The nuclear receptors of Biomphalaria glabrata and Lottia gigantea: Implications for developing new model organisms

© 2015 Kaur et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedNuclear receptors (NRs) are transcription regulators involved in an array of diverse physiological functions including key roles in endocrine and metabolic function. The aim of this study was to identify nuclear receptors in the fully sequenced genome of the gastropod snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni and compare these to known vertebrate NRs, with a view to assessing the snail's potential as a invertebrate model organism for endocrine function, both as a prospective new test organism and to elucidate the fundamental genetic and mechanistic causes of disease. For comparative purposes, the genome of a second gastropod, the owl limpet, Lottia gigantea was also investigated for nuclear receptors. Thirty-nine and thirty-three putative NRs were identified from the B. glabrata and L. gigantea genomes respectively, based on the presence of a conserved DNA-binding domain and/or ligand-binding domain. Nuclear receptor transcript expression was confirmed and sequences were subjected to a comparative phylogenetic analysis, which demonstrated that these molluscs have representatives of all the major NR subfamilies (1-6). Many of the identified NRs are conserved between vertebrates and invertebrates, however differences exist, most notably, the absence of receptors of Group 3C, which includes some of the vertebrate endocrine hormone targets. The mollusc genomes also contain NR homologues that are present in insects and nematodes but not in vertebrates, such as Group 1J (HR48/DAF12/HR96). The identification of many shared receptors between humans and molluscs indicates the potential for molluscs as model organisms; however the absence of several steroid hormone receptors indicates snail endocrine systems are fundamentally different.The National Centre for the Replacement, Refinement and Reduction of Animals in Research, Grant Ref:G0900802 to CSJ, LRN, SJ & EJR [www.nc3rs.org.uk]

Molecular modelling of the GIR1 branching ribozyme gives new insight into evolution of structurally related ribozymes

Twin-ribozyme introns contain a branching ribozyme (GIR1) followed by a homing endonuclease (HE) encoding sequence embedded in a peripheral domain of a group I splicing ribozyme (GIR2). GIR1 catalyses the formation of a lariat with 3 nt in the loop, which caps the HE mRNA. GIR1 is structurally related to group I ribozymes raising the question about how two closely related ribozymes can carry out very different reactions. Modelling of GIR1 based on new biochemical and mutational data shows an extended substrate domain containing a GoU pair distinct from the nucleophilic residue that dock onto a catalytic core showing a different topology from that of group I ribozymes. The differences include a core J8/7 region that has been reduced and is complemented by residues from the pre-lariat fold. These findings provide the basis for an evolutionary mechanism that accounts for the change from group I splicing ribozyme to the branching GIR1 architecture. Such an evolutionary mechanism can be applied to other large RNAs such as the ribonuclease P

A novel approach to modelling water transport and drug diffusion through the stratum corneum

<p>Abstract</p> <p>Background</p> <p>The potential of using skin as an alternative path for systemically administering active drugs has attracted considerable interest, since the creation of novel drugs capable of diffusing through the skin would provide a great step towards easily applicable -and more humane- therapeutic solutions. However, for drugs to be able to diffuse, they necessarily have to cross a permeability barrier: the <it>stratum corneum </it>(SC), the uppermost set of skin layers. The precise mechanism by which drugs penetrate the skin is generally thought to be diffusion of molecules through this set of layers following a "tortuous pathway" around corneocytes, i.e. impermeable dead cells.</p> <p>Results</p> <p>In this work, we simulate water transport and drug diffusion using a three-dimensional porous media model. Our numerical simulations show that diffusion takes place through the SC regardless of the direction and magnitude of the fluid pressure gradient, while the magnitude of the concentrations calculated are consistent with experimental studies.</p> <p>Conclusions</p> <p>Our results support the possibility for designing arbitrary drugs capable of diffusing through the skin, the time-delivery of which is solely restricted by their diffusion and solubility properties.</p

Zwanzig-Mori projection operators and EEG dynamics: deriving a simple equation of motion

We present a macroscopic theory of electroencephalogram (EEG) dynamics based on the laws of motion that govern atomic and molecular motion. The theory is an application of Zwanzig-Mori projection operators. The result is a simple equation of motion that has the form of a generalized Langevin equation (GLE), which requires knowledge only of macroscopic properties. The macroscopic properties can be extracted from experimental data by one of two possible variational principles. These variational principles are our principal contribution to the formalism. Potential applications are discussed, including applications to the theory of critical phenomena in the brain, Granger causality and Kalman filters

Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification

The study of functional RNAs of various sizes and structures requires efficient methods for their synthesis and purification. Here, 23 group I intron variants ranging in length from 246 to 341 nucleotides—some containing exons—were subjected to a native purification technique previously applied only to shorter RNAs (<160 nucleotides). For the RNAs containing both exons, we adjusted the original purification protocol to allow for purification of radiolabeled molecules. The resulting RNAs were used in folding assays on native gel electrophoresis and in self-splicing assays. The intron-only RNAs were subjected to the regular native purification scheme, assayed for folding and employed in crystallization screens. All RNAs that contained a 3′ overhang of one nucleotide were efficiently cleaved off from the support and were at least 90% pure after the non-denaturing purification. A representative subset of these RNAs was shown to be folded and self-splicing after purification. Additionally, crystals were grown for a 286 nucleotide long variant of the Clostridium botulinum intron. These results demonstrate the suitability of the native affinity purification method for the preparation of group I introns. We hope these findings will stimulate a broader application of this strategy to the preparation of other large RNA molecules

RNAi Screen of DAF-16/FOXO Target Genes in C. elegans Links Pathogenesis and Dauer Formation

The DAF-16/FOXO transcription factor is the major downstream output of the insulin/IGF1R signaling pathway controlling C. elegans dauer larva development and aging. To identify novel downstream genes affecting dauer formation, we used RNAi to screen candidate genes previously identified to be regulated by DAF-16. We used a sensitized genetic background [eri-1(mg366); sdf-9(m708)], which enhances both RNAi efficiency and constitutive dauer formation (Daf-c). Among 513 RNAi clones screened, 21 displayed a synthetic Daf-c (SynDaf) phenotype with sdf-9. One of these genes, srh-100, was previously identified to be SynDaf, but twenty have not previously been associated with dauer formation. Two of the latter genes, lys-1 and cpr-1, are known to participate in innate immunity and six more are predicted to do so, suggesting that the immune response may contribute to the dauer decision. Indeed, we show that two of these genes, lys-1 and clc-1, are required for normal resistance to Staphylococcus aureus. clc-1 is predicted to function in epithelial cohesion. Dauer formation exhibited by daf-8(m85), sdf-9(m708), and the wild-type N2 (at 27°C) were all enhanced by exposure to pathogenic bacteria, while not enhanced in a daf-22(m130) background. We conclude that knockdown of the genes required for proper pathogen resistance increases pathogenic infection, leading to increased dauer formation in our screen. We propose that dauer larva formation is a behavioral response to pathogens mediated by increased dauer pheromone production

Molecular Time-Course and the Metabolic Basis of Entry into Dauer in Caenorhabditis elegans

When Caenorhabditis elegans senses dauer pheromone (daumone), signaling inadequate growth conditions, it enters the dauer state, which is capable of long-term survival. However, the molecular pathway of dauer entry in C. elegans has remained elusive. To systematically monitor changes in gene expression in dauer paths, we used a DNA microarray containing 22,625 gene probes corresponding to 22,150 unique genes from C. elegans. We employed two different paths: direct exposure to daumone (Path 1) and normal growth media plus liquid culture (Path 2). Our data reveal that entry into dauer is accomplished through the multi-step process, which appears to be compartmentalized in time and according to metabolic flux. That is, a time-course of dauer entry in Path 1 shows that dauer larvae formation begins at post-embryonic stage S4 (48 h) and is complete at S6 (72 h). Our results also suggest the presence of a unique adaptive metabolic control mechanism that requires both stage-specific expression of specific genes and tight regulation of different modes of fuel metabolite utilization to sustain the energy balance in the context of prolonged survival under adverse growth conditions. It is apparent that worms entering dauer stage may rely heavily on carbohydrate-based energy reserves, whereas dauer larvae utilize fat or glyoxylate cycle-based energy sources. We created a comprehensive web-based dauer metabolic database for C. elegans (www.DauerDB.org) that makes it possible to search any gene and compare its relative expression at a specific stage, or evaluate overall patterns of gene expression in both paths. This database can be accessed by the research community and could be widely applicable to other related nematodes as a molecular atlas

RNAi in the regulation of mammalian viral infections

Although RNA interference (RNAi) is known to play an important part in defense against viruses of invertebrates, its contribution to mammalian anti-viral defense has been a matter of dispute. This is surprising because all components of the RNAi machinery necessary for robust RNAi-mediated restriction of viruses are conserved in mammals, and the introduction of synthetic small interfering RNAs (siRNAs) into cells efficiently silences the replication of viruses that contain siRNA complementary sequences in those cells. Here, I discuss the reasons for the dispute, and review the evidence that RNAi is a part of the physiological defense of mammalian cells against viral infections

DAF-16 and Δ9 Desaturase Genes Promote Cold Tolerance in Long-Lived Caenorhabditis elegans age-1 Mutants

In Caenorhabditis elegans, mutants of the conserved insulin/IGF-1 signalling (IIS) pathway are long-lived and stress resistant due to the altered expression of DAF-16 target genes such as those involved in cellular defence and metabolism. The three Δ9 desaturase genes, fat-5, fat-6 and fat-7, are included amongst these DAF-16 targets, and it is well established that Δ9 desaturase enzymes play an important role in survival at low temperatures. However, no assessment of cold tolerance has previously been reported for IIS mutants. We demonstrate that long-lived age-1(hx546) mutants are remarkably resilient to low temperature stress relative to wild type worms, and that this is dependent upon daf-16. We also show that cold tolerance following direct transfer to low temperatures is increased in wild type worms during the facultative, daf-16 dependent, dauer stage. Although the cold tolerant phenotype of age-1(hx546) mutants is predominantly due to the Δ9 desaturase genes, additional transcriptional targets of DAF-16 are also involved. Surprisingly, survival of wild type adults following a rapid temperature decline is not dependent upon functional daf-16, and cellular distributions of a DAF-16::GFP fusion protein indicate that DAF-16 is not activated during low temperature stress. This suggests that cold-induced physiological defences are not specifically regulated by the IIS pathway and DAF-16, but expression of DAF-16 target genes in IIS mutants and dauers is sufficient to promote cross tolerance to low temperatures in addition to other forms of stress