CTP biosynthesis is carried out by two pathways: salvage and de novo. CTPsyn catalyzes the latter. The study of CTPsyn activity in mammalian cells began in the 1970s, and various fascinating discoveries were made regarding the role of CTPsyn in cancer and development. However, its ability to fit into a cellular serpent-like structure, termed 'cytoophidia,' was only discovered a decade ago by three independent groups of scientists. Although the self-assembly of CTPsyn into a filamentous structure is evolutionarily conserved, the enzyme activity upon this self-assembly varies in different species. CTPsyn is required for cellular development and homeostasis. Changes in the expression of CTPsyn cause developmental changes in Drosophila melanogaster. A high level of CTPsyn activity and formation of cytoophidia are often observed in rapidly proliferating cells such as in stem and cancer cells. Meanwhile, the deficiency of CTPsyn causes severe immunodeficiency leading to immunocompromised diseases caused by bacteria, viruses, and parasites, making CTPsyn an attractive therapeutic target. Here, we provide an overview of the role of CTPsyn in cellular and disease perspectives along with its potential as a drug target.
Alzheimer's disease (AD) is the most common neurological ailment worldwide. Its process comprises the unique aggregation of extracellular senile plaques composed of amyloid-beta (Aβ) in the brain. Aβ42 is the most neurotoxic and aggressive of the Aβ42 isomers released in the brain. Despite much research on AD, the complete pathophysiology of this disease remains unknown. Technical and ethical constraints place limits on experiments utilizing human subjects. Thus, animal models were used to replicate human diseases. The Drosophila melanogaster is an excellent model for studying both physiological and behavioural aspects of human neurodegenerative illnesses. Here, the negative effects of Aβ42-expression on a Drosophila AD model were investigated through three behavioural assays followed by RNA-seq. The RNA-seq data was verified using qPCR. AD Drosophila expressing human Aβ42 exhibited degenerated eye structures, shortened lifespan, and declined mobility function compared to the wild-type Control. RNA-seq revealed 1496 genes that were differentially expressed from the Aβ42-expressing samples against the control. Among the pathways that were identified from the differentially expressed genes include carbon metabolism, oxidative phosphorylation, antimicrobial peptides, and longevity-regulating pathways. While AD is a complicated neurological condition whose aetiology is influenced by a number of factors, it is hoped that the current data will be sufficient to give a general picture of how Aβ42 influences the disease pathology. The discovery of molecular connections from the current Drosophila AD model offers fresh perspectives on the usage of this Drosophila which could aid in the discovery of new anti-AD medications.
Drosophila melanogaster glutathione S-transferase D3 (DmGSTD3) has a shorter amino acid sequence as compared to other GSTs known in the fruit flies. This is due to the 15 amino acid N-terminal truncation in which normally active amino acid residue is located. The work has made use of homology modeling to visualize the arrangement of amino acid side chains in the glutathione (GSH) substrate cavity. The identified amino acids were then replaced with amino acids without functional groups in the side chains and the mutants were analyzed kinetically. Homology modeling revealed that the side chains of Y89 and Y97 were shown facing toward the substrate cavity proposing their possible role in catalyzing the conjugation. Y97A and Y89A GSH gave large changes in Km (twofold increase), Vmax (fivefold reduction), and Kcat /Km values for GSH suggesting their significant role in the conjugation reaction. The replacement at either positions has not affected the affinity of the enzyme toward 1-chloro-2,4-dinitrobenzene as no significant change in values of Kmax was observed. The replacement, however, had significantly reduced the catalytic efficiency of both mutants with (Kcat /Km )(GSH) and (Kcat /Km )(CDNB) of eight- and twofold reduction. The recombinant DmGSTD3 has shown no activity toward 1,2-dichloro-4-nitrobenzene, 2,4-hexadienal, 2,4-heptadienal, p-nitrobenzyl chloride, ethacrynic acid, and sulfobromophthalein. Therefore, it was evident that DmGSTD3 has made use of distal amino acids Y97 and Y89 for GSH conjugation.
Alzheimer's disease (AD) is the most common neurodegenerative condition in civilizations worldwide. The distinctive occurrence of amyloid-beta (Aβ) accumulation into insoluble fibrils is part of the disease pathophysiology with Aβ42 being the most toxic and aggressive Aβ species. The polyphenol, p-Coumaric acid (pCA), has been known to boost a number of therapeutic benefits. Here, pCA's potential to counteract the negative effects of Aβ42 was investigated. First, pCA was confirmed to reduce Aβ42 fibrillation using an in vitro activity assay. The compound was next examined on Aβ42-exposed PC12 neuronal cells and was found to significantly decrease Aβ42-induced cell mortality. pCA was then examined using an AD Drosophila melanogaster model. Feeding of pCA partially reversed the rough eye phenotype, significantly lengthened AD Drosophila's lifespan, and significantly enhanced the majority of the AD Drosophila's mobility in a sex-dependent manner. The findings of this study suggest that pCA may have therapeutic benefits for AD.
The steady decline of physiological function and increased vulnerability to age-related disorders are two features of the complicated biological process of ageing. As a key organ for nutrient absorption, metabolism, and immunological regulation, the gut plays a major part in the ageing process. Drosophila melanogaster, a well-established model organism, has emerged as a significant tool for exploring the intricate rapport between the gut and ageing. Through the use of Drosophila models, the physiological and molecular elements of the gut-brain axis have been thoroughly explored. These models have also provided insights into the mechanisms by which gut health impacts ageing and age-related illnesses. Drosophila's gut microbiota experience dysbiosis with age which has been linked to age-related diseases. To prevent this and promote healthy ageing in Drosophila, gut microbiota modification methods, such as dietary restriction in tandem with time-restricted feeding, administration of pro-, pre- and synbiotics, as well as pharmaceutical interventions have been generated with positive impacts. The article also covers the drawbacks and difficulties of investigating the gut via the Drosophila. Thus, with an emphasis on the lessons discovered from Drosophila research, this review provides an extensive description of the current studies on the role of the gut-brain axis in ageing and health.
The ability to cope with infection by a parasite is one of the major challenges for any host species and is a major driver of evolution. Parasite pressure differs between habitats. It is thought to be higher in tropical regions compared to temporal ones. We infected Drosophila melanogaster from two tropical (Malaysia and Zimbabwe) and two temperate populations (the Netherlands and North Carolina) with the generalist entomopathogenic fungus Beauveria bassiana to examine if adaptation to local parasite pressures led to differences in resistance. Contrary to previous findings we observed increased survival in temperate populations. This, however, is not due to increased resistance to infection per se, but rather the consequence of a higher general vigor of the temperate populations. We also assessed transcriptional response to infection within these flies eight and 24 hours after infection. Only few genes were induced at the earlier time point, most of which are involved in detoxification. In contrast, we identified more than 4,000 genes that changed their expression state after 24 hours. This response was generally conserved over all populations with only few genes being uniquely regulated in the temperate populations. We furthermore found that the American population was transcriptionally highly diverged from all other populations concerning basal levels of gene expression. This was particularly true for stress and immune response genes, which might be the genetic basis for their elevated vigor.
Insect resistance to toxins exerts not only a great impact on our economy, but also on the ecology of many species. Resistance to one toxin is often associated with cross-resistance to other, sometimes unrelated, chemicals. In this study, we investigated mushroom toxin resistance in the fruit fly Drosophila melanogaster (Meigen). This fruit fly species does not feed on mushrooms in nature and may thus have evolved cross-resistance to α-amanitin, the principal toxin of deadly poisonous mushrooms, due to previous pesticide exposure. The three Asian D. melanogaster stocks used in this study, Ama-KTT, Ama-MI, and Ama-KLM, acquired α-amanitin resistance at least five decades ago in their natural habitats in Taiwan, India, and Malaysia, respectively. Here we show that all three stocks have not lost the resistance phenotype despite the absence of selective pressure over the past half century. In response to α-amanitin in the larval food, several signs of developmental retardation become apparent in a concentration-dependent manner: higher pre-adult mortality, prolonged larva-to-adult developmental time, decreased adult body size, and reduced adult longevity. In contrast, female fecundity nearly doubles in response to higher α-amanitin concentrations. Our results suggest that α-amanitin resistance has no fitness cost, which could explain why the resistance has persisted in all three stocks over the past five decades. If pesticides caused α-amanitin resistance in D. melanogaster, their use may go far beyond their intended effects and have long-lasting effects on ecosystems.
We present a robot that enables high-content studies of alert adult Drosophila by combining operations including gentle picking; translations and rotations; characterizations of fly phenotypes and behaviors; microdissection; or release. To illustrate, we assessed fly morphology, tracked odor-evoked locomotion, sorted flies by sex, and dissected the cuticle to image neural activity. The robot's tireless capacity for precise manipulations enables a scalable platform for screening flies' complex attributes and behavioral patterns.
The circadian clock regulates vital aspects of physiology including protein synthesis and oxidative stress response. In this investigation, we performed a proteome-wide scrutiny of rhythmic protein accrual in Drosophila melanogaster on exposure to rotenone, rotenone + hesperidin and hesperidin in D. melanogaster. Total protein from fly samples collected at 6 h intervals over the 24 h period was subjected to two-dimensional gel electrophoresis and mass spectrometry. Bioinformatics tool, Protein ANalysis THrough Evolutionary Relationships classification system was used to the determine the biological processes of the proteins of altered abundance. Conspicuous variations in the proteome (151 proteins) of the flies exposed to oxidative stress (by rotenone treatment) and after alleviating oxidative stress (by hesperidin treatment) were observed during the 24 h cycle. Significantly altered levels of abundance of a wide variety of proteins under oxidative stress (rotenone treatment) and under alleviation of oxidative stress (rotenone + hesperidin treatment) and hesperidin (alone) treatment were observed. These proteins are involved in metabolism, muscle activity, heat shock response, redox homeostasis, protein synthesis/folding/degradation, development, ion-channel/cellular transport, and gustatory and olfactory function of the flies. Our data indicates that numerous cellular processes are involved in the temporal regulation of proteins and widespread modulations happen under rotenone treatment and, action of hesperidin could also be seen on these categories of proteins.
Ageing is a phenomenon where the accumulation of all the stresses that alter the functions of living organisms, halter them from maintaining their physiological balance and eventually lead to death. The emergence of epigenetic tremendously contributed to the knowledge of ageing. Epigenetic changes in cells or tissues like deoxyribonucleic acid (DNA) methylation, modification of histone proteins, transcriptional modification and also the involvement of non-coding DNA has been documented to be associated with ageing. In order to study ageing, scientists have taken advantage of several potential organisms to aid them in their study. Drosophila melanogaster has been an essential model in establishing current understanding of the mechanism of ageing as they possess several advantages over other competitors like having homologues to more than 75% of human disease genes, having 50% of Drosophila genes are homologues to human genes and most importantly they are genetically amenable. Here, we would like to summarise the extant knowledge about ageing and epigenetic process and the role of Drosophila as an ideal model to study epigenetics in association with ageing process.
Southeast Asian populations of the fruit fly Drosophila melanogaster differ from ancestral African and derived European populations by several morphological characteristics. It has been argued that this morphological differentiation could be the result of an early colonization of Southeast Asia that predated the migration of D. melanogaster to Europe after the last glacial period (around 10,000 years ago). To investigate the colonization process of Southeast Asia, we collected nucleotide polymorphism data for more than 200 X-linked fragments and 50 autosomal loci from a population of Malaysia. We analyzed this new single nucleotide polymorphism data set jointly with already existing data from an African and a European population by employing an Approximate Bayesian Computation approach. By contrasting different demographic models of these three populations, we do not find any evidence for an early divergence between the African and the Asian populations. Rather, we show that Asian and European populations of D. melanogaster share a non-African most recent common ancestor that existed about 2,500 years ago.
CTP synthase (CTPSyn) is an essential metabolic enzyme, synthesizing precursors required for nucleotides and phospholipids production. Previous studies have also shown that CTPSyn is elevated in various cancers. In many organisms, CTPSyn compartmentalizes into filaments called cytoophidia. In Drosophila melanogaster, only its isoform C (CTPSynIsoC) forms cytoophidia. In the fruit fly's testis, cytoophidia are normally seen in the transit amplification regions close to its apical tip, where the stem-cell niche is located, and development is at its most rapid. Here, we report that CTPSynIsoC overexpression causes the lengthening of cytoophidia throughout the entirety of the testicular body. A bulging apical tip is found in approximately 34% of males overexpressing CTPSynIsoC. Immunostaining shows that this bulged phenotype is most likely due to increased numbers of both germline cells and spermatocytes. Through a microRNA (miRNA) overexpression screen, we found that ectopic miR-975 concurrently increases both the expression levels of CTPSyn and the length of its cytoophidia. The bulging testes phenotype was also recovered at a penetration of approximately 20%. However, qPCR assays reveal that CTPSynIsoC and miR-975 overexpression each provokes a differential response in expression of a number of cancer-related genes, indicating that the shared CTPSyn upregulation seen in either case is likely the cause of observed testicular overgrowth. This study presents the first instance of consequences of miRNA-asserted regulation upon CTPSyn in D. melanogaster, and further reaffirms the enzyme's close ties to germline cells overgrowth.
Accruing evidences imply that circadian organization of biochemical, endocrinological, cellular and physiological processes contribute to wellness of organisms and in the development of pathologies such as malignancy, sleep and endocrine disorders. Oxidative stress is known to mediate a number of diseases and it is notable to comprehend the orchestration of circadian clock of a model organism of circadian biology, Drosophila melanogaster, under oxidative stress. We investigated the nexus between circadian clock and oxidative stress susceptibility by exposing D. melanogaster to hydrogen peroxide (H2O2) or rotenone; the reversibility of rhythms following exposure to Bacopa monnieri extract (ayurvedic medicine rich in antioxidants) was also investigated. Abolishment of 24h rhythms in physiological response (negative geotaxis), oxidative stress markers (protein carbonyl and thiobarbituric acid reactive substances) and antioxidants (superoxide dismutase, catalase, glutathione-S-transferase and reduced glutathione) were observed under oxidative stress. Furthermore, abolishment of per mRNA rhythm in H2O2 treated wild type flies and augmented susceptibility to oxidative stress in clock mutant (cry(b)) flies connotes the role of circadian clock in reactive oxygen species (ROS) homeostasis. Significant reversibility of rhythms was noted following B. monnieri treatment in wild type flies than cry(b) flies. Our experimental approach revealed a relationship involving oxidative stress and circadian clock in fruit fly and the utility of Drosophila model in screening putative antioxidative phytomedicines prior to their use in mammalian systems.
In Drosophila, protein trap strategies provide powerful approaches for the generation of tagged proteins expressed under endogenous control. Here, we describe expression and functional analysis to evaluate new Ubx and hth protein trap lines generated by the Cambridge Protein Trap project. Both protein traps exhibit spatial and temporal expression patterns consistent with the reported endogenous pattern in the embryo. In imaginal discs, Ubx-YFP is expressed throughout the haltere and 3rd leg imaginal discs, while Hth-YFP is expressed in the proximal regions of haltere and wing discs but not in the pouch region. The Ubx (CPTI000601) line is semilethal as a homozygote. No T3/A1 to T2 transformations were observed in the embryonic cuticle or the developing midgut. The homozygous survivors, however, exhibit a weak haltere phenotype with a few wing-like marginal bristles on the haltere capitellum. Although hth (CPTI000378) is completely lethal as a homozygote, the hth (CPTI000378) /hth (C1) genotype is viable. Using a hth deletion (Df(3R)BSC479) we show that hth (CPTI000378) /Df(3R)BSC479 adults are phenotypically normal. No transformations were observed in hth (CPTI000378), hth (CPTI000378) /hth (C1), or hth (CPTI000378) /Df(3R)BSC479 embryonic cuticles. We have successfully characterised the Ubx-YFP and Hth-YFP protein trap lines demonstrating that the tagged proteins show appropriate expression patterns and produce at least partially functional proteins.
Mutant lethal giant larvae (lgl) flies (Drosophila melanogaster) are known to develop epithelial tumors with invasive characteristics. The present study has been conducted to investigate the influence of melatonin (0.025 mM) on behavioral responses of lgl mutant flies as well as on biochemical indices (redox homeostasis, carbohydrate and lipid metabolism, transaminases, and minerals) in hemolymph, and head and intestinal tissues. Behavioral abnormalities were quantitatively observed in lgl flies but were found normalized among melatonin-treated lgl flies. Significantly decreased levels of lipid peroxidation products and antioxidants involved in redox homeostasis were observed in hemolymph and tissues of lgl flies, but had restored close to normalcy in melatonin-treated flies. Carbohydrates including glucose, trehalose, and glycogen were decreased and increased in the hemolymph and tissues of lgl and melatonin-treated lgl flies, respectively. Key enzymes of carbohydrate metabolism showed a significant increment in their levels in lgl mutants but had restored close to wild-type baseline levels in melatonin-treated flies. Variables of lipid metabolism showed significantly inverse levels in hemolymph and tissues of lgl flies, while normalization of most of these variables was observed in melatonin-treated mutants. Lipase, chitinase, transaminases, and alkaline phosphatase showed an increment in their activities and minerals exhibited decrement in lgl flies; reversal of changes was observed under melatonin treatment. The impairment of cognition, disturbance of redox homeostasis and metabolic reprogramming in lgl flies, and restoration of normalcy in all these cellular and behavioral processes indicate that melatonin could act as oncostatic and cytoprotective agents in Drosophila.
Palm fruit juice (PFJ) containing oil palm phenolics is obtained as a by-product from oil palm (Elaeis guineensis) fruit milling. It contains shikimic acid, soluble fibre and various phenolic acids including p-hydroxybenzoic acid and three caffeoylshikimic acid isomers. PFJ has also demonstrated beneficial health properties in various biological models. Increasing concentrations of PFJ and different PFJ fractions were used to assess growth dynamics and possible anti-ageing properties in fruit flies (Drosophila melanogaster) genotype w1118. Microarray gene expression analysis was performed on whole fruit fly larvae and their fat bodies, after the larvae were fed a control Standard Brandeis Diet (SBD) with or without PFJ. Transcripts from Affymetrix GeneChips were utilised to identify the possible mechanisms involved, with genes having fold changes > |1.30| and p
Alzheimer's disease (AD) is the most widespread neurodegenerative disorder worldwide. Its pathogenesis involves two hallmarks: aggregation of amyloid beta (Aβ) and occurrence of neurofibrillary tangles (NFTs). The mechanism behind the disease is still unknown. This has prompted the use of animal models to mirror the disease. The fruit fly, Drosophila melanogaster has garnered considerable attention as an organism to recapitulate human disorders. With the ability to monopolise a multitude of traditional and novel genetic tools, Drosophila is ideal for studying not only cellular aspects but also physiological and behavioural traits of human neurodegenerative diseases. Here, we discuss the use of the Drosophila model in understanding AD pathology and the insights gained in discovering drug therapies for AD.
Background. Diurnal rhythms of protein synthesis controlled by the biological clock underlie the rhythmic physiology in the fruit fly, Drosophila melanogaster. In this study, we conducted a proteome-wide investigation of rhythmic protein accumulation in D. melanogaster. Materials and Methods. Total protein collected from fly samples harvested at 4 h intervals over the 24 h period were subjected to two-dimensional gel electrophoresis, trypsin digestion and MS/MS analysis. Protein spots/clusters were identified with MASCOT search engine and Swiss-Prot database. Expression of proteins was documented as percentage of volume contribution using the Image Master 2D Platinum software. Results. A total of 124 protein spots/clusters were identified using MS/MS analysis. Significant variation in the expression of 88 proteins over the 24-h period was observed. A relatively higher number of proteins was upregulated during the night compared to the daytime. The complexity of temporal regulation of the D. melanogaster proteome was further reflected from functional annotations of the differently expressed proteins, with those that were upregulated at night being restricted to the heat shock proteins and proteins involved in metabolism, muscle activity, protein synthesis/folding/degradation and apoptosis, whilst those that were overexpressed in the daytime were apparently involved in metabolism, muscle activity, ion-channel/cellular transport, protein synthesis/folding/degradation, redox homeostasis, development and transcription. Conclusion. Our data suggests that a wide range of proteins synthesized by the fruit fly, D. melanogaster, is under the regulation of the biological clock.
In Drosophila, the Glycerol-3-phosphate dehydrogenase (Gpdh) enzyme plays an active role in many pathways, including the glycerol metabolic pathway and the alphaglycerophosphate cycle. It is also important for ethanol metabolism, as well as flight muscle development. Recent years have exposed small RNAs as a major posttranscriptional regulator of multiple metabolic-pathway genes. Of the many kinds of these RNAs at work, micro RNAs (miRNAs) are the most widely implicated and well understood. However, the roles they may play in regulating Gpdh has never been shown in any model organism. In this study, a pasha-mutant D. melanogaster strain was found to express only 25% of the Gpdh levels typical of their wild type counterparts. Such mutants lack the ability to produce pasha, a protein integral during miRNA-processing, and as a consequence do not produce mature miRNAs. As miRNA-centric regulation often culminates in the depletion of their targets, the concurrent downregulation of Gpdh observed in their absence here therefore alludes to two possibilities: one, that rather than being explicitly bound and repressed by miRNAs, Gpdh expression relies on their action upon an upstream Gpdh-antagonist; or two, that Gpdh may come under the regulation of another class of miRNA-like elements called mirtrons, which do not require pasha to be processed into their functional form. The preliminary findings in this study further highlights the imperative nature of miRNAs in regulating metabolic processes and subsequently, ensuring proper organismal development and its continued survival.
Alzheimer's disease (AD) is a progressive disease and one of the most common forms of neurodegenerative disorders. Emerging evidence is supporting the use of various strategies that modulate gut microbiota to exert neurological and psychological changes. This includes the utilisation of probiotics as a natural and dietary intervention for brain health. Here, we showed the potential AD-reversal effects of Lactobacillus probiotics through feeding to our Drosophila melanogaster AD model. The administration of Lactobacillus strains was able to rescue the rough eye phenotype (REP) seen in AD-induced Drosophila, with a more prominent effect observed upon the administration of Lactobacillus plantarum DR7 (DR7). Furthermore, we analysed the gut microbiota of the AD-induced Drosophila and found elevated levels of Wolbachia. The administration of DR7 restored the gut microbiota diversity of AD-induced Drosophila with a significant reduction in Wolbachia's relative abundance, accompanied by an increase of Stenotrophomonas and Acetobacter. Through functional predictive analyses, Wolbachia was predicted to be positively correlated with neurodegenerative disorders, such as Parkinson's, Huntington's and Alzheimer's diseases, while Stenotrophomonas was negatively correlated with these neurodegenerative disorders. Altogether, our data exhibited DR7's ability to ameliorate the AD effects in our AD-induced Drosophila. Thus, we propose that Wolbachia be used as a potential biomarker for AD.