Stoichiometric partially-protonated states in hydroxide perovskites: the jeanbandyite enigma revisited

© 2017 The Mineralogical Society. This document is the author’s final accepted version of the journal article. You are advised to consult the published version if you wish to cite from it

Pathogen Entrapment by Transglutaminase—A Conserved Early Innate Immune Mechanism

Clotting systems are required in almost all animals to prevent loss of body fluids after injury. Here, we show that despite the risks associated with its systemic activation, clotting is a hitherto little appreciated branch of the immune system. We compared clotting of human blood and insect hemolymph to study the best-conserved component of clotting systems, namely the Drosophila enzyme transglutaminase and its vertebrate homologue Factor XIIIa. Using labelled artificial substrates we observe that transglutaminase activity from both Drosophila hemolymph and human blood accumulates on microbial surfaces, leading to their sequestration into the clot. Using both a human and a natural insect pathogen we provide functional proof for an immune function for transglutaminase (TG). Drosophila larvae with reduced TG levels show increased mortality after septic injury. The same larvae are also more susceptible to a natural infection involving entomopathogenic nematodes and their symbiotic bacteria while neither phagocytosis, phenoloxidase or—as previously shown—the Toll or imd pathway contribute to immunity. These results firmly establish the hemolymph/blood clot as an important effector of early innate immunity, which helps to prevent septic infections. These findings will help to guide further strategies to reduce the damaging effects of clotting and enhance its beneficial contribution to immune reactions

Eikosanoid biyosentezi inhibitörlerinin Galleria mellonella (Linnaeus, 1758) (Lepidoptera: Pyralidae) larvalarının hemolenf protein profiline etkisi

Eikosanoidlerin böceklerin hücresel, humoral immün savunma reaksiyonlarına ve strese karşı tepkilerine aracılık ettiği bilinmektedir. Bu aracı moleküllerin işlevi farklı eikosanoid biyosentezi inhibitörleri tarafından spesifik olarak önlenebilir. Bu inhibitörlerin böceklerde total hemolenf protein bileşimi üzerine etkileri detaylı olarak çalışılmamıştır. Eikosanoidlerin böceklerde stress faktörlerine karşı sentezlenen ve diğer savunma reaksiyonlarından sorumlu proteinler üzerinde etkili olarak fizyolojik dengenin sağlanma-sına aracılık ettiği düşünülmüştür. Bu sebeple Büyük bal mumu güvesi Galleria mellonella (Linnaeus, 1758) (Lepidoptera: Pyralidae) larvaları farklı etki mekanizmalarına sahip eikosanoid biyosentezi inhibitörleri eskuletin, deksametazon ve fenidonu % 0.001, 0.01, 0.1 ve 1.0 oranında içeren yapay besinler ile beslenmiştir. Larvaların eskuletin ile beslenmesi 45 kDa protein bileşiminde (belirlenen 16 protein bandından biri) uygulanan inhibitörün dozuna bağlı olarak önemli değişime sebep olmuştur. Diğer başlıca hemolenf proteinlerinden lipoforinler (ApoLP-I) ve depo proteinleri üzerinde denenen eikosanoid biyosentezi inhibitörlerinin önemli bir etkisi olmamıştır. Deksametazon ve fenidon hemolenf proteinlerinde önemli bir değişikliğe sebep olmamıştır. Bu çalışmanın sonuçları eikosanoidlerin, en azından lipoksijenaz ürünlerinin, böcek dokularında yapısal ve işlevsel olarak protein bileşimi üzerinde etkili olduğunu göstermiştir. Eikosanoid biyosentezi inhibitörlerinin doğrudan zararlı böceklerin kontrolünde kullanılması yaygın olmasa da bu çalışmadan elde edilen sonuçlar böceklerde eikosanoidlerin fizyolojik sinyal sistemlerindeki rollerinin anlaşılmasına katkıda bulunmaktadır.Eicosanoids mediate insects cellular and humoral immune reactions and stress responses. Function of these mediators can be specifically blocked using different eicosanoid biosynthesis inhibitors (EBIs). Effects of EBIs on total haemolymph protein composition have not been extensively studied in insects. We posed the hypothesis that eicosanoids also mediate physiological homeostasis by regulating protein profiles involved in stress response and other defensive reactions. To test this idea, we reared greater wax moth Galleria mellonella (Linnaeus,1758) (Lepidoptera: Pyralidae) larvae on artificial diets containing 0.001, 0.01, 0.1 or 1.0% of specific EBIs with different mode of action: Esculetin, dexamethasone and phenidone. Feeding larvae with esculetin caused significantly dose-dependent changes in 45 kDa protein fraction (one of 16 proteins detected) using sodium dodecyl-sulphate polyacrylamide gradient gel electrophoresis followed by silver staining. Other main haemolymph proteins, lipophorins (ApoLP-I) and storage proteins, were not affected by EBIs treatments. Dexamethasone and phenidone caused no significant differences in detected protein fractions. We infer from these findings that eicosanoids, at least lipoxygenase products, have been implicated in the protein composition of insect tissues as structural and functional concept. Although it has not yet been possible to use directly EBIs for insect pest control, our results bring new data to understand physiological signaling systems in insects

Monitoring the effect of pathogenic nematodes on locomotion of Drosophila larvae

One of the key factors that determine the interaction between hosts and their parasites is the frequency of their interactions, which depends on the locomotory behavior of both parts. To address host behavior we used natural infections involving insect pathogenic nematodes and Drosophila melanogaster larvae as hosts. Using a modified version of a recently described method (FIMTrack) to assess several parameters in larger sets of animals, we initially detected specific differences in larval food searching when comparing Drosophila strains. These differences were further influenced by the presence of nematodes. Given a choice, Drosophila larvae clearly avoided nematodes irrespective of their genetic background. Our newly developed methods will be useful to test candidate genes and pathways involved in host/pathogen interactions in general and to assess specific parameters of their interaction

Analysis of the acute response of Galleria mellonella larvae to potassium nitrate

Potassium nitrate (E252) is widely used as a food preservative and has applications in the treatment of high blood pressure however high doses are carcinogenic. Larvae of Galleria mellonella were administered potassium nitrate to establish whether the acute effects in larvae correlated with those evident in mammals. Intra-haemocoel injection of potassium nitrate resulted in a significant increase in the density of circulating haemocytes and a small change in the relative proportions of haemocytes but haemocytes showed a reduced fungicidal ability. Potassium nitrate administration resulted in increased superoxide dismutase activity and in the abundance of a range of proteins associated with mitochondrial function (e.g. mitochondrial aldehyde dehydrogenase, putative mitochondrial Mn superoxide dismutase), metabolism (e.g. triosephosphate isomerase, glyceraldehyde 3 phosphate dehydrogenase) and nitrate metabolism (e.g. aliphatic nitrilase, glutathione S-transferase). A strong correlation exists between the toxicity of a range of food preservatives when tested in G. mellonella larvae and rats. In this work a correlation between the effect of potassium nitrate in larvae and mammals is shown and opens the way to the utilization of insects for studying the in vivo acute and chronic toxicity of xenobiotics

Caffeine administration alters the behaviour and development of Galleria mellonella larvae

The effect of feeding caffeine on the behaviour and neural proteome of Galleria mellonella larvae was assessed. Caffeine was administered to larvae by force feeding and the metabolites theobromine and theophylline were subsequently detected by RP-HPLC analysis. Administration of caffeine to larvae resulted in reduced movement and a reduction in the formation of pupae. The production of the muscle relaxant theophylline may contribute to the reduction in larval movement. Analysis of the changes in proteome of the brain and surrounding tissues of caffeine fed larvae revealed an increase in the abundance of immune related proteins such as immune-related Hdd1 (6.28 fold increase) and hemolin (1.68 fold increase), ATPase associated proteins such as H + transporting ATP synthase O subunit isoform 1 (1.87 fold increase) and H + transporting ATP synthase delta subunit (1.53 fold increase) and proteins indicative of brain trauma such as troponin T transcript variant B, partial (1.55 fold increase). Proteins involved in development and protein degradation such as SUMO-activating enzyme subunit 1 (3.08 fold decrease) and chitin deacetylase, partial (3.67 fold decrease) were decreased in abundance. The results presented here indicate that caffeine is metabolised in a similar way in G. mellonella larvae to that in mammals and results in a variety of behavioural and developmental alterations. Utilisation of insects for studying the effects of caffeine and other neuroactive compounds may offer new insights into their mode of action and reduce the need to use mammals for this type of analysis