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Science Advances Dec 2023Melanization and Toll pathway activation are essential innate immune mechanisms in insects, which result in the generation of reactive compounds and antimicrobial...
Melanization and Toll pathway activation are essential innate immune mechanisms in insects, which result in the generation of reactive compounds and antimicrobial peptides, respectively, to kill pathogens. These two processes are mediated by phenoloxidase (PO) and Spätzle (Spz) through an extracellular network of serine proteases. While some proteases have been identified in in genetic studies, the exact order of proteolytic activation events remains controversial. Here, we reconstituted the serine protease framework in by biochemical methods. This system comprises 10 proteases, i.e., ModSP, cSP48, Grass, Psh, Hayan-PA, Hayan-PB, Sp7, MP1, SPE and Ser7, which form cascade pathways that recognize microbial molecular patterns and virulence factors, and generate PO1, PO2, and Spz from their precursors. Furthermore, the serpin Necrotic negatively regulates the immune response progression by inhibiting ModSP and Grass. The biochemical approach, when combined with genetic analysis, is crucial for addressing problems that long stand in this important research field.
Topics: Animals; Drosophila; Drosophila melanogaster; Drosophila Proteins; Serine Endopeptidases; Serine Proteases
PubMed: 38117884
DOI: 10.1126/sciadv.adk2756 -
Food and Chemical Toxicology : An... Jul 20245-hydroxymethylfurfural is a common byproduct in food. However, its effect on growth and development remains incompletely understood. This study investigated the...
5-hydroxymethylfurfural is a common byproduct in food. However, its effect on growth and development remains incompletely understood. This study investigated the developmental toxicity of 5-HMF to Drosophila larvae. The growth and development of Drosophila melanogaster fed with 5-50 mM 5-HMF was monitored, and its possible mechanism was explored. It was found that 5-HMF prolonged the developmental cycle of Drosophila melanogaster (25 mM and 50 mM). After 5-HMF intake, the level of reactive oxygen species in the third instar larvae increased by 1.23-1.40 fold, which increased the level of malondialdehyde and caused changes in antioxidant enzymes. Moreover, the nuclear factor erythroid-2 related factor 2 antioxidant signaling pathway and the expression of heat shock protein genes were affected. At the same time, 5-HMF disrupted the glucose and lipid metabolism in the third instar larvae, influencing the expression level of key genes in the insulin signal pathway. Furthermore, 5-HMF led to intestinal oxidative stress, and up-regulated the expression of the pro-apoptotic gene, consequently impacting intestinal health. In short, 5-HMF causes oxidative stress, disturbs glucose and lipid metabolism and induces intestinal damage, damaging related signaling pathways, and ultimately affecting the development of Drosophila melanogaster.
Topics: Animals; Drosophila melanogaster; Larva; Furaldehyde; Oxidative Stress; Reactive Oxygen Species; Lipid Metabolism; Signal Transduction; Drosophila Proteins; Glucose
PubMed: 38754806
DOI: 10.1016/j.fct.2024.114738 -
Neuroscience Research May 2024DSCAM (Down syndrome cell adhesion molecule) is a unique neuronal adhesion protein with extensively documented multifaceted functionalities. DSCAM also has interesting... (Review)
Review
DSCAM (Down syndrome cell adhesion molecule) is a unique neuronal adhesion protein with extensively documented multifaceted functionalities. DSCAM also has interesting properties in vertebrates and invertebrates, respectively. In Drosophila species, particularly, Dscam exhibits remarkable genetic diversity, with tens of thousands of splicing isoforms that modulate the specificity of neuronal wiring. Interestingly, this splice variant diversity of Dscam is absent in vertebrates. DSCAM plays a pivotal role in mitigating excessive adhesion between identical cell types, thereby maintaining the structural and functional coherence of neural networks. DSCAM contributes to the oversight of selective intercellular interactions such as synaptogenesis; however, the precise regulatory mechanisms underlying the promotion and inhibition of cell adhesion involved remain unclear. In this review, we aim to delineate the distinct molecules that interact with DSCAM and their specific roles within the biological landscapes of Drosophila and vertebrates. By integrating these comparative insights, we aim to elucidate the multifunctional nature of DSCAM, particularly its capacity to facilitate or deter intercellular adhesion.
Topics: Animals; Cell Adhesion Molecules; Drosophila; Vertebrates; Drosophila Proteins; Humans; Cell Adhesion; Neurons
PubMed: 38141781
DOI: 10.1016/j.neures.2023.12.005 -
Cell Reports Oct 2023Accepting or rejecting a mate is one of the most crucial decisions a female will make, especially when faced with food shortage. Previous studies have identified the...
Accepting or rejecting a mate is one of the most crucial decisions a female will make, especially when faced with food shortage. Previous studies have identified the core neural circuity from sensing male courtship or mating status to decision-making for sexual receptivity in Drosophila females, but how hunger and satiety states modulate female receptivity is poorly understood. Here, we identify the neural circuit and its neuromodulation underlying the hunger modulation of female receptivity. We find that adipokinetic hormone receptor (AkhR)-expressing neurons inhibit sexual receptivity in a starvation-dependent manner. AkhR neurons are octopaminergic and act on a subset of Octβ1R-expressing LH421 neurons. Knocking down Octβ1R expression in LH421 neurons eliminates starvation-induced suppression of female receptivity. We further find that LH421 neurons inhibit the sex-promoting pC1 neurons via GABA-resistant to dieldrin (Rdl) signaling. pC1 neurons also integrate courtship stimulation and mating status and thus serve as a common integrator of multiple internal and external cues for decision-making.
Topics: Animals; Female; Male; Drosophila; Drosophila melanogaster; Drosophila Proteins; Hunger; Sexual Behavior, Animal; Neural Pathways; Courtship
PubMed: 37819758
DOI: 10.1016/j.celrep.2023.113243 -
Life Science Alliance May 2024Ciliary defects cause several ciliopathies, some of which have late onset, suggesting cilia are actively maintained. Still, we have a poor understanding of the...
Ciliary defects cause several ciliopathies, some of which have late onset, suggesting cilia are actively maintained. Still, we have a poor understanding of the mechanisms underlying their maintenance. Here, we show r IFT88 (IFT88/nompB) continues to move along fully formed sensory cilia. We further identify Inactive, a TRPV channel subunit involved in hearing and negative-gravitaxis behaviour, and a yet uncharacterised Guanylyl Cyclase 2d (Gucy2d/CG34357) as IFT88 cargoes. We also show IFT88 binding to the cyclase´s intracellular part, which is evolutionarily conserved and mutated in several degenerative retinal diseases, is important for the ciliary localisation of Gucy2d. Finally, acute knockdown of both IFT88 and Gucy2d in ciliated neurons of adult flies caused defects in the maintenance of cilium function, impairing hearing and negative-gravitaxis behaviour, but did not significantly affect ciliary ultrastructure. We conclude that the sensory ciliary function underlying hearing in the adult fly requires an active maintenance program which involves IFT88 and at least two of its signalling transmembrane cargoes, Gucy2d and Inactive.
Topics: Animals; Drosophila; Cilia; Drosophila melanogaster; Drosophila Proteins; Hearing
PubMed: 38373798
DOI: 10.26508/lsa.202302289 -
Brain : a Journal of Neurology Jun 2024Aggregation of the RNA-binding protein TAR DNA binding protein (TDP-43) is a hallmark of TDP-proteinopathies including amyotrophic lateral sclerosis (ALS) and...
Aggregation of the RNA-binding protein TAR DNA binding protein (TDP-43) is a hallmark of TDP-proteinopathies including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As TDP-43 aggregation and dysregulation are causative of neuronal death, there is a special interest in targeting this protein as a therapeutic approach. Previously, we found that TDP-43 extensively co-aggregated with the dual function protein GEF (guanine exchange factor) and RNA-binding protein rho guanine nucleotide exchange factor (RGNEF) in ALS patients. Here, we show that an N-terminal fragment of RGNEF (NF242) interacts directly with the RNA recognition motifs of TDP-43 competing with RNA and that the IPT/TIG domain of NF242 is essential for this interaction. Genetic expression of NF242 in a fruit fly ALS model overexpressing TDP-43 suppressed the neuropathological phenotype increasing lifespan, abolishing motor defects and preventing neurodegeneration. Intracerebroventricular injections of AAV9/NF242 in a severe TDP-43 murine model (rNLS8) improved lifespan and motor phenotype, and decreased neuroinflammation markers. Our results demonstrate an innovative way to target TDP-43 proteinopathies using a protein fragment with a strong affinity for TDP-43 aggregates and a mechanism that includes competition with RNA sequestration, suggesting a promising therapeutic strategy for TDP-43 proteinopathies such as ALS and FTD.
Topics: Animals; Amyotrophic Lateral Sclerosis; DNA-Binding Proteins; Mice; Humans; Disease Models, Animal; Guanine Nucleotide Exchange Factors; Phenotype; Drosophila; Mice, Transgenic; Drosophila Proteins; Male
PubMed: 38739752
DOI: 10.1093/brain/awae078 -
Proceedings of the National Academy of... Aug 2023Cell-type-specific tools facilitate the identification and functional characterization of the distinct cell types that form the complexity of neuronal circuits. A large...
Cell-type-specific tools facilitate the identification and functional characterization of the distinct cell types that form the complexity of neuronal circuits. A large collection of existing genetic tools in relies on enhancer activity to label different subsets of cells and has been extremely useful in analyzing functional circuits in adults. However, these enhancer-based GAL4 lines often do not reflect the expression of nearby gene(s) as they only represent a small portion of the full gene regulatory elements. While genetic intersectional techniques such as the split-GAL4 system further improve cell-type-specificity, it requires significant time and resources to screen through combinations of enhancer expression patterns. Here, we use existing developmental single-cell RNA sequencing (scRNAseq) datasets to select gene pairs for split-GAL4 and provide a highly efficient and predictive pipeline (scMarco) to generate cell-type-specific split-GAL4 lines at any time during development, based on the native gene regulatory elements. These gene-specific split-GAL4 lines can be generated from a large collection of coding intronic MiMIC/CRIMIC lines or by CRISPR knock-in. We use the developing visual system as a model to demonstrate the high predictive power of scRNAseq-guided gene-specific split-GAL4 lines in targeting known cell types, annotating clusters in scRNAseq datasets as well as in identifying novel cell types. Lastly, the gene-specific split-GAL4 lines are broadly applicable to any other tissue. Our work opens new avenues for generating cell-type-specific tools for the targeted manipulation of distinct cell types throughout development and represents a valuable resource for the community.
Topics: Animals; Transcription Factors; Drosophila; Drosophila Proteins; Genetic Techniques; Sequence Analysis, RNA; Drosophila melanogaster
PubMed: 37523539
DOI: 10.1073/pnas.2307451120 -
Protein Expression and Purification Nov 2023SIRT6 and SIRT7, as members of the Sirtuins family, are indispensable for the growth and development of Drosophila. They play crucial roles in maintaining genome...
SIRT6 and SIRT7, as members of the Sirtuins family, are indispensable for the growth and development of Drosophila. They play crucial roles in maintaining genome stability, regulating metabolic senescence, and controlling tumorigenesis. To investigate their involvement in the Drosophila life cycle, we focused on describing the expression and purification of recombinant Drosophila SIRT6 and SIRT7 proteins. Subsequently, these proteins were utilized for generating polyclonal antibodies against Drosophila SIRT6 and SIRT7. The recombinant expression plasmid was introduced into E. coli cells to enable the production of SIRT6 and SIRT7 proteins. Following immunizations of New Zealand white rabbits and guinea pigs with the recombinant proteins as antigens, specific polyclonal antisera against both proteins were obtained. After purification, the specificity of SIRT6 and SIRT7 was confirmed using ELISA and western blot analyses, demonstrating strong specificity. These antibodies hold promise for the development of detection assays required for further research.
Topics: Animals; Guinea Pigs; Rabbits; Antibodies; Drosophila; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Sirtuins; Drosophila Proteins
PubMed: 37460032
DOI: 10.1016/j.pep.2023.106338 -
Genetics Aug 2023In the last decade, researchers using Drosophila melanogaster have made extraordinary progress in uncovering the mysteries underlying learning and memory. This progress...
In the last decade, researchers using Drosophila melanogaster have made extraordinary progress in uncovering the mysteries underlying learning and memory. This progress has been propelled by the amazing toolkit available that affords combined behavioral, molecular, electrophysiological, and systems neuroscience approaches. The arduous reconstruction of electron microscopic images resulted in a first-generation connectome of the adult and larval brain, revealing complex structural interconnections between memory-related neurons. This serves as substrate for future investigations on these connections and for building complete circuits from sensory cue detection to changes in motor behavior. Mushroom body output neurons (MBOn) were discovered, which individually forward information from discrete and non-overlapping compartments of the axons of mushroom body neurons (MBn). These neurons mirror the previously discovered tiling of mushroom body axons by inputs from dopamine neurons and have led to a model that ascribes the valence of the learning event, either appetitive or aversive, to the activity of different populations of dopamine neurons and the balance of MBOn activity in promoting avoidance or approach behavior. Studies of the calyx, which houses the MBn dendrites, have revealed a beautiful microglomeruluar organization and structural changes of synapses that occur with long-term memory (LTM) formation. Larval learning has advanced, positioning it to possibly lead in producing new conceptual insights due to its markedly simpler structure over the adult brain. Advances were made in how cAMP response element-binding protein interacts with protein kinases and other transcription factors to promote the formation of LTM. New insights were made on Orb2, a prion-like protein that forms oligomers to enhance synaptic protein synthesis required for LTM formation. Finally, Drosophila research has pioneered our understanding of the mechanisms that mediate permanent and transient active forgetting, an important function of the brain along with acquisition, consolidation, and retrieval. This was catalyzed partly by the identification of memory suppressor genes-genes whose normal function is to limit memory formation.
Topics: Animals; Drosophila melanogaster; Drosophila; Memory, Long-Term; Drosophila Proteins; Dopaminergic Neurons
PubMed: 37212449
DOI: 10.1093/genetics/iyad085 -
Molecular & Cellular Proteomics : MCP Aug 2023Reproductive traits often evolve rapidly between species. Understanding the causes and consequences of this rapid divergence requires characterization of female and male...
Reproductive traits often evolve rapidly between species. Understanding the causes and consequences of this rapid divergence requires characterization of female and male reproductive proteins and their effect on fertilization success. Species in the Drosophila virilis clade exhibit rampant interspecific reproductive incompatibilities, making them ideal for studies on diversification of reproductive proteins and their role in speciation. Importantly, the role of intraejaculate protein abundance and allocation in interspecific divergence is poorly understood. Here, we identify and quantify the transferred male ejaculate proteome using multiplexed isobaric labeling of the lower female reproductive tract before and immediately after mating using three species of the virilis group. We identified over 200 putative male ejaculate proteins, many of which show differential abundance between species, suggesting that males transfer a species-specific allocation of seminal fluid proteins during copulation. We also identified over 2000 female reproductive proteins, which contain female-specific serine-type endopeptidases that showed differential abundance between species and elevated rates of molecular evolution, similar to that of some male seminal fluid proteins. Our findings suggest that reproductive protein divergence can also manifest in terms of species-specific protein abundance patterns.
Topics: Animals; Male; Female; Drosophila; Proteomics; Reproduction; Biological Evolution; Drosophila Proteins
PubMed: 37391044
DOI: 10.1016/j.mcpro.2023.100610