Effects of Isoform-selective Phosphatidylinositol 3-Kinase Inhibitors on Osteoclasts: ACTIONS ON CYTOSKELETAL ORGANIZATION,SURVIVAL, AND RESORPTION*

Phosphatidylinositol 3-kinases (PI3K) participate in numerous signaling pathways, and control distinct biological functions. Studies using pan-PI3K inhibitors suggest roles for PI3K in osteoclasts, but little is known about specific PI3K isoforms in these cells. Our objective was to determine effects of isoform-selective PI3K inhibitors on osteoclasts. The following inhibitors were investigated (targets in parentheses): wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (pan-p110), PIK75 (α), GDC0941 (α, δ), TGX221 (β), AS252424 (γ), and IC87114 (δ). In addition, we characterized a new potent and selective PI3Kδ inhibitor, GS-9820, and explored roles of PI3K isoforms in regulating osteoclast function. Osteoclasts were isolated from long bones of neonatal rats and rabbits. Wortmannin, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, GDC0941, IC87114, and GS-9820 induced a dramatic retraction of osteoclasts within 15–20 min to 65–75% of the initial area. In contrast, there was no significant retraction in response to vehicle, PIK75, TGX221, or AS252424. Moreover, wortmannin and GS-9820, but not PIK75 or TGX221, disrupted actin belts. We examined effects of PI3K inhibitors on osteoclast survival. Whereas PIK75, TGX221, and GS-9820 had no significant effect on basal survival, all blocked RANKL-stimulated survival. When studied on resorbable substrates, osteoclastic resorption was suppressed by wortmannin and inhibitors of PI3Kβ and PI3Kδ, but not other isoforms. These data are consistent with a critical role for PI3Kδ in regulating osteoclast cytoskeleton and resorptive activity. In contrast, multiple PI3K isoforms contribute to the control of osteoclast survival. Thus, the PI3Kδ isoform, which is predominantly expressed in cells of hematopoietic origin, is an attractive target for anti-resorptive therapeutics.     

Sequencing,Annotation, and Characterization of the Influenza Ferret Infectome

Ferrets have become an indispensable tool in the understanding of influenza virus virulence and pathogenesis. Furthermore, ferrets are the preferred preclinical model for influenza vaccine and therapeutic testing. Here we characterized the influenza infectome during the different stages of the infectious process in ferrets with and without prior specific immunity to influenza. RNA from lung tissue and lymph nodes from infected and naïve animals was subjected to next-generation sequencing, followed by de novo data assembly and annotation of the resulting sequences; this process generated a library comprising 13,202 ferret mRNAs. Gene expression profiles during pandemic H1N1 (pdmH1N1) influenza virus infection were analyzed by digital gene expression and solid support microarrays. As expected during primary infection, innate immune responses were triggered in the lung tissue; meanwhile, in the lymphoid tissue, genes encoding antigen presentation and maturation of effector cells of adaptive immunity increased dramatically. After 5 days postinfection, the innate immune gene expression was replaced by the adaptive immune response, which correlates with viral clearance. Reinfection with homologous pandemic influenza virus resulted in a diminished innate immune response, early adaptive immune gene regulation, and a reduction in clinical severity. The fully annotated ferret infectome will be a critical aid to the understanding of the molecular events that regulate disease severity and host-influenza virus interactions among seasonal, pandemic, and highly pathogenic avian influenzas.     

Natural history and behaviour of Dunatothrips aneurae Mound (Thysanoptera: Phlaeothripidae), a phyllode‐gluing thrips with facultative pleometrosis

Thrips of the genus Dunatothrips (Thysanoptera: Phlaeothripidae) construct domiciles by tying phyllodes of Australian Acacia trees together with silk‐like glue. Females often co‐found domiciles (pleometrosis), an apparently cooperative behaviour that potentially provides insights into social evolution. However, little is known about their basic natural history, limiting the scope for testable predictions. Here, we address this crucial prerequisite step by investigating some key outstanding questions in the most common species, D. aneurae, on its host, Acacia aneura. We detail distribution in space and time, mating, dispersal, domicile building and defence. Dunatothrips aneurae was distributed in loosely reproductively synchronized patches, and tended to prefer east‐facing, terminal phyllodes on thin‐phyllode A. aneura varieties. Mature domiciles contained middens, concentrated areas of waste, suggesting active maintenance of domiciles and the potential for the division of labour. We observed inbreeding and outbreeding. Dunatothrips aneurae males engaged in short, truncated matings with sisters before dispersing locally, mating with females in nearby immature domiciles; longer distance dispersal, although it must happen, is still undocumented. Males and females mated multiply. Lone females required male presence to initiate domiciles, constructed them without male help and lost wings on nesting by abscission. Silk production occurred well before egg laying. Aggression or defence appeared to be entirely absent. Taken together, these observations suggest that research into co‐founding behaviour should focus on: (1) local crowding; (2) lack of aggression; and (3) potential division of labour with respect to egg production, silk production and domicile maintenance. These results should provide a springboard for questions on the potential evolution of cooperation in this species. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 802‐816.     

Reconstruction of Certain Phylogenetic Networks from Their Tree-Average Distances

Trees are commonly utilized to describe the evolutionary history of a collection of biological species, in which case the trees are called phylogenetic trees. Often these are reconstructed from data by making use of distances between extant species corresponding to the leaves of the tree. Because of increased recognition of the possibility of hybridization events, more attention is being given to the use of phylogenetic networks that are not necessarily trees. This paper describes the reconstruction of certain such networks from the tree-average distances between the leaves. For a certain class of phylogenetic networks, a polynomial-time method is presented to reconstruct the network from the tree-average distances. The method is proved to work if there is a single reticulation cycle.     

Diversity of Planktonic and Attached Bacterial Communities in a Phenol-Contaminated Sandstone Aquifer

Polluted aquifers contain indigenous microbial communities with the potential for in situ bioremediation. However, the effect of hydrogeochemical gradients on in situ microbial communities (especially at the plume fringe, where natural attenuation is higher) is still not clear. In this study, we used culture-independent techniques to investigate the diversity of in situ planktonic and attached bacterial communities in a phenol-contaminated sandstone aquifer. Within the upper and lower plume fringes, denaturing gradient gel electrophoresis profiles indicated that planktonic community structure was influenced by the steep hydrogeochemical gradient of the plume rather than the spatial location in the aquifer. Under the same hydrogeochemical conditions (in the lower plume fringe, 30 m below ground level), 16S rRNA gene cloning and sequencing showed that planktonic and attached bacterial communities differed markedly and that the attached community was more diverse. The 16S rRNA gene phylogeny also suggested that a phylogenetically diverse bacterial community operated at this depth (30 mbgl), with biodegradation of phenolic compounds by nitrate-reducing Azoarcus and Acidovorax strains potentially being an important process. The presence of acetogenic and sulphate-reducing bacteria only in the planktonic clone library indicates that some natural attenuation processes may occur preferentially in one of the two growth phases (attached or planktonic). Therefore, this study has provided a better understanding of the microbial ecology of this phenol-contaminated aquifer, and it highlights the need for investigating both planktonic and attached microbial communities when assessing the potential for natural attenuation in contaminated aquifers.     

Terrestrial support of pelagic consumers: patterns and variability revealed by a multilake study

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Evidence of an increased incidence of day 3 parasitaemia in Suriname: an indicator of the emerging resistance of Plasmodium falciparum to artemether

The emerging resistance to artemisinin derivatives that has been reported in South-East Asia led us to assess the efficacy of artemether-lumefantrine as the first line therapy for uncomplicated Plasmodium falciparum infections in Suriname. This drug assessment was performed according to the recommendations of the World Health Organization in 2011. The decreasing number of malaria cases in Suriname, which are currently limited to migrating populations and gold miners, precludes any conclusions on artemether efficacy because adequate numbers of patients with 28-day follow-up data are difficult to obtain. Therefore, a comparison of day 3 parasitaemia in a 2011 study and in a 2005/2006 study was used to detect the emergence of resistance to artemether. The prevalence of day 3 parasitaemia was assessed in a study in 2011 and was compared to that in a study in 2005/2006. The same protocol was used in both studies and artemether-lumefantrine was the study drug. Of 48 evaluable patients in 2011, 15 (31%) still had parasitaemia on day 3 compared to one (2%) out of 45 evaluable patients in 2005/2006. Overall, 11 evaluable patients in the 2011 study who were followed up until day 28 had negative slides and similar findings were obtained in all 38 evaluable patients in the 2005/2006 study. The significantly increased incidence of parasite persistence on day 3 may be an indication of emerging resistance to artemether.     

Influence of plant growth regulators on shoot proliferation and secondary metabolite production in micropropagated Huernia hystrix

Although the effectiveness of topolins in plant tissue culture systems has recently been highlighted, there is a dearth of information on their interactions with auxins in relation to shoot organogenesis and secondary metabolite production. The current study evaluated the role of topolins singly or in combination with an auxin in comparison to 6-benzyladenine (BA) on shoot proliferation and secondary metabolite production of Huernia hystrix, a medicinal and ornamental stem-succulent of the endemic flora of southern Africa. Meta-topolin (mT) was more effective in improving shoot proliferation and phenolic production compared to BA. In general, the exogenous addition of α-naphthalene acetic acid (NAA) significantly increased shoot proliferation. The highest number of regenerated shoots (12.2 ± 0.98 shoots per explant) was recorded with medium containing 20 μM mT supplemented with 10 μM NAA and was three-times higher when compared to the treatments with cytokinin only. This suggests a synergistic interaction of auxin with cytokinin. On the other hand, supplementation with low NAA concentrations resulted in reduced in vitro flavonoid production in most cases, when compared to treatments with cytokinin only. Moreover, differences in cytokinin concentrations (even when used in combination with NAA equimolar concentrations) significantly affected secondary metabolite production in some cases. The current findings highlighted the differential effects of auxin-cytokinin interactions on shoot proliferation and the production of secondary metabolites in H. hystrix.     

Analysis of heterologous regulatory and coding regions in algal chloroplasts

The basic photosynthetic apparatus is highly conserved across all photosynthetic organisms, and this conservation can be seen in both protein composition and amino acid sequence. Conservation of regulatory elements also seems possible in chloroplast genes, as many mRNA untranslated regions (UTRs) appear to have similar structural elements. The D1 protein of Photosystem II (psbA gene) is a highly conserved core reaction center protein that shows very similar regulation from cyanobacteria through higher plants. We engineered full and partial psbA genes from a diverse set of photosynthetic organisms into a psbA deficient strain of Chlamydomonas reinhardtii. Analysis of D1 protein accumulation and photosynthetic growth revealed that coding sequences and promoters are interchangeable even between anciently diverged species. On the other hand functional recognition of 5′ UTRs is limited to closely related organisms. Furthermore transformation of heterologous promoters and 5′ UTRs from the atpA, tufA and psbD genes conferred psbA mRNA accumulation but not translation. Overall, our results show that heterologous D1 proteins can be expressed and complement Photosystem II function in green algae, while RNA regulatory elements appear to be very specific and function only from closely related species. Nonetheless, there is great potential for the expression of heterologous photosynthetic coding sequences for studying and modifying photosynthesis in C. reinhardtii chloroplasts.