulting shell-coiling direction is a well described genetically tractable trait, inherited through a maternal effect. However, the ‘chirality gene’ is still unknown. In L. stagnalis, clockwise (dextral) coiling is the dominant genotype, therefore snails with homozygote genotype ‘DD’ or heterozygote ‘Dd’ both produce dextral offspring, whereas those with the homozygote recessive genotype ‘dd’ have anticlockwise (sinistral) coiling offspring. To further the Davison research group’s ongoing characterisation of the chirality gene in L. stagnalis, this project focussed on gene expression patterns exhibited between chiral genotypes. Differential gene expression was explored via a candidate gene approach, performing quantitative real-time PCR (qPCR) experiments on specific genes of interest, and also a transcriptomic sweep, utilising next generation sequencing. To enable accurate quantification of gene expression by relative qPCR, first, stable endogenous control genes had to be established. In light of general failings of the previously published control genes to meet the criteria for appropriate use of qPCR, five genes were verified for use as stable endogenous controls in L. stagnalis embryo, ovotestis and foot tissue, for the accurate comparison of gene expression between and within chiral genotypes. These endogenous control genes will enable other researchers of L. stagnalis to rapidly identify stable controls for relative qPCR experiments. qPCR experiments were performed to compare gene expression of 13 candidate genes between chiral genotypes in the single-cell embryo, ovotestis and foot tissues. Significant differential expression was observed between chiral genotypes only in the diaphanous related formin gene, Ldia2, and two actin-related protein genes, Larp2/3 1a and Larp2/3 3. A frameshift mutation in the sinistral copy of Ldia2, discovered by the Davison research group, has identified Ldia2 as the primary candidate for the causal gene in LR asymmetry determination in L. stagnalis. In support of this, Ldia2 mRNA was found to be dramatically underrepresented in the sinistral one cell embryo and significantly reduced in the sinistral ovotestis tissue, yet not in the somatic foot tissue. Ldia2 was also the only gene found to be overrepresented in the embryo tissue relative to the ovotestis and foot tissue, providing further support for the functional importance the gene in early development. The expression level of Ldia2 in the heterozygote genotype groups was calculated to be halfway between that of the homozygote groups, indicating equal expression dominance of the alleles at the chirality locus. The expression pattern observed in the actin-related proteins was less clear and will require further analysis to infer any true biological meaning. However due to the close interaction of actin-related proteins and formins the differential expression observed in the embryo tissue provides functional support for the role of Ldia2 in chiral dimorphism. Next generation transcriptome sequencing methods were employed to gain a transcriptome-wide scan of patterns of gene expression in the ovotestis tissue of snails of differing chiral genotype. A comparative analysis was initiated trialling a novel reduced-representation sequencing method, expression RAD sequencing (eRAD) and traditional RNA Seq. eRAD applies the method of restriction-site associated DNA Sequencing (RADSeq) to the transcriptome by utilising double-stranded complementary DNA (cDNA) in place of genomic DNA. Due to delays in sequencing, the RNA Seq data was not received in sufficient time to perform the comparative assessment within this thesis. Consequently, only the eRAD data is presented here. The eRAD data failed to identify reliable differences in gene expression between chiral genotypes, although did provide a transcriptomic resource of de novo assembled contigs, which has been verified through further analyses. Overall the lack of differential expression identified between chiral genotypes in both the qPCR and eRAD analyses has indicated that the sinistral morph of L. stagnalis does not exhibit a large-scale loss of gene function and pleiotropic effects on gene expression. Therefore, the negative consequences of chiral reversal in L. stagnalis, such as the low hatch rate observed in sinistral broods, may all result from the single chirality gene polymorphism

The genome sequence of the lunar underwing, Omphaloscelis lunosa (Haworth, 1809)

We present a genome assembly from an individual female Omphaloscelis lunosa (the Lunar Underwing; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 661.9 megabases in span. Most of the assembly is scaffolded into 32 chromosomal pseudomolecules, including the W and Z sex chromosomes. The mitochondrial genome has also been assembled and is 15.47 kilobases in length. Gene annotation of this assembly on Ensembl identified 18,931 protein coding genes

Metagenetic analysis of patterns of distribution and diversity of marine meiobenthic eukaryotes

AimMeiofaunal communities that inhabit the marine benthos offer unique opportunities to simultaneously study the macroecology of numerous phyla that exhibit different life-history strategies. Here, we ask: (1) if the macroecology of meiobenthic communities is explained mainly by dispersal constraints or by environmental conditions; and (2) if levels of meiofaunal diversity surpass existing estimates based on morphological taxonomy. LocationUK and mainland European coast. MethodsNext-generation sequencing techniques (NGS; Roche 454 FLX platform) using 18S nuclear small subunit ribosomal DNA (rDNA) gene. Pyrosequences were analysed using AmpliconNoise followed by chimera removal using Perseus. ResultsRarefaction curves revealed that sampling saturation was only reached at 15% of sites, highlighting that the bulk of meiofaunal diversity is yet to be discovered. Overall, 1353 OTUs were recovered and assigned to 23 different phyla. The majority of sampled sites had c. 60-70 unique operational taxonomic units (OTUs) per site, indicating high levels of beta diversity. The environmental parameters that best explained community structure were seawater temperature, geographical distance and sediment size, but most of the variability (R-2=70%-80%) remains unexplained. Main conclusionsHigh percentages of endemic OTUs suggest that meiobenthic community composition is partly niche-driven, as observed in larger organisms, but also shares macroecological features of microorganisms by showing high levels of cosmopolitanism (albeit on a much smaller scale). Meiobenthic communities exhibited patterns of isolation by distance as well as associations between niche, latitude and temperature, indicating that meiobenthic communities result from a combination of niche assembly and dispersal processes. Conversely, isolation-by-distance patterns were not identified in the featured protists, suggesting that animals and protists adhere to radically different macroecological processes, linked to life-history strategies.Natural Environment Research Council (NERC) [NE/E001505/1, NE/F001266/1, MGF-167]; Portuguese Foundation for Science and Technology (FCT) [SFRH/BD/27413/2006, SFRH/BPD/80447/2014]; EPSRC [EP/H003851/1]; BBSRC CASE studentship; Unilever; Biotechnology and Biological Sciences Research Council [987347]; Engineering and Physical Sciences Research Council [EP/H003851/1]; Natural Environment Research Council [NE/F001290/1, NE/F001266/1, NE/E001505/1, NBAF010002]info:eu-repo/semantics/publishedVersio

Tuberculosis Treatment in HIV Infected Ugandans with CD4 Counts >350 Cells/mm3 Reduces Immune Activation with No Effect on HIV Load or CD4 Count

Both HIV and TB cause a state of heightened immune activation. Immune activation in HIV is associated with progression to AIDS. Prior studies, focusing on persons with advanced HIV, have shown no decline in markers of cellular activation in response to TB therapy alone.) and pulmonary TB. HIV load, CD4 count, and markers of immune activation (CD38 and HLA-DR on CD4 and CD8 T cells) were measured prior to starting, during, and for 6 months after completion of standard 6 month anti-tuberculosis (TB) therapy in 38 HIV infected Ugandans with smear and culture confirmed pulmonary TB.Expression of CD38, and co-expression of CD38 and HLA-DR, on CD8 cells declined significantly within 3 months of starting standard TB therapy in the absence of anti-retroviral therapy, and remained suppressed for 6 months after completion of therapy. In contrast, HIV load and CD4 count remained unchanged throughout the study period.

Formin is associated with left-right asymmetry in the pond snail and the frog

While components of the pathway that establishes left-right asymmetry have been identified in diverse animals, from vertebrates to flies, it is striking that the genes involved in the first symmetry-breaking step remain wholly unknown in the most obviously chiral animals, the gastropod snails. Previously, research on snails was used to show that left-right signalling of Nodal, downstream of symmetry-breaking, may be an ancestral feature of the Bilateria. Here we report that a disabling mutation in one copy of a tandemly duplicated, diaphanous-related formin is perfectly associated with symmetry-breaking in the pond snail. This is supported by the observation that an anti-formin drug treatment converts dextral snail embryos to a sinistral phenocopy, and in frogs, drug inhibition or over-expression by microinjection of formin has a chirality-randomizing effect in early (pre-cilia) embryos. Contrary to expectations based on existing models, we discovered asymmetric gene expression in 2 and 4 cell snail embryos, preceding morphological asymmetry. As the formin-actin filament has been shown to be part of an asymmetry-breaking switch in vitro, together these results are consistent with the view that animals with diverse bodyplans may derive their asymmetries from the same intracellular chiral elements

Formin is associated with left-right asymmetry in the pond snail and the frog

While components of the pathway that establishes left-right asymmetry have been identified in diverse animals, from vertebrates to flies, it is striking that the genes involved in the first symmetry-breaking step remain wholly unknown in the most obviously chiral animals, the gastropod snails. Previously, research on snails was used to show that left-right signalling of Nodal, downstream of symmetry-breaking, may be an ancestral feature of the Bilateria. Here we report that a disabling mutation in one copy of a tandemly duplicated, diaphanous-related formin is perfectly associated with symmetry-breaking in the pond snail. This is supported by the observation that an anti-formin drug treatment converts dextral snail embryos to a sinistral phenocopy, and in frogs, drug inhibition or over-expression by microinjection of formin has a chirality-randomizing effect in early (pre-cilia) embryos. Contrary to expectations based on existing models, we discovered asymmetric gene expression in 2 and 4 cell snail embryos, preceding morphological asymmetry. As the formin-actin filament has been shown to be part of an asymmetry-breaking switch in vitro, together these results are consistent with the view that animals with diverse bodyplans may derive their asymmetries from the same intracellular chiral elements

Red Aesthetics, Intermediality and the Use of Posters in Chinese Cinema after 1949

Abstract: This article focuses on the aesthetic and affective techniques of saturation through which posters legitimated the Party-State in Mao’s China by closing the gap between everyday experience and political ideology. Propaganda posters were designed to put into practice the principle of unity, as conceptua- lised by Mao Zedong. The argument posits that while the “poster” is normally a printed edition of a painting or design intended for mass distribution in this way, the term may fairly be deployed to capture other cultural objects that function as “posters”, in that they provide public, political information that expresses or con- structs a political self in aesthetic form. This approach requires a metonymic understanding of a visual field in which cultural objects are interrelated and mutually reinforcing. The essay draws on recent in-depth interviews with poster artists of the 1960s and 1970s