Tolerance occurs when, following an initial experience with a substance, more of the substance is required subsequently to induce identical behavioral effects. Tolerance is not well-understood, and numerous researchers have turned to model organisms, particularly Drosophila melanogaster, to unravel its mechanisms. Flies have high translational relevance for human alcohol responses, and there is substantial overlap in disease-causing genes between flies and humans, including those associated with Alcohol Use Disorder. Numerous Drosophila tolerance mutants have been described; however, approaches used to identify and characterize these mutants have varied across time and labs and have mostly disregarded any impact of initial resistance/sensitivity to ethanol on subsequent tolerance development. Here, we analyzed our own, as well as data published by other labs to uncover an inverse correlation between initial ethanol resistance and tolerance phenotypes. This inverse correlation suggests that initial resistance phenotypes can explain many \'perceived\' tolerance phenotypes, thus classifying such mutants as \'secondary\' tolerance mutants. Additionally, we show that tolerance should be measured as a relative increase in time to sedation between an initial and second exposure rather than an absolute change in time to sedation. Finally, based on our analysis, we provide a method for using a linear regression equation to assess the residuals of potential tolerance mutants. These residuals provide predictive insight into the likelihood of a mutant being a \'primary\' tolerance mutant, where a tolerance phenotype is not solely a consequence of initial resistance, and we offer a framework for understanding the relationship between initial resistance and tolerance.

The Wnt/Wingless signaling pathway plays critical roles in metazoan development and energy metabolism, but its role in regulating lipid homeostasis remains not fully understood. Here, we report that the activation of canonical Wnt/Wg signaling promotes lipolysis while concurrently inhibiting lipogenesis and fatty acid β-oxidation in both larval and adult adipocytes, as well as cultured S2R+ cells, in Drosophila. Using RNA-sequencing and CUT&RUN (Cleavage Under Targets & Release Using Nuclease) assays, we identified a set of Wnt target genes responsible for intracellular lipid homeostasis. Notably, active Wnt signaling directly represses the transcription of these genes, resulting in decreased de novo lipogenesis and fatty acid β-oxidation, but increased lipolysis. These changes lead to elevated free fatty acids and reduced triglyceride (TG) accumulation in adipocytes with active Wnt signaling. Conversely, downregulation of Wnt signaling in the fat body promotes TG accumulation in both larval and adult adipocytes. The attenuation of Wnt signaling also increases the expression of specific lipid metabolism-related genes in larval adipocytes, wing discs, and adult intestines. Taken together, these findings suggest that Wnt signaling-induced transcriptional repression plays an important role in regulating lipid homeostasis by enhancing lipolysis while simultaneously suppressing lipogenesis and fatty acid β-oxidation.

The rising global incidence of cancer makes it the second leading cause of death worldwide. Over the past decades, significant progress has been made in both basic knowledge and the discovery of new therapeutic approaches. However, the complexity of mechanisms related to tumor development requires the use of sophisticated and adapted research tools. Among these, the fruitfly Drosophila melanogaster represents a powerful genetic model with numerous practical and conceptual advantages. Indeed, the conservation of genes implicated in cancer between this insect and mammals places Drosophila as a crucial genetic tool for understanding the fundamental mechanisms governing tumorigenesis and identifying new therapeutic targets. This review aims to describe this original model and demonstrate its relevance for studying cancer biology.

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BACKGROUND: Restraining or slowing ageing hallmarks at the cellular level have been proposed as a route to increased organismal lifespan and healthspan. Consequently, there is great interest in anti-ageing drug discovery. However, this currently requires laborious and lengthy longevity analysis. Here, we present a novel screening readout for the expedited discovery of compounds that restrain ageing of cell populations in vitro and enable extension of in vivo lifespan.
METHODS: Using Illumina methylation arrays, we monitored DNA methylation changes accompanying long-term passaging of adult primary human cells in culture. This enabled us to develop, test, and validate the CellPopAge Clock, an epigenetic clock with underlying algorithm, unique among existing epigenetic clocks for its design to detect anti-ageing compounds in vitro. Additionally, we measured markers of senescence and performed longevity experiments in vivo in Drosophila, to further validate our approach to discover novel anti-ageing compounds. Finally, we bench mark our epigenetic clock with other available epigenetic clocks to consolidate its usefulness and specialisation for primary cells in culture.
RESULTS: We developed a novel epigenetic clock, the CellPopAge Clock, to accurately monitor the age of a population of adult human primary cells. We find that the CellPopAge Clock can detect decelerated passage-based ageing of human primary cells treated with rapamycin or trametinib, well-established longevity drugs. We then utilise the CellPopAge Clock as a screening tool for the identification of compounds which decelerate ageing of cell populations, uncovering novel anti-ageing drugs, torin2 and dactolisib (BEZ-235). We demonstrate that delayed epigenetic ageing in human primary cells treated with anti-ageing compounds is accompanied by a reduction in senescence and ageing biomarkers. Finally, we extend our screening platform in vivo by taking advantage of a specially formulated holidic medium for increased drug bioavailability in Drosophila. We show that the novel anti-ageing drugs, torin2 and dactolisib (BEZ-235), increase longevity in vivo.
CONCLUSIONS: Our method expands the scope of CpG methylation profiling to accurately and rapidly detecting anti-ageing potential of drugs using human cells in vitro, and in vivo, providing a novel accelerated discovery platform to test sought after anti-ageing compounds and geroprotectors.

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Quantitative immunoassays, such as the traditional enzyme-linked immunosorbent assay (ELISA), are used to determine concentrations of an antigen in a matrix of unknown antigen concentration. Magnetic immunoassays, such as the Luminex xMAP technology, allow for the simultaneous detection of multiple analytes and offer heightened sensitivity, specificity, low sample volume requirements, and high-throughput capabilities. Here, we describe a quantitative immunoassay using the Luminex MAGPIX® System to determine the antigen concentration from liquid samples with unknown concentrations. In detail, we describe a newly developed assay for determining production yields of Drosophila S2-produced Marburg virus (MARV) glycoprotein in insect-cell-culture-derived supernatant. The potential applications of this assay could extend to the quantification of viral antigens in fluids derived from both in vitro and in vivo models infected with live MARV, thereby providing additional applications for virological research.

In human Alzheimer\'s disease (AD), the aggregation of tau protein is considered a significant hallmark, along with amyloid-beta. The formation of neurofibrillary tangles due to aberrant phosphorylation of tau disrupts microtubule stability, leading to neuronal toxicity, dysfunction, and subsequent cell death. Nesfatin-1 is a neuropeptide primarily known for regulating appetite and energy homeostasis. However, the function of Nesfatin-1 in a neuroprotective role has not been investigated. In this study, we aimed to elucidate the effect of Nesfatin-1 on tau pathology using the Drosophila model system. Our findings demonstrate that Nesfatin-1 effectively mitigates the pathological phenotypes observed in Drosophila human Tau overexpression models. Nesfatin-1 overexpression rescued the neurodegenerative phenotypes in the adult fly\'s eye and bristle. Additionally, Nesfatin-1 improved locomotive behavior, neuromuscular junction formation, and lifespan in the hTau AD model. Moreover, Nesfatin-1 controls tauopathy by reducing the protein level of hTau. Overall, this research highlights the potential therapeutic applications of Nesfatin-1 in ameliorating the pathological features associated with Alzheimer\'s disease.

Pyridoxamine (PM) is one of the natural vitamins B6 (VB6) and functions as an endogenous inhibitor for the formation of AGEs (advanced glycation end products). The AGEs are implicated in aging, diabetes, and various neuropsychiatric disease, including schizophrenia, Alzheimer\'s disease, and Parkinson\'s disease. However, it is unclear whether the absence of PM per se accumulates AGEs in vivo and causes behavioral dysfunctions. To address these points, we raised PM-deficient fruit flies, Drosophila melanogaster, with the sterilized defined medium. Flies reared in a PM-deficient medium accumulated AGEs and reduced lifespan, impaired gustatory response, sleep, courtship behavior, and olfactory learning. These results suggest that PM suppresses AGE accumulation in vivo and is required for regulating innate and empirical behaviors.

The basement membrane (BM) is an extracellular matrix that plays important roles in animal development. A spatial heterogeneity in composition and structural properties of the BM provide cells with vital cues for morphogenetic processes such as cell migration or cell polarization. Here, using the Drosophila egg chamber as a model system, we show that the BM becomes heterogeneous during development, with a reduction in Collagen IV density at the posterior pole and differences in the micropattern of aligned fiber-like structures. We identified two AdamTS matrix proteases required for the proper elongated shape of the egg chamber, yet the molecular mechanisms by which they act are different. Stall is required to establish BM heterogeneity by locally limiting Collagen IV protein density, whereas AdamTS-A alters the micropattern of fiber-like structures within the BM at the posterior pole. Our results suggest that AdamTS proteases control BM heterogeneity required for organ shape.