Genome-Wide Association Analysis of Radiation Resistance in Drosophila melanogaster

Background

Ionizing radiation is genotoxic to cells. Healthy tissue toxicity in patients and radiation resistance in tumors present common clinical challenges in delivering effective radiation therapies. Radiation response is a complex, polygenic trait with unknown genetic determinants. The Drosophila Genetic Reference Panel (DGRP) provides a model to investigate the genetics of natural variation for sensitivity to radiation.

Methods and Findings

Radiation response was quantified in 154 inbred DGRP lines, among which 92 radiosensitive lines and 62 radioresistant lines were classified as controls and cases, respectively. A case-control genome-wide association screen for radioresistance was performed. There are 32 single nucleotide polymorphisms (SNPs) associated with radio resistance at a nominal p<10⁻⁵; all had modest effect sizes and were common variants with the minor allele frequency >5%. All the genes implicated by those SNP hits were novel, many without a known role in radiation resistance and some with unknown function. Variants in known DNA damage and repair genes associated with radiation response were below the significance threshold of p<10⁻⁵ and were not present among the significant hits. No SNP met the genome-wide significance threshold (p = 1.49×10⁻⁷), indicating a necessity for a larger sample size.

Conclusions

Several genes not previously associated with variation in radiation resistance were identified. These genes, especially the ones with human homologs, form the basis for exploring new pathways involved in radiation resistance in novel functional studies. An improved DGRP model with a sample size of at least 265 lines and ideally up to 793 lines is recommended for future studies of complex traits.     

Conus venom peptides: correlating chemistry and behavior

Chemical communication in scarab beetles involves female-released long-distance sex pheromones. Electrophysiological recordings using tungsten microelectrodes demonstrated two types of olfactory receptor neurons in the scarab beetle Anomala cuprea, each specific for one of the two pheromone components (R)-buibuilactone and (R)-japonilure, respectively. No neurons were found that responded specifically to enantiomers of the pheromone compounds, i.e. (S)-buibuilactone and (S)-japonilure. Pheromone receptor neurons are present in high numbers on both the male and the female antenna, with a lower sensitivity in the females. As in bark beetles and moths, the pheromone receptor neurons in A. cuprea are very sensitive and selective. The difference in response thresholds between (R)- and (S)-enantiomers is almost three orders of magnitude. Pheromone receptor neurons are found in sensilla placodea located in a defined area on each lamella in the antennal club. (R)-buibuilactone and (R)-japonilure neurons are always found in different sensilla. Both types of sensilla contain two neurons, with the pheromone-sensitive neuron displaying a high spike amplitude and the second neuron, not responding to any of the tested compounds, always with a lower spike amplitude. Accepted: 19 December 1998     

Possible Influence of B Chromosomes on Genes Included in Immune Response and Parasite Burden in Apodemus flavicollis

Genetic background underlying wild populations immune response to different parasites is still not well understood. We studied immune response to multiple infections and to competition between different parasite species at different developmental stages in population of yellow-necked mouse, Apodemus flavicollis. Quantitative real-time PCR was used to investigate associations of MHC II-DRB, IL-10 and Tgf-β genes expressions with presence of intestinal parasites at different developmental stages. Furthermore, we were interested whether the host related characteristics (sex, age, body condition, presence of B chromosomes or expression of other genes) or characteristics of present parasites (number of adult parasites of each identified species, egg count of each parasite genus, total number of nematode individuals) affect differential expression of the studied genes. A significant invert association between the expression of MHC II-DRB and Tgf-β gene was found, which together with absence of IL-10 association confirmed modified Th2 as the main type of immune response to nematode infections. Effect of recorded parasites and parasite life-cycle stage on expression levels of MHC II-DRB gene was detected only through interactions with host-related characteristics such as sex, age, and the presence of B chromosomes. The presence of B chromosomes is associated with lower expression level of Tgf-β gene. Although the influence of host genetic background on parasite infection has already been well documented, this is the first study in mammals that gave presence of B chromosomes on immune response full consideration.     

Nitric Oxide Protects against Infection-Induced Neuroinflammation by Preserving the Stability of the Blood-Brain Barrier

Nitric oxide (NO) generated by inducible NO synthase (iNOS) is critical for defense against intracellular pathogens but may mediate inflammatory tissue damage. To elucidate the role of iNOS in neuroinflammation, infections with encephalitogenic Trypanosoma brucei parasites were compared in inos^-/- and wild-type mice. Inos^-/- mice showed enhanced brain invasion by parasites and T cells, and elevated protein permeability of cerebral vessels, but similar parasitemia levels. Trypanosome infection stimulated T cell- and TNF-mediated iNOS expression in perivascular macrophages. NO nitrosylated and inactivated pro-inflammatory molecules such as NF-κΒp65, and reduced TNF expression and signalling. iNOS-derived NO hampered both TNF- and T cell-mediated parasite brain invasion. In inos^-/- mice, TNF stimulated MMP, including MMP9 activity that increased cerebral vessel permeability. Thus, iNOS-generated NO by perivascular macrophages, strategically located at sites of leukocyte brain penetration, can serve as a negative feed-back regulator that prevents unlimited influx of inflammatory cells by restoring the integrity of the blood-brain barrier.     

muO conotoxins inhibit NaV channels by interfering with their voltage sensors in domain-2

The muO-conotoxins MrVIA and MrVIB are 31-residue peptides from Conus marmoreus, belonging to the O-superfamily of conotoxins with three disulfide bridges. They have attracted attention because they are inhibitors of tetrodotoxin-insensitive voltage-gated sodium channels (Na(V)1.8) and could therefore serve as lead structure for novel analgesics. The aim of this study was to elucidate the molecular mechanism by which muO-conotoxins affect Na(V) channels. Rat Na(V)1.4 channels and mutants thereof were expressed in mammalian cells and were assayed with the whole-cell patch-clamp method. Unlike for the M-superfamily mu-conotoxin GIIIA from Conus geographus, channel block by MrVIA was strongly diminished after activating the Na(V) channels by depolarizing voltage steps. Searching for the source of this voltage dependence, the gating charges in all four-voltage sensors were reduced by site-directed mutagenesis showing that alterations of the voltage sensor in domain-2 have the strongest impact on MrVIA action. These results, together with previous findings that the effect of MrVIA depends on the structure of the pore-loop in domain-3, suggest a functional similarity with scorpion beta-toxins. In fact, MrVIA functionally competed with the scorpion beta-toxin Ts1 from Tityus serrulatus, while it did not show competition with mu-GIIIA. Ts1 and mu-GIIIA did not compete either. Thus, similar to scorpion beta-toxins, muO-conotoxins are voltage-sensor toxins targeting receptor site-4 on Na(V) channels. They "block" Na(+) flow most likely by hindering the voltage sensor in domain-2 from activating and, hence, the channel from opening.     

Warming and water deficit impact leaf photosynthesis and decrease forage quality and digestibility of a C4 tropical grass

Global warming is predicted to cause more intense extreme events such as heat waves, flooding and severe droughts, producing significant effects on agriculture. In tropics, climate change will severely impact livestock production affecting water availability, forage quality and food for cattle. We investigated the isolated and combined effects of soil water deficit (wS) and + 2°C increase in canopy temperature (eT) on leaf gas exchange, chlorophyll fluorescence, carbohydrate content, forage quality and in vitro dry matter digestibility (IVDMD) of a field‐grown C4 tropical forage grass Panicum maximum Jacq. using a temperature‐free air‐controlled enhancement (T‐FACE) system. The wS and eT treatments showed no effects on photosystem II photochemistry. However, wS under ambient temperature decreased net photosynthesis rate (A), stomatal conductance (g_s) and maximum rate of carboxylation of Rubisco (V_cmax), leading to a reduced starch content in leaves. A 16% reduction in leaf dry mass (LDM) and reduction in forage quality by increasing fibers, reducing crude protein (CP) and decreasing the IVDMD was also observed by effect of wS. Warming under adequate soil moisture (eT) significantly increased LDM by 25% but reduced the forage quality, increasing the lignin content and reducing starch, CP and digestibility. The combined wSeT treatment reduced A, g_s, V_cmax and the forage quality. When compared to control, the lignin content in leaves increased by 43, 28 and 17% in wS, eT and wSeT, respectively, causing a significant reduction in IVDMD. We concluded that despite physiological mechanisms to acclimate to warming, both warming and water deficit will impair the quality and digestibility of C4 tropical pastures.     

Neuroactive diol and acyloin metabolites from cone snail-associated bacteria

The bacterium Gordonia sp. 647 W.R.1a.05 was cultivated from the venom duct of the cone snail, Conus circumcisus. The Gordonia sp. organic extract modulated the action potential of mouse dorsal root ganglion neurons. Assay-guided fractionation led to the identification of the new compound circumcin A (1) and 11 known analogs (2–12). Two of these compounds, kurasoin B (7) and soraphinol A (8), were active in a human norepinephrine transporter assay with K_i values of 2575 and 867 nM, respectively. No neuroactivity had previously been reported for compounds in this structural class. Gordonia species have been reproducibly isolated from four different cone snail species, indicating a consistent association between these organisms.     

Helicobacter pylori urease activates blood platelets through a lipoxygenase‐mediated pathway

The bacterium Helicobacter pylori causes peptic ulcers and gastric cancer in human beings by mechanisms yet not fully understood. H. pylori produces urease which neutralizes the acidic medium permitting its survival in the stomach. We have previously shown that ureases from jackbean, soybean or Bacillus pasteurii induce blood platelet aggregation independently of their enzyme activity by a pathway requiring platelet secretion, activation of calcium channels and lipoxygenase‐derived eicosanoids. We investigated whether H. pylori urease displays platelet‐activating properties and defined biochemical pathways involved in this phenomenon. For that the effects of purified recombinant H. pylori urease (HPU) added to rabbit platelets were assessed turbidimetrically. ATP secretion and production of lipoxygenase metabolites by activated platelets were measured. Fluorescein‐labelled HPU bound to platelets but not to erythrocytes. HPU induced aggregation of rabbit platelets (ED₅₀ 0.28 μM) accompanied by ATP secretion. No correlation was found between platelet activation and ureolytic activity of HPU. Platelet aggregation was blocked by esculetin (12‐lipoxygenase inhibitor) and enhanced ～3‐fold by indomethacin (cyclooxygenase inhibitor). A metabolite of 12‐lipoxygenase was produced by platelets exposed to HPU. Platelet responses to HPU did not involve platelet‐activating factor, but required activation of verapamil‐inhibitable calcium channels. Our data show that purified H. pylori urease activates blood platelets at submicromolar concentrations. This property seems to be common to ureases regardless of their source (plant or bacteria) or quaternary structure (single, di‐ or tri‐chain proteins). These properties of HPU could play an important role in pathogenesis of gastrointestinal and associated cardiovascular diseases caused by H. pylori.     

Plasma membrane lipids in the resurrection plant Ramonda serbica following dehydration and rehydration

Plants of Ramonda serbica were dehydrated to 3.6% relative water content (RWC) by withholding water for 3 weeks, afterwards the plants were rehydrated for 1 week to 93.8% RWC. Plasma membranes were isolated from leaves using a two-phase aqueous polymer partition system. Compared with well-hydrated (control) leaves, dehydrated leaves suffered a reduction of about 75% in their plasma membrane lipid content, which returned to the control level following rewatering. Also the lipid to protein ratio decreased after dehydration, almost regaining the initial value after rehydration. Lipids extracted from the plasma membrane of fully-hydrated leaves were characterized by a high level of free sterols and a much lower level of phospholipids. Smaller amounts of cerebrosides, acylated steryl glycosides and steryl glycosides were also detected. The main phospholipids of control leaves were phosphatidylcholine and phosphatidylethanolamine, whereas sitosterol was the free sterol present in the highest amount. Following dehydration, leaf plasma membrane lipids showed a constant level of free sterols and a reduction in phospholipids compared with the well-hydrated leaves. Both phosphatidylcholine and phosphatidylethanolamine decreased following dehydration, their molar ratio remaining unchanged. Among free sterols, the remarkably high cholesterol level present in the control leaves (about 14 mol%) increased 2-fold as a result of dehydration. Dehydration caused a general decrease in the unsaturation level of individual phospholipids and total lipids as well. Upon rehydration the lipid composition of leaf plasma membranes restored very quickly approaching the levels of well-hydrated leaves.