Dispensable role of Drosophila ortholog of LRRK2 kinase activity in survival of dopaminergic neurons

Background

Parkinson's disease (PD) is the most prevalent incurable neurodegenerative movement disorder. Mutations in LRRK2 are associated with both autosomal dominant familial and sporadic forms of PD. LRRK2 encodes a large putative serine/threonine kinase with GTPase activity. Increased LRRK2 kinase activity plays a critical role in pathogenic LRRK2 mutant-induced neurodegeneration in vitro. Little is known about the physiological function of LRRK2.

Results

We have recently identified a Drosophila line with a P-element insertion in an ortholog gene of human LRRK2 (dLRRK). The insertion results in a truncated Drosophila LRRK variant with N-terminal 1290 amino acids but lacking C-terminal kinase domain. The homozygous mutant fly develops normally with normal life span as well as unchanged number and pattern of dopaminergic neurons. However, dLRRK mutant flies were selectively sensitive to hydrogen peroxide induced stress but not to paraquat, rotenone and β-mercaptoethanol induced stresses.

Conclusion

Our results indicate that inactivation of dLRRK kinase activity is not essential for fly development and suggest that inhibition of LRRK activity may serve as a potential treatment of PD. However, dLRRK kinase activity likely plays a role in protecting against oxidative stress.     

Unfertilized frog eggs die by apoptosis following meiotic exit

Background

GGAs (Golgi-localised, γ-ear containing, ADP ribosylation factor-binding) are a family of clathrin adaptors that sort a number of biologically important transmembrane proteins into clathrin-coated vesicles. Knockout and knockdown studies to determine GGA function are confounded by the fact that there are 3 GGA genes in mammalian cells. Thus Drosophila melanogaster is a useful model system to study tissue expression profiles and knockdown phenotypes as there is a single GGA ortholog.

Results

Here we have quantified protein expression in Drosophila and show that there is >3-fold higher expression of GGA in male flies relative to female flies. In female flies the majority of GGA expression is in the head. In male flies GGA is not only expressed at high levels in the head but there is a gender specific increased expression which is due to the abundant expression of GGA in the testes. Using a highly specific antibody we have localised endogenous GGA protein in testes squashes, and visualised it in somatic and germ line cells. We show that GGA is expressed during multiple stages of sperm development, and co-stains with a marker of the trans-Golgi Network. This is most striking at the acroblast of early spermatids. In spite of the high expression of GGA in testes, knocking down its expression by >95% using transgenic RNAi fly lines did not affect male fertility. Therefore spermatogenesis in the male flies appears to progress normally with <5% GGA, most likely because alternative adaptors may be able to substitute partially or completely for the function of GGA. We also identify 'cueball' as a novel cargo for GGA, and mutants of cueball have been shown to have a male sterility phenotype.

Conclusion

In Drosophila we have uncovered a potential role for GGA in the testes of male flies. The gender specific higher expression of GGA, its specific enrichment in testes and its localisation to developing spermatocytes and at the acroblast of spermatids supports a role for GGA function in Drosophila spermatogenesis, even though spermatogenesis still occurs when GGA expression is depleted to <5% of control.     

Fine-scale genetic breaks driven by historical range dynamics and ongoing density-barrier effects in the estuarine seaweed Fucus ceranoides L.

Background

In the laboratory, the Drosophila melanogaster heat shock protein Hsp90 can buffer the phenotypic effects of genetic variation. Laboratory experiments either manipulate Hsp90 activity pharmacologically, or they induce mutations with strong effects in the gene Hsp83, the single-copy fly gene encoding Hsp90. It is unknown whether observations from such laboratory experiments are relevant in the wild.

Results

We here study naturally occurring mutations in Hsp83, and their effects on fitness and phenotypic buffering in flies derived from wild populations. We examined more than 4500 flies from 42 Drosophila populations distributed world-wide for insertions or deletions of mobile DNA in or near the Hsp83 gene. The insertions we observed occur at low population frequencies, and reduce Hsp83 gene expression. In competition experiments, mutant flies performed much more poorly than wild-type flies. Mutant flies were also significantly less fecund and shorter-lived than wild-type flies, as well as less well buffered against cryptic deleterious variation, as we show through inbreeding experiments. Specifically, in Hsp83 mutant flies female fecundity dropped to much lower levels after inbreeding than in wild-type flies. At even slightly elevated temperatures, inbred mutant Hsp83 populations went extinct, whereas inbred wild-type populations persisted.

Conclusions

Our work shows that Hsp90, a regulator of the stress response and of signaling, helps buffer deleterious variation in fruit flies derived from wild population, and that its buffering role becomes even more important under heat stress.     

Genome-wide acceleration of protein evolution in flies (Diptera)

Background

The rate of molecular evolution varies widely between proteins, both within and among lineages. To what extent is this variation influenced by genome-wide, lineage-specific effects? To answer this question, we assess the rate variation between insect lineages for a large number of orthologous genes.

Results

When compared to the beetle Tribolium castaneum, we find that the stem lineage of flies and mosquitoes (Diptera) has experienced on average a 3-fold increase in the rate of evolution. Pairwise gene comparisons between Drosophila and Tribolium show a high correlation between evolutionary rates of orthologous proteins.

Conclusion

Gene specific divergence rates remain roughly constant over long evolutionary times, modulated by genome-wide, lineage-specific effects. Among the insects analysed so far, it appears that the Tribolium genes show the lowest rates of divergence. This has the practical consequence that homology searches for human genes yield significantly better matches in Tribolium than in Drosophila. We therefore suggest that Tribolium is better suited for comparisons between phyla than the widely employed dipterans.     

Isolation and characterization of a wheat IF2 homolog required for innate immunity to stripe rust

Key message

The wheat eIF2 homolog, TaIF2, is induced by the stripe rust pathogen CYR 32 at an early stage of inoculation and is related to the innate immunity resistance level in wheat.

Abstract

The initiation of translation represents a critical control point in the regulation of gene expression in all organisms. We previously identified an upregulated EST S186 (EL773056) from an SSH-cDNA library of the Shaanmai 139 strain of wheat (Triticum aestivum) infected with Puccinia striiformis (Pst). In the present work, we isolated a cDNA clone and identified it as a wheat IF2 homolog. This cDNA consisted of 1,314 nucleotides and contained an open reading frame of 795 nucleotides encoding a polypeptide of 254 amino acids. The amino acids represent a conserved domain in EF-Tu, mtIF2-II, and mtIF2-Ivc. The alignment result showed that it maybe a partial cDNA of the initiation factor 2/eukaryotic initiation factor 5B (IF2/eIF5B) superfamily gene. Paradoxically, results of a Swiss-model analysis suggesting a low QMEAN Z-score implied that it was a membrane protein. Quantitative RT-PCR studies confirmed that the wheat eIF2 (TaIF2) homolog was differentially expressed in three near-isogenic lines. Critical time points for the induction of resistance by inoculation with Pst CYR32 in YrSM139-1B + YrSM139-2D immune resistance genotype occurred at 1 and 3 dpi (days post-infection). RNAi test showed that the inoculated BSMV-IF2 leaves of Shaanmai 139 showed obvious cell death after 15 days of inoculation with CYR 32. qRT-PCR analysis of the target gene in cDNA samples isolated from BSMV-IF2-Pst, BSMV-0-Pst and Pst infected leaves confirmed that the expression of TaIF2 is suppressed by BSMV-IF2 at 3 dpi. This suggested that TaIF2/eIF5B plays an important role in the mechanism of innate immunity to stripe rust pathogen.     

Metabolism as means for hypoxia adaptation: metabolic profiling and flux balance analysis

Background  

Cellular hypoxia is a component of many diseases, but mechanisms of global hypoxic adaptation and resistance are not completely understood. Previously, a population of Drosophila flies was experimentally selected over several generations to survive a chronically hypoxic environment. NMR-based metabolomics, combined with flux-balance simulations of genome-scale metabolic networks, can generate specific hypotheses for global reaction fluxes within the cell. We applied these techniques to compare metabolic activity during acute hypoxia in muscle tissue of adapted versus "na?ve" control flies.     

Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity

Background

Eosinophils are involved in various inflammatory processes including allergic inflammation during which angiogenesis has been documented. Angiogenesis is most likely connected to the hypoxia which characterizes inflamed tissues. Eosinophils produce VEGF and are pro-angiogenic. However, to the best of our knowledge no study has been performed to verify the existence of a direct link between eosinophils, hypoxia and angiogenesis in allergic inflammation.

Objective

To characterize eosinophil function and angiogenic potential under hypoxic conditions.

Methods

Human peripheral blood eosinophils were cultured in normoxic or hypoxic conditions with or without cytokines. Viability and apoptosis were assessed by Annexin V/PI staining. Anti- or pro-apoptotic protein levels, HIF-1α levels and MAPK phosphorylation were analyzed by immunoblot analysis. Angiogenic mediator release was evaluated by ELISA.

Results

Hypoxic eosinophils were more viable than normoxic ones after up to three days. In addition in hypoxia, anti-apoptotic Bcl-XL protein levels increased more than pro-apoptotic Bax levels. Hypoxia increased VEGF and IL-8 release. In hypoxic eosinophils high levels of HIF-1α were observed, particularly in the presence of GM-CSF. MAPK, particularly ERK1/2 inhibitors, decreased hypoxia-mediated VEGF release and HIF-1α expression.

Conclusion

Eosinophils respond to hypoxia by up-regulation of survival and of some of their pro-angiogenic functions indicating a correlation between eosinophilic inflammation and angiogenesis.     

Proline accumulation has prevalence over polyamines in nodules of Medicago sativa in symbiosis with Sinorhizobium meliloti during the initial response to salinity

Background and aims

Polyamines are cationic molecules that play an important role in the plant response to environmental stresses. The aim of this work is to determine the role of these compounds in the response to salinity of Medicago sativa plants in symbiosis with the soil bacteria Sinorhizobium meliloti.

Methods

M. sativa plants inoculated with S. meliloti were subjected to 100 and 150 mM NaCl treatments. The concentration of nodular polyamines was determined in relation to the nitrogen fixation parameters, proline accumulation, and oxidative damage. In addition, polyamines concentrations were analyzed in different nodular fractions as well as the effect of exogenous polyamines in the nodulation response.

Results

The concentration of nodular polyamines decreased by the salinity in correlation with the nitrogenase activity after 2 and 4 weeks of salt treatment while spermine accumulated after 6 weeks. On the contrary, proline accumulation was induced by the salinity at all time points. The analysis of different nodular fractions showed the highest polyamines concentration in bacteroids being homospermidine the most abundant.

Conclusion

Proline accumulation had prevalence over polyamines at the earliest response to salinity probably due to nitrogen limitation under salt stress conditions and the existence of a common precursor for both compounds in the nodule. Nevertheless, after long salt exposure, spermine was also accumulated. The analysis of different nodular fractions indicated the bacteroidal origin of polyamines in nodules being homoespermidine, one of the most abundant.     

A molecular diffusion based utility model for Drosophila larval phototaxis

Background

Generally, utility based decision making models focus on experimental outcomes. In this paper we propose a utility model based on molecular diffusion to simulate the choice behavior of Drosophila larvae exposed to different light conditions.

Methods

In this paper, light/dark choice-based Drosophila larval phototaxis is analyzed with our molecular diffusion based model. An ISCEM algorithm is developed to estimate the model parameters.

Results

By applying this behavioral utility model to light intensity and phototaxis data, we show that this model fits the experimental data very well.

Conclusions

Our model provides new insights into decision making mechanisms in general. From an engineering viewpoint, we propose that the model could be applied to a wider range of decision making practices.     

Identification and application of amino acids as chelators in phytoremediation of rare earth elements lanthanum and yttrium

Aims

This study aimed to identify amino acids that could act as chelators in enhancing absorption and translocation capabilities of a rare earth element (REE) lanthanum and yttrium in a non-hyperaccumulator plant.

Methods

We analysed correlations between amino acid, La and Y concentrations in xylem saps of the REE hyperaccumulator Phytolacca americana L., to identify functional amino acids in long-distance transportation processes. These were used as chelators to observe the efficacy of La uptake and translocation in REE non-hyperaccumulator tomato seedlings. Pot culture experiments were conducted using modified Hoagland solution artificially contaminated with REEs.

Results

Eighteen xylem sap amino acids were identified and measured in the xylem sap, using reversed-phase high-performance liquid chromatography. Aspartic acid, asparagine, histidine and glutamic acid may be related to xylem La and Y long-distance transportion in P. americana L. Extraneous aspartic acid, asparagine, histidine and glutamic acid enhanced La absorption in the whole tomato, compared with La alone. Moreover, the whole tomato La content increased by 449 μg and 139 μg in the presence of aspartic acid and asparagine as compared to P. americana L. Tryptophan insignificantly affected La uptake and translocation in tomato seedlings.

Conclusions

Aspartic acid and asparagine could potentially promote remediation of La contamination in soil when used as a chelator.     

Loss of the bloom syndrome helicase increases DNA ligase 4-independent genome rearrangements and tumorigenesis in aging Drosophila

Background

The BLM DNA helicase plays a vital role in maintaining genome stability. Mutations in BLM cause Bloom syndrome, a rare disorder associated with cancer predisposition and premature aging. Humans and mice with blm mutations have increased frequencies of spontaneous mutagenesis, but the molecular basis of this increase is not well understood. In addition, the effect of aging on spontaneous mutagenesis in blm mutants has not been characterized. To address this, we used a lacZ reporter system in wild-type and several mutant strains of Drosophila melanogaster to analyze mechanisms of mutagenesis throughout their lifespan.

Results

Our data show that Drosophila lacking BLM have an elevated frequency of spontaneous genome rearrangements that increases with age. Although in normal flies most genome rearrangements occur through DNA ligase 4-dependent classical end joining, most rearrangements that accumulate during aging in blm mutants do not require DNA ligase 4, suggesting the influence of an alternative end-joining mechanism. Adult blm mutants also display reduced lifespan and ligase 4-independent enhanced tumorigenesis in mitotically active tissues.

Conclusions

These results suggest that Drosophila BLM suppresses error-prone alternative end-joining repair of DNA double-strand breaks that can result in genome instability and tumor formation during aging. In addition, since loss of BLM significantly affects lifespan and tumorigenesis, the data provide a link between error-prone end joining, genome rearrangements, and tumor formation in a model metazoan.     

Bone marrow-derived fibroblast growth factor-2 induces glial cell proliferation in the regenerating peripheral nervous system

Backgound

Amyotrophic lateral sclerosis (ALS) is progressive neurodegenerative disease characterized by the loss of motor function. Several ALS genes have been identified as their mutations can lead to familial ALS, including the recently reported RNA-binding protein fused in sarcoma (Fus). However, it is not clear how mutations of Fus lead to motor neuron degeneration in ALS. In this study, we present a Drosophila model to examine the toxicity of Fus, its Drosophila orthologue Cabeza (Caz), and the ALS-related Fus mutants.

Results

Our results show that the expression of wild-type Fus/Caz or FusR521G induced progressive toxicity in multiple tissues of the transgenic flies in a dose- and age-dependent manner. The expression of Fus, Caz, or FusR521G in motor neurons significantly impaired the locomotive ability of fly larvae and adults. The presynaptic structures in neuromuscular junctions were disrupted and motor neurons in the ventral nerve cord (VNC) were disorganized and underwent apoptosis. Surprisingly, the interruption of Fus nuclear localization by either deleting its nuclear localization sequence (NLS) or adding a nuclear export signal (NES) blocked Fus toxicity. Moreover, we discovered that the loss of caz in Drosophila led to severe growth defects in the eyes and VNCs, caused locomotive disability and NMJ disruption, but did not induce apoptotic cell death.

Conclusions

These data demonstrate that the overexpression of Fus/Caz causes in vivo toxicity by disrupting neuromuscular junctions (NMJs) and inducing apoptosis in motor neurons. In addition, the nuclear localization of Fus is essential for Fus to induce toxicity. Our findings also suggest that Fus overexpression and gene deletion can cause similar degenerative phenotypes but the underlying mechanisms are likely different.     

Evaluation of reference genes for real-time RT-PCR expression studies in the plant pathogen Pectobacterium atrosepticum

Background

Translation initiation factors of the 4E and 4G protein families mediate resistance to several RNA plant viruses in the natural diversity of crops. Particularly, a single point mutation in melon eukaryotic translation initiation factor 4E (eIF4E) controls resistance to Melon necrotic spot virus (MNSV) in melon. Identification of allelic variants within natural populations by EcoTILLING has become a rapid genotype discovery method.

Results

A collection of Cucumis spp. was characterised for susceptibility to MNSV and Cucumber vein yellowing virus (CVYV) and used for the implementation of EcoTILLING to identify new allelic variants of eIF4E. A high conservation of eIF4E exonic regions was found, with six polymorphic sites identified out of EcoTILLING 113 accessions. Sequencing of regions surrounding polymorphisms revealed that all of them corresponded to silent nucleotide changes and just one to a non-silent change correlating with MNSV resistance. Except for the MNSV case, no correlation was found between variation of eIF4E and virus resistance, suggesting the implication of different and/or additional genes in previously identified resistance phenotypes. We have also characterized a new allele of eIF4E from Cucumis zeyheri, a wild relative of melon. Functional analyses suggested that this new eIF4E allele might be responsible for resistance to MNSV.

Conclusion

This study shows the applicability of EcoTILLING in Cucumis spp., but given the conservation of eIF4E, new candidate genes should probably be considered to identify new sources of resistance to plant viruses. Part of the methodology described here could alternatively be used in TILLING experiments that serve to generate new eIF4E alleles.     

Effects of bone marrow-derived cells on monocrotaline- and hypoxia-induced pulmonary hypertension in mice

Background

Bone marrow -derived cells (BMDCs) can either limit or contribute to the process of pulmonary vascular remodeling. Whether the difference in their effects depends on the mechanism of pulmonary hypertension (PH) remains unknown.

Objectives

We investigated the effect of BMDCs on PH induced in mice by either monocrotaline or exposure to chronic hypoxia.

Methods

Intravenous administration of the active monocrotaline metabolite (monocrotaline pyrrole, MCTp) to C57BL/6 mice induced PH within 15 days, due to remodeling of small distal vessels. Three days after the MCTp injection, the mice were injected with BMDCs harvested from femurs and tibias of donor mice treated with 5-fluorouracil (3.5 mg IP/animal) to deplete mature cells and to allow proliferation of progenitor cells.

Results

BMDCs significantly attenuated PH as assessed by reductions in right ventricular systolic pressure (20 ± 1 mmHg vs. 27 ± 1 mmHg, P ≤ 0.01), right ventricle weight/left ventricle+septum weight ratio (0.29 ± 0.02 vs. 0.36 ± 0.01, P ≤ 0.03), and percentage of muscularized vessels (26.4% vs. 33.5%, P ≤ 0.05), compared to control animals treated with irradiated BMDCs. Tracking cells from constitutive GFP-expressing male donor mice with anti-GFP antibodies or chromosome Y level measurement by quantitative real-time PCR showed BMDCs in the lung. In contrast, chronically hypoxic mice subjected to the same procedure failed to show improvement in PH.

Conclusion

These results show that BMDCs limit pulmonary vascular remodeling induced by vascular injury but not by hypoxia.