Metabolic profiling of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> metamorphosis: a new insight into the central metabolic pathways

Introduction

Metamorphosis is a complicated process in which cell proliferation, differentiation, and death are orchestrated to form the mature structures of insects. In Drosophila, this process is controlled by ecdysone, a steroid hormone responsible for tissue remodeling and organogenesis that gives rise to the adult fly.

Objective

By using a metabolomics approach, this study aimed to elucidate global changes in the central metabolic pathways in Drosophila throughout metamorphosis and then further examine the effects of temperature and origin on metabolic profiles.

Methods

Targeted and non-targeted metabolic profiling of time-course samples from Drosophila were constructed to cover a wide range of cellular metabolites during metamorphosis.

Results

This was the first wide-scale metabolomics study of Drosophila metamorphosis focusing on central metabolism. The abundance of detected metabolites changed drastically and correlated strongly with the development of Drosophila pupae. In non-stress conditions, temperature affected the developmental time, but the metabolic state at a certain stage of metamorphosis remained stable. Between D. melanogaster Canton S and Oregon R, similar metabolic profiles throughout metamorphosis was observed. However, there were still differences in purine and pyrimidine metabolism at an early stage in the pupal period, which was matched by differences in ecdysteroid levels.

Conclusion

This study supported the strength of metabolomics in the field of developmental biology. The results provided a general view on the metabolic profile of Drosophila during metamorphosis, which provides basic 3 knowledge for future metabolomics studies using Drosophila.

     

Testis-specific ATP synthase peripheral stalk subunits required for tissue-specific mitochondrial morphogenesis in <Emphasis Type="Italic">Drosophila</Emphasis>

Background

In Drosophila early post-meiotic spermatids, mitochondria undergo dramatic shaping into the Nebenkern, a spherical body with complex internal structure that contains two interwrapped giant mitochondrial derivatives. The purpose of this study was to elucidate genetic and molecular mechanisms underlying the shaping of this structure.

Results

The knotted onions (knon) gene encodes an unconventionally large testis-specific paralog of ATP synthase subunit d and is required for internal structure of the Nebenkern as well as its subsequent disassembly and elongation. Knon localizes to spermatid mitochondria and, when exogenously expressed in flight muscle, alters the ratio of ATP synthase complex dimers to monomers. By RNAi knockdown we uncovered mitochondrial shaping roles for other testis-expressed ATP synthase subunits.

Conclusions

We demonstrate the first known instance of a tissue-specific ATP synthase subunit affecting tissue-specific mitochondrial morphogenesis. Since ATP synthase dimerization is known to affect the degree of inner mitochondrial membrane curvature in other systems, the effect of Knon and other testis-specific paralogs of ATP synthase subunits may be to mediate differential membrane curvature within the Nebenkern.

     

Total and active microbial communities and <Emphasis Type="Italic">phoD</Emphasis> as affected by phosphate depletion and pH in soil

Background and Aims

Soil microbial communities contribute to organic phosphorus cycling in a variety of ways, including secretion of the PhoD alkaline phosphatase. We sampled a long-term grassland fertilization trial in Switzerland characterized by a natural pH gradient. We examined the effects of phosphate depletion and pH on total and active microbial community structures and on the structure and composition of the total and active phoD-harboring community.

Methods

Archaeal, bacterial and fungal communities were investigated using T-RFLP and phoD-harboring members of these communities were identified by 454-sequencing.

Results

Phosphate depletion decreased total, resin-extractable and organic phosphorus and changed the structure of all active microbial communities, and of the total archaeal and phoD-harboring communities. Organic carbon, nitrogen and phosphorus increased with pH, and the structures of all total and active microbial communities except the total fungal community differed between the two pH levels. phoD-harboring members were affiliated to Actinomycetales, Bacilliales, Gloeobacterales, Planctomycetales and Rhizobiales.

Conclusions

Our results suggest that pH and associated soil factors are important determinants of microbial and phoD-harboring community structures. These associated factors include organic carbon and total nitrogen, and to a lesser degree phosphorus status, and active communities are more responsive than total communities. Key players in organic P mineralization are affiliated to phyla that are known to be important in organic matter decomposition.

     

Efficient expression of functional human coagulation factor IX in stably-transfected <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> S2 cells; comparison with the mammalian CHO system

Objective

To compare stably-transfected Drosophila melanogaster S2 and mammalian Chinese hamster ovary (CHO) cells for the expression and secretion efficiency of biologically-active human coagulation factor IX (hFIX).

Result

Selection of an hFIX-expressing cell line derived from stably-transfected S2 cells was performed over 2 weeks, while the same procedure required 2 months for stably-transfected CHO cells. Furthermore, the selected S2 cell line was superior in producing of total hFIX protein (70 % increase), biologically-active hFIX (35 % increase), and specific hFIX activity (20 % increase) relative to the selected CHO cell line. Enrichment for functional, fully γ-carboxylated hFIX species via barium citrate adsorption demonstrated that up to 90 % of the hFIX expressed by S2 cells was γ-carboxylated versus 79 % of CHO-expressed hFIX. Inhibition of N-glycosylation by tunicamycin indicated that N-glycosylation of S2-expressed hFIX had occurred to a similar extent as in the CHO-produced hFIX.

Conclusion

The Drosophila S2 cell system is an attractive candidate for the efficient production of recombinant hFIX as it has the potential of significantly reducing the cell development time, while producing functional hFIX.

     

Histone acetyltransferase CBP-related H3K23 acetylation contributes to courtship learning in <Emphasis Type="Italic">Drosophila</Emphasis>

Background

Histone modifications are critical in regulating neuronal processes. However, the impacts of individual histone modifications on learning and memory are elusive. Here, we investigated the contributions of histone H3 lysine modifications to learning and memory in Drosophila by using histone lysine-to-alanine mutants.

Results

Behavioural analysis indicated that compared to the H3WT group, mutants overexpressing H3K23A displayed impaired courtship learning. Chromatin immunoprecipitation analysis of H3K23A mutants showed that H3K23 acetylation (H3K23ac) levels were decreased on learning-related genes. Knockdown of CREB-binding protein (CBP) decreased H3K23ac levels, attenuated the expression of learning-related genes, led to a courtship learning defect and altered development of the mushroom bodies. A decline in courtship learning ability was observed in both larvae and adult treatments with ICG-001. Furthermore, treatment of Drosophila overexpressing mutated H3K23A with a CBP inhibitor did not aggravate the learning defect.

Conclusions

H3K23ac, catalysed by the acetyltransferases dCBP, contributes to Drosophila learning, likely by controlling the expression of specific genes. This is a novel epigenetic regulatory mechanism underlying neuronal behaviours.

     

Expression of <Emphasis Type="Italic">Ciona intestinalis</Emphasis> AOX causes male reproductive defects in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background

Mitochondrial alternative respiratory-chain enzymes are phylogenetically widespread, and buffer stresses affecting oxidative phosphorylation in species that possess them. However, they have been lost in the evolutionary lineages leading to vertebrates and arthropods, raising the question as to what survival or reproductive disadvantages they confer. Recent interest in using them in therapy lends a biomedical dimension to this question.

Methods

Here, we examined the impact of the expression of Ciona intestinalis alternative oxidase, AOX, on the reproductive success of Drosophila melanogaster males. Sperm-competition assays were performed between flies carrying three copies of a ubiquitously expressed AOX construct, driven by the α-tubulin promoter, and wild-type males of the same genetic background.

Results

In sperm-competition assays, AOX conferred a substantial disadvantage, associated with decreased production of mature sperm. Sperm differentiation appeared to proceed until the last stages, but was spatially deranged, with spermatozoids retained in the testis instead of being released to the seminal vesicle. High AOX expression was detected in the outermost cell-layer of the testis sheath, which we hypothesize may disrupt a signal required for sperm maturation.

Conclusions

AOX expression in Drosophila thus has effects that are deleterious to male reproductive function. Our results imply that AOX therapy must be developed with caution.

     

Identification of a novel <Emphasis Type="Italic">Drosophila</Emphasis> gene, <Emphasis Type="Italic">beltless</Emphasis>, using injectable embryonic and adult RNA interference (RNAi)

Background

RNA interference (RNAi) is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly) gene (corresponding to a putative gene CG5652/GM06434), that we named beltless based on an embryonic loss-of-function phenotype.

Results

Beltless mRNA is expressed in all developmental stages except in 0–6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp) beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless) of the abdominal segments A2–A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1)RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1)RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts.

Conclusions

We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF) NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should elucidate the role and mechanism of action of beltless during Drosophila development and in adults, including in the adult nervous system.

     

Mutational analysis of COL1A1 and COL1A2 genes among Estonian osteogenesis imperfecta patients

Background

Osteogenesis imperfecta (OI) is a rare bone disorder. In 90% of cases, OI is caused by mutations in the COL1A1/2 genes, which code procollagen α1 and α2 chains. The main aim of the current research was to identify the mutational spectrum of COL1A1/2 genes in Estonian patients. The small population size of Estonia provides a unique chance to explore the collagen I mutational profile of 100% of OI families in the country.

Methods

We performed mutational analysis of peripheral blood gDNA of 30 unrelated Estonian OI patients using Sanger sequencing of COL1A1 and COL1A2 genes, including all intron-exon junctions and 5′UTR and 3′UTR regions, to identify causative OI mutations.

Results

We identified COL1A1/2 mutations in 86.67% of patients (26/30). 76.92% of discovered mutations were located in the COL1A1 (n = 20) and 23.08% in the COL1A2 (n = 6) gene. Half of the COL1A1/2 mutations appeared to be novel. The percentage of quantitative COL1A1/2 mutations was 69.23%. Glycine substitution with serine was the most prevalent among missense mutations. All qualitative mutations were situated in the chain domain of pro-α1/2 chains.

Conclusion

Our study shows that among the Estonian OI population, the range of collagen I mutations is quite high, which agrees with other described OI cohorts of Northern Europe. The Estonian OI cohort differs due to the high number of quantitative variants and simple missense variants, which are mostly Gly to Ser substitutions and do not extend the chain domain of COL1A1/2 products.

     

An integrated approach of bioinformatic prediction and in vitro analysis identified that miR-34a targets <Emphasis Type="Italic">MET</Emphasis> and <Emphasis Type="Italic">AXL</Emphasis> in triple-negative breast cancer

Background

Breast cancer is the most prevalent cancer among women, and AXL and MET are the key genes in the PI3K/AKT/mTOR pathway as critical elements in proliferation and invasion of cancer cells. MicroRNAs (miRNAs) are small non-coding RNAs regulating the expression of genes.

Methods

Bioinformatic approaches were used to find a miRNA that simultaneously targets both AXL and MET 3′-UTRs. The expression of target miRNA was evaluated in triple-negative (MDA-MB-231) and HER2-overexpressing (SK-BR-3) breast cancer cell lines as well as normal breast cells, MCF-10A, using quantitative real-time PCR. Then, the miRNA was overexpressed in normal and cancer cell lines using a lentiviral vector system. Afterwards, effects of overexpressed miRNA on the expression of AXL and MET genes were evaluated using quantitative real-time PCR.

Results

By applying bioinformatic software and programs, miRNAs that target the 3′-UTR of both AXL and MET mRNAs were determined, and according to the scores, miR-34a was selected for further analyses. The expression level of miR-34a in MDA-MB-231 and SK-BR-3 was lower than that of MCF-10A. Furthermore, AXL and MET expression in SK-BR-3 and MDA-MB-231 was lower and higher, respectively, than that of MCF-10A. After miR-34a overexpression, MET and AXL were downregulated in MDA-MB-231. In addition, MET was downregulated in SK-BR-3, while AXL was upregulated in this cell line.

Conclusions

These findings may indicate that miR-34a is an oncogenic miRNA, downregulated in the distinct breast cancer subtypes. It also targets MET and AXL 3′-UTRs in triple-negative breast cancer. Therefore, it can be considered as a therapeutic target in this type of breast cancer.

     

Various <Emphasis Type="Italic">Wolbachia</Emphasis> genotypes differently influence host <Emphasis Type="Italic">Drosophila</Emphasis> dopamine metabolism and survival under heat stress conditions

Background

One of the most widespread prokaryotic symbionts of invertebrates is the intracellular bacteria of Wolbachia genus which can be found in about 50% of insect species. Wolbachia causes both parasitic and mutualistic effects on its host that include manipulating the host reproductive systems in order to increase their transmission through the female germline, and increasing the host fitness. One of the mechanisms, promoting adaptation in biological organisms, is a non-specific neuroendocrine stress reaction. In insects, this reaction includes catecholamines, dopamine, serotonin and octopamine, which act as neurotransmitters, neuromodulators and neurohormones. The level of dopamine metabolism correlates with heat stress resistance in Drosophila adults.

Results

To examine Wolbachia effect on Drosophila survival under heat stress and dopamine metabolism we used five strains carrying the nuclear background of interbred Bi90 strain and cytoplasmic backgrounds with different genotype variants of Wolbachia (produced by 20 backcrosses of Bi90 males with appropriate source of Wolbachia). Non-infected Bi90 strain (treated with tetracycline for 3 generations) was used as a control group. We demonstrated that two of five investigated Wolbachia variants promote changes in Drosophila heat stress resistance and activity of enzymes that produce and degrade dopamine, alkaline phosphatase and dopamine-dependent arylalkylamine N-acetyltransferase. What is especially interesting, wMelCS genotype of Wolbachia increases stress resistance and the intensity of dopamine metabolism, whereas wMelPop strain decreases them. wMel, wMel2 and wMel4 genotypes of Wolbachia do not show any effect on the survival under heat stress or dopamine metabolism. L-DOPA treatment, known to increase the dopamine content in Drosophila, levels the difference in survival under heat stress between all studied groups.

Conclusions

The genotype of symbiont determines the effect that the symbiont has on the stress resistance of the host insect.

     

Developmental nicotine exposure affects larval brain size and the adult dopaminergic system of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background

Pregnant women may be exposed to nicotine if they smoke or use tobacco products, nicotine replacement therapy, or via e-cigarettes. Prenatal nicotine exposure has been shown to have deleterious effects on the nervous system in mammals including changes in brain size and in the dopaminergic system. The genetic and molecular mechanisms for these changes are not well understood. A Drosophila melanogaster model for these effects of nicotine exposure could contribute to faster identification of genes and molecular pathways underlying these effects. The purpose of this study was to determine if developmental nicotine exposure affects the nervous system of Drosophila melanogaster, focusing on changes to brain size and the dopaminergic system at two developmental stages.

Results

We reared flies on control or nicotine food from egg to 3rd instar larvae or from egg to adult and determined effectiveness of the nicotine treatment. We used immunohistochemistry to visualize the whole brain and dopaminergic neurons, using tyrosine hydroxylase as the marker. We measured brain area, tyrosine hydroxylase fluorescence, and counted the number of dopaminergic neurons in brain clusters.We detected an increase in larval brain hemisphere area, a decrease in tyrosine hydroxylase fluorescence in adult central brains, and a decrease in the number of neurons in the PPM3 adult dopaminergic cluster. We tested involvement of Dα7, one of the nicotinic acetylcholine receptor subunits, and found it was involved in eclosion, as previously described, but not involved in brain size.

Conclusions

We conclude that developmental nicotine exposure in Drosophila melanogaster affects brain size and the dopaminergic system. Prenatal nicotine exposure in mammals has also been shown to have effects on brain size and in the dopaminergic system. This study further establishes Drosophila melanogaster as model organism to study the effects of developmental nicotine exposure. The genetic and molecular tools available for Drosophila research will allow elucidation of the mechanisms underlying the effects of nicotine exposure during development.

     

<Emphasis Type="Italic">In silico</Emphasis> characterization and Molecular modeling of double-strand break repair protein <Emphasis Type="Italic">MRE11</Emphasis> from <Emphasis Type="Italic">Phoenix dactylifera</Emphasis> v deglet nour

Background

DNA double-strand breaks (DSBs) are highly cytotoxic and mutagenic. MRE11 plays an essential role in repairing DNA by cleaving broken ends through its 3′ to 5′ exonuclease and single-stranded DNA endonuclease activities.

Methods

The present study aimed to in silico characterization and molecular modeling of MRE11 from Phoenix dactylifera L cv deglet nour (DnMRE11) by various bioinformatic approaches. To identify DnMRE11 cDNA, assembled contigs from our cDNA libraries were analysed using the Blast2GO2.8 program.

Results

The DnMRE11 protein length was 726 amino acids. The results of HUMMER show that DnMRE11 is formed by three domains: the N-terminal core domain containing the nuclease and capping domains, the C-terminal half containing the DNA binding and coiled coil region. The structure of DnMRE11 is predicted using the Swiss-Model server, which contains the nuclease and capping domains. The obtained model was verified with the structure validation programs such as ProSA and QMEAN servers for reliability. Ligand binding studies using COACH indicated the interaction of DnMRE11 protein with two Mn²⁺ ions and dAMP. The ConSurf server predicted that residues of the active site and Nbs binding site have high conservation scores between plant species.

Conclusions

A model structure of DnMRE11 was constructed and validated with various bioinformatics programs which suggested the predicted model to be satisfactory. Further validation studies were conducted by COACH analysis for active site ligand prediction, and revealed the presence of six ligands binding sites and two ligands (2 Mn²⁺ and dAMP).

     

Improved process conditions for increasing expression of MHC class II protein from a stable <Emphasis Type="Italic">Drosophila</Emphasis> S2 cell line

Objectives

To investigate the effects of operational process conditions on expression of MHC class II protein from a stable Drosophila S2 cell line.

Results

When the Drosophila S2 cells were grown in vented orbitally shaken TubeSpin bioreactor 600 containers, cell growth was improved three-fold and the yield of recombinant major histocompatibility (MHC) class II protein (HLA-DR1_2xHis) increased four-fold over the levels observed for the same cells cultivated in roller bottles (RB) without vented caps. Culturing in RB with vented caps while increasing the rotation speed from 6 rpm to 18 rpm also improved cell growth five-fold and protein productivity three-fold which is comparable to the levels observed in the orbitally shaken containers. Protein activity was found to be almost identical between the two vessel systems tested.

Conclusions

Optimized cell culture conditions and a more efficient vessel type can enhance gas transfer and mixing and lead to substantial improvement of recombinant product yields from S2 cells.

     

A thermostable pyrimidine nucleoside phosphorylase from <Emphasis Type="Italic">Brevibacillus borstelensis</Emphasis> LK01 for synthesizing halogenated nucleosides

Objective

To isolate a thermostable pyrimidine nucleoside phosphorylase (PyNP) from mesophilic bacteria by gene mining.

Results

BbPyNP from Brevibacillus borstelensis LK01 was isolated by gene mining. BbPyNP had a highest 60% identity with that of reported PyNPs. BbPyNP could catalyze the phosphorolysis of thymidine, 2′-deoxyuridine, uridine and 5-methyuridine. BbPyNP had good thermostability and retained 73% of its original activity after 2 h incubation at 50 °C. BbPyNP had the highest activity at an optimum alkaline pH of 8.5. BbPyNP was stable from pH 7 to 9.8. Under preliminary optimized conditions, the biosynthesis of various 5-halogenated pyrimidine nucleosides by BbPyNP reached the yield of 61–84%.

Conclusion

An efficient approach was estimated in isolating thermostable PyNP from mesophilic bacteria.