Biological activity of SeMNPV, AcMNPV, and three AcMNPV deletion mutants against Spodoptera exigua larvae (Lepidoptera: noctuidae)

Virulence and speed of action, as related to dose, are important effectiveness-determining properties of insect-pathogenic biocontrol agents. We used the droplet-feeding bioassay to compare dose responses between two wild-type baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), and three deletion mutants of AcMNPV in S. exigua larvae. In each mutant one gene was deleted by genetic engineering: pp34, coding for the polyhedral membrane; egt, coding for ecdysteroid UDP-glucosyltransferase; or p10, coding for fibrillar structures in infected insect cells. SeMNPV had the lowest median lethal dose (LD(50)) as well as the highest speed of action (LT(50)) of all viruses investigated. In our comparative bioassays the only significant effect of gene deletions in AcMNPV was a slightly lower speed of action for the p10 deletion mutant. Otherwise, wild-type and recombinant AcMNPVs had similar biological activities. Our results suggest, in contrast to what is generally assumed, that gene deletions in AcMNPV for improved insecticidal activity should be critically assessed in each host system prior to further implementation as a control agent. Insertion of foreign genes coding for entomotoxins is less questionable and more promising in this respect.     

Apyrase Activity and Platelet Aggregation Inhibitors in the Tick Ornithodoros Savignyi (Acari: Argasidae)

Ticks are ectoparasites that cause considerable damage to their hosts while feeding. The feeding process is facilitated by anti-haemostatic factors present in the tick saliva. Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) is a platelet aggregation inhibitor found in most haematophagous organisms studied. The present study describes the identification and characterization of such an activity in the tick Ornithodoros savignyi. The enzyme conformed to many properties common to apyrases. These included a low substrate specificity, dependence on bivalent metal ions for activity and insensitivity to the classical ATPase inhibitors. Heat denaturation studies, pH optima and similar effects of inhibitors on the enzyme's ATP and ADP hydrolysing activities supported its classification as an apyrase. Salivary gland extracts inhibited the platelet aggregation induced by ADP, collagen and thrombin and disaggregated aggregated platelets. The results suggest the presence of two or more anti-platelet factors present in the salivary glands of this tick species.     

The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking

Ciliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary compartment.     

Nuttalliella namaqua: a living fossil and closest relative to the ancestral tick lineage: implications for the evolution of blood-feeding in ticks

Ticks are monophyletic and composed of the hard (Ixodidae) and soft (Argasidae) tick families, as well as the Nuttalliellidae, a family with a single species, Nuttalliella namaqua. Significant biological differences in lifestyle strategies for hard and soft ticks suggest that various blood-feeding adaptations occurred after their divergence. The phylogenetic relationships between the tick families have not yet been resolved due to the lack of molecular data for N. namaqua. This tick possesses a pseudo-scutum and apical gnathostoma as observed for ixodids, has a leathery cuticle similar to argasids and has been considered the evolutionary missing link between the two families. Little knowledge exists with regard to its feeding biology or host preferences. Data on its biology and systematic relationship to the other tick families could therefore be crucial in understanding the evolution of blood-feeding behaviour in ticks. Live specimens were collected and blood meal analysis showed the presence of DNA for girdled lizards from the Cordylid family. Feeding of ticks on lizards showed that engorgement occurred rapidly, similar to argasids, but that blood meal concentration occurs via malpighian excretion of water. Phylogenetic analysis of the 18S nuclear and 16S mitochondrial genes indicate that N. namaqua grouped basal to the main tick families. The data supports the monophyly of all tick families and suggests the evolution of argasid-like blood-feeding behaviour in the ancestral tick lineage. Based on the data and considerations from literature we propose an origin for ticks in the Karoo basin of Gondwanaland during the late Permian. The nuttalliellid family almost became extinct during the End Permian event, leaving N. namaqua as the closest living relative to the ancestral tick lineage and the evolutionary missing link between the tick families.     

<Emphasis Type="Italic">Scoredist</Emphasis>: A simple and robust protein sequence distance estimator

Background  

Distance-based methods are popular for reconstructing evolutionary trees thanks to their speed and generality. A number of methods exist for estimating distances from sequence alignments, which often involves some sort of correction for multiple substitutions. The problem is to accurately estimate the number of true substitutions given an observed alignment. So far, the most accurate protein distance estimators have looked for the optimal matrix in a series of transition probability matrices, e.g. the Dayhoff series. The evolutionary distance between two aligned sequences is here estimated as the evolutionary distance of the optimal matrix. The optimal matrix can be found either by an iterative search for the Maximum Likelihood matrix, or by integration to find the Expected Distance. As a consequence, these methods are more complex to implement and computationally heavier than correction-based methods. Another problem is that the result may vary substantially depending on the evolutionary model used for the matrices. An ideal distance estimator should produce consistent and accurate distances independent of the evolutionary model used.     

Improved profile HMM performance by assessment of critical algorithmic features in SAM and HMMER

Background  

Profile hidden Markov model (HMM) techniques are among the most powerful methods for protein homology detection. Yet, the critical features for successful modelling are not fully known. In the present work we approached this by using two of the most popular HMM packages: SAM and HMMER. The programs' abilities to build models and score sequences were compared on a SCOP/Pfam based test set. The comparison was done separately for local and global HMM scoring.     

Thermodynamic Characterization of ppGpp Binding to EF-G or IF2 and of Initiator tRNA Binding to Free IF2 in the Presence of GDP, GTP, or ppGpp

In addition to their natural substrates GDP and GTP, the bacterial translational GTPases initiation factor (IF) 2 and elongation factor G (EF-G) interact with the alarmone molecule guanosine tetraphosphate (ppGpp), which leads to GTPase inhibition. We have used isothermal titration calorimetry to determine the affinities of ppGpp for IF2 and EF-G at a temperature interval of 5-25 °C. We find that ppGpp has a higher affinity for IF2 than for EF-G (1.7-2.8 μM K_dversus 9.1-13.9 μM K_d at 10-25 °C), suggesting that during stringent response in vivo, IF2 is more responsive to ppGpp than to EF-G. We investigated the effects of ppGpp, GDP, and GTP on IF2 interactions with fMet-tRNA^fMet demonstrating that IF2 binds to initiator tRNA with submicromolar K_d and that affinity is altered by the G nucleotides only slightly. This—in conjunction with earlier reports on IF2 interactions with fMet-tRNA^fMet in the context of the 30S initiation complex, where ppGpp was suggested to strongly inhibit fMet-tRNA^fMet binding and GTP was suggested to strongly promote fMet-tRNA^fMet binding—sheds new light on the mechanisms of the G-nucleotide-regulated fMet-tRNA^fMet selection.     

Mutations in C8orf37, encoding a ciliary protein, are associated with autosomal-recessive retinal dystrophies with early macular involvement

Cone-rod dystrophy (CRD) and retinitis pigmentosa (RP) are clinically and genetically overlapping heterogeneous retinal dystrophies. By using homozygosity mapping in an individual with autosomal-recessive (ar) RP from a consanguineous family, we identified three sizeable homozygous regions, together encompassing 46 Mb. Next-generation sequencing of all exons, flanking intron sequences, microRNAs, and other highly conserved genomic elements in these three regions revealed a homozygous nonsense mutation (c.497T>A [p.Leu166^∗]) in C8orf37, located on chromosome 8q22.1. This mutation was not present in 150 ethnically matched control individuals, single-nucleotide polymorphism databases, or the 1000 Genomes database. Immunohistochemical studies revealed C8orf37 localization at the base of the primary cilium of human retinal pigment epithelium cells and at the base of connecting cilia of mouse photoreceptors. C8orf37 sequence analysis of individuals who had retinal dystrophy and carried conspicuously large homozygous regions encompassing C8orf37 revealed a homozygous splice-site mutation (c.156−2A>G) in two siblings of a consanguineous family and homozygous missense mutations (c.529C>T [p.Arg177Trp]; c.545A>G [p.Gln182Arg]) in siblings of two other consanguineous families. The missense mutations affect highly conserved amino acids, and in silico analyses predicted that both variants are probably pathogenic. Clinical assessment revealed CRD in four individuals and RP with early macular involvement in two individuals. The two CRD siblings with the c.156−2A>G mutation also showed unilateral postaxial polydactyly. These results underline the importance of disrupted ciliary processes in the pathogenesis of retinal dystrophies.