Vitamin B6 deficiency cooperates with oncogenic Ras to induce malignant tumors in Drosophila.

Vitamin B6 is a water-soluble vitamin which possesses antioxidant properties. Its catalytically active form, pyridoxal 5'-phosphate (PLP), is a crucial cofactor for DNA and amino acid metabolism. The inverse correlation between vitamin B6 and cancer risk has been observed in several studies, although dietary vitamin B6 intake sometimes failed to confirm this association. However, the molecular link between vitamin B6 and cancer remains elusive. Previous work has shown that vitamin B6 deficiency causes chromosome aberrations (CABs) in Drosophila and human cells, suggesting that genome instability may correlate the lack of this vitamin to cancer. Here we provide evidence in support of this hypothesis. Firstly, we show that PLP deficiency, induced by the PLP antagonists 4-deoxypyridoxine (4DP) or ginkgotoxin (GT), promoted tumorigenesis in eye larval discs transforming benign RasV12 tumors into aggressive forms. In contrast, PLP supplementation reduced the development of tumors. We also show that low PLP levels, induced by 4DP or by silencing the sgllPNPO gene involved in PLP biosynthesis, worsened the tumor phenotype in another Drosophila cancer model generated by concomitantly activating RasV12 and downregulating Discs-large (Dlg) gene. Moreover, we found that RasV12 eye discs from larvae reared on 4DP displayed CABs, reactive oxygen species (ROS) and low catalytic activity of serine hydroxymethyltransferase (SHMT), a PLP-dependent enzyme involved in thymidylate (dTMP) biosynthesis, in turn required for DNA replication and repair. Feeding RasV12 4DP-fed larvae with PLP or ascorbic acid (AA) plus dTMP, rescued both CABs and tumors. The same effect was produced by overexpressing catalase in RasV12 DlgRNAi 4DP-fed larvae, thus allowing to establish a relationship between PLP deficiency, CABs, and cancer. Overall, our data provide the first in vivo demonstration that PLP deficiency can impact on cancer by increasing genome instability, which is in turn mediated by ROS and reduced dTMP levels.

Pan-neuronal expression of human mutant SOD1 in Drosophila impairs survival and motor performance, induces early neuroinflammation and chromosome aberrations.

Several mutations in the SOD1 gene encoding for the antioxidant enzyme Superoxide Dismutase 1, are associated with amyotrophic lateral sclerosis, a rare and devastating disease characterized by motor neuron degeneration and patients' death within 2-5 years from diagnosis. Motor neuron loss and related symptomatology manifest mostly in adult life and, to date, there is still a gap of knowledge on the precise cellular and molecular events preceding neurodegeneration. To deepen our awareness of the early phases of the disease, we leveraged two Drosophila melanogaster models pan-neuronally expressing either the mutation A4V or G85R of the human gene SOD1 (hSOD1A4V or hSOD1G85R). We demonstrate that pan-neuronal expression of the hSOD1A4V or hSOD1G85R pathogenic construct impairs survival and motor performance in transgenic flies. Moreover, protein and transcript analysis on fly heads indicates that mutant hSOD1 induction stimulates the glial marker Repo, up-regulates the IMD/Toll immune pathways through antimicrobial peptides and interferes with oxidative metabolism. Finally, cytological analysis of larval brains demonstrates hSOD1-induced chromosome aberrations. Of note, these parameters are found modulated in a timeframe when neurodegeneration is not detected. The novelty of our work is twofold: we have expressed for the first time hSOD1 mutations in all neurons of Drosophila and confirmed some ALS-related pathological phenotypes in these flies, confirming the power of SOD1 mutations in generating ALS-like phenotypes. Moreover, we have related SOD1 pathogenesis to chromosome aberrations and antimicrobial peptides up-regulation. These findings were unexplored in the SOD1-ALS field.

A Closer Look at Histamine in Drosophila.

The present work intends to provide a closer look at histamine in Drosophila. This choice is motivated firstly because Drosophila has proven over the years to be a very simple, but powerful, model organism abundantly assisting scientists in explaining not only normal functions, but also derangements that occur in higher organisms, not excluding humans. Secondly, because histamine has been demonstrated to be a pleiotropic master molecule in pharmacology and immunology, with increasingly recognized roles also in the nervous system. Indeed, it interacts with various neurotransmitters and controls functions such as learning, memory, circadian rhythm, satiety, energy balance, nociception, and motor circuits, not excluding several pathological conditions. In view of this, our review is focused on the knowledge that the use of Drosophila has added to the already vast histaminergic field. In particular, we have described histamine's actions on photoreceptors sustaining the visual system and synchronizing circadian rhythms, but also on temperature preference, courtship behavior, and mechanosensory transmission. In addition, we have highlighted the pathophysiological consequences of mutations on genes involved in histamine metabolism and signaling. By promoting critical discussion and further research, our aim is to emphasize and renew the importance of histaminergic research in biomedicine through the exploitation of Drosophila, hopefully extending the scientific debate to the academic, industry, and general public audiences.

Benzenesulfonamide derivatives as Vibrio cholerae carbonic anhydrases inhibitors: a computational-aided insight in the structural rigidity-activity relationships.

Vibrio cholerae causes life-threatening infections in low-income countries due to the rise of antibacterial resistance. Innovative pharmacological targets have been investigated and carbonic anhydrases (CAs, EC: 4.2.1.1) encoded by V. cholerae (VchCAs) emerged as a valuable option. Recently, we developed a large library of para- and meta-benzenesulfonamides characterised by moieties with a different flexibility degree as CAs inhibitors. Stopped flow-based enzymatic assays showed strong inhibition of VchαCA for this library, while lower affinity was detected against the other isoforms. In particular, cyclic urea 9c emerged for a nanomolar inhibition of VchαCA (KI = 4.7 nM) and high selectivity with respect to human isoenzymes (SI≥ 90). Computational studies revealed the influence of moiety flexibility on inhibitory activity and isoform selectivity and allowed accurate SARs. However, although VchCAs are involved in the bacterium virulence and not in its survival, we evaluated the antibacterial activity of such compounds, resulting in no direct activity.

Delineating Purinergic Signaling in Drosophila.

Simplistic models can aid in discovering what is important in the context of normal and pathological behavior. First recognized as a genetic model more than 100 years ago, to date, fruit flies (Drosophila melanogaster) still remain an astonishingly good laboratory stand-in for scientists to study development and physiology and to investigate the molecular mechanisms of human diseases. This is because fruit flies indeed represent a simplistic model. Furthermore, about 75% of human disease-related genes have their counterparts in the Drosophila genome, added to the fact that fruit flies are inexpensive and extremely easy to maintain, being invertebrates and, moreover, lacking any ethical concern issues. Purinergic signaling is, by definition, mediated by extracellular purinergic ligands, among which ATP represents the prototype molecule. A key feature that has progressively emerged when dissecting the purinergic mechanisms is the multilayer and dynamic nature of the signaling sustained by purinergic ligands. Indeed, these last are sequentially metabolized by several different ectonucleotidases, which generate the ligands that simultaneously activate several different purinergic receptors. Since significant purinergic actions have also been described in Drosophila, the aim of the present work is to provide a comprehensive picture of the purinergic events occurring in fruit flies.

Vitamin B6 rescues insulin resistance and glucose-induced DNA damage caused by reduced activity of Drosophila PI3K.

The insulin signaling pathway controls cell growth and metabolism, thus its deregulation is associated with both cancer and diabetes. Phosphatidylinositol 3-kinase (PI3K) contributes to the cascade of phosphorylation events occurring in the insulin pathway by activating the protein kinase B (PKB/AKT), which phosphorylates several substrates, including those involved in glucose uptake and storage. PI3K inactivating mutations are associated with insulin resistance while activating mutations are identified in human cancers. Here we show that RNAi-induced depletion of the Drosophila PI3K catalytic subunit (Dp110) results in diabetic phenotypes such as hyperglycemia, body size reduction, and decreased glycogen content. Interestingly, we found that hyperglycemia produces chromosome aberrations (CABs) triggered by the accumulation of advanced glycation end-products and reactive oxygen species. Rearing PI3KRNAi flies in a medium supplemented with pyridoxal 5'-phosphate (PLP; the catalytically active form of vitamin B6) rescues DNA damage while, in contrast, treating PI3KRNAi larvae with the PLP inhibitor 4-deoxypyridoxine strongly enhances CAB frequency. Interestingly, PLP supplementation rescues also diabetic phenotypes. Taken together, our results provide a strong link between impaired PI3K activity and genomic instability, a crucial relationship that needs to be monitored not only in diabetes due to impaired insulin signaling but also in cancer therapies based on PI3K inhibitors. In addition, our findings confirm the notion that vitamin B6 is a good natural remedy to counteract insulin resistance and its complications.

Benzenesulfonamides with different rigidity-conferring linkers as carbonic anhydrase inhibitors: an insight into the antiproliferative effect on glioblastoma, pancreatic, and breast cancer cells.

Among the chemotypes studied for selective inhibition of tumour-associated carbonic anhydrases (CAs), SLC-0111, a ureido-bearing benzenesulfonamide CA IX inhibitor, displayed promising antiproliferative effects in cancer cells in vitro and in vivo, being in Phase Ib/II clinical development. To explore the structural characteristics required for better discrimination of less conserved regions of the enzyme, we investigate the incorporation of the urea linker into an imidazolidin-2-one cycle, a modification already explored previously for obtaining CA inhibitors. This new library of compounds inhibited potently four different hCAs in the nanomolar range with a different isoform selectivity profile compared to the lead SLC-0111. Several representative CA IX inhibitors were tested for their efficacy to inhibit the proliferation of glioblastoma, pancreatic, and breast cancer cells expressing CA IX, in hypoxic conditions. Unlike previous literature data on SLC-149, a structurally related sulphonamide to compounds investigated here, our data reveal that these derivatives possess promising anti-proliferative effects, comparable to those of SLC-0111.

Transposable element activation promotes neurodegeneration in a Drosophila model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant disorder with progressive motor dysfunction and cognitive decline. The disease is caused by a CAG repeat expansion in the IT15 gene, which elongates a polyglutamine stretch of the HD protein, Huntingtin. No therapeutic treatments are available, and new pharmacological targets are needed. Retrotransposons are transposable elements (TEs) that represent 40% and 30% of the human and Drosophila genomes and replicate through an RNA intermediate. Mounting evidence suggests that mammalian TEs are active during neurogenesis and may be involved in diseases of the nervous system. Here we show that TE expression and mobilization are increased in a Drosophila melanogaster HD model. By inhibiting TE mobilization with Reverse Transcriptase inhibitors, polyQ-dependent eye neurodegeneration and genome instability in larval brains are rescued and fly lifespan is increased. These results suggest that TE activation may be involved in polyQ-induced neurotoxicity and a potential pharmacological target.

The Genetics of Diabetes: What We Can Learn from Drosophila.

Diabetes mellitus is a heterogeneous disease characterized by hyperglycemia due to impaired insulin secretion and/or action. All diabetes types have a strong genetic component. The most frequent forms, type 1 diabetes (T1D), type 2 diabetes (T2D) and gestational diabetes mellitus (GDM), are multifactorial syndromes associated with several genes' effects together with environmental factors. Conversely, rare forms, neonatal diabetes mellitus (NDM) and maturity onset diabetes of the young (MODY), are caused by mutations in single genes. Large scale genome screenings led to the identification of hundreds of putative causative genes for multigenic diabetes, but all the loci identified so far explain only a small proportion of heritability. Nevertheless, several recent studies allowed not only the identification of some genes as causative, but also as putative targets of new drugs. Although monogenic forms of diabetes are the most suited to perform a precision approach and allow an accurate diagnosis, at least 80% of all monogenic cases remain still undiagnosed. The knowledge acquired so far addresses the future work towards a study more focused on the identification of diabetes causal variants; this aim will be reached only by combining expertise from different areas. In this perspective, model organism research is crucial. This review traces an overview of the genetics of diabetes and mainly focuses on Drosophila as a model system, describing how flies can contribute to diabetes knowledge advancement.

Fly for ALS: Drosophila modeling on the route to amyotrophic lateral sclerosis modifiers.

Amyotrophic lateral sclerosis (ALS) is a rare, devastating disease, causing movement impairment, respiratory failure and ultimate death. A plethora of genetic, cellular and molecular mechanisms are involved in ALS signature, although the initiating causes and progressive pathological events are far from being understood. Drosophila research has produced seminal discoveries for more than a century and has been successfully used in the past 25 years to untangle the process of ALS pathogenesis, and recognize potential markers and novel strategies for therapeutic solutions. This review will provide an updated view of several ALS modifiers validated in C9ORF72, SOD1, FUS, TDP-43 and Ataxin-2 Drosophila models. We will discuss basic and preclinical findings, illustrating recent developments and novel breakthroughs, also depicting unsettled challenges and limitations in the Drosophila-ALS field. We intend to stimulate a renewed debate on Drosophila as a screening route to identify more successful disease modifiers and neuroprotective agents.

Material Design for Optimal Postbuckling Behaviour of Composite Shells.

Lightweight thin-walled structures are crucial for many engineering applications. Advanced manufacturing methods are enabling the realization of composite materials with spatially varying material properties. Variable angle tow fibre composites are a representative example, but also nanocomposites are opening new interesting possibilities. Taking advantage of these tunable materials requires the development of computational design methods. The failure of such structures is often dominated by buckling and can be very sensitive to material configuration and geometrical imperfections. This work is a review of the recent computational developments concerning the optimisation of the response of composite thin-walled structures prone to buckling, showing how baseline products with unstable behaviour can be transformed in stable ones operating safely in the post-buckling range. Four main aspects are discussed: mechanical and discrete models for composite shells, material parametrization and objective function definition, solution methods for tracing the load-displacement path and assessing the imperfection sensitivity, structural optimisation algorithms. A numerical example of optimal material design for a curved panel is also illustrated.

Where and Why Modeling Amyotrophic Lateral Sclerosis.

Over the years, researchers have leveraged a host of different in vivo models in order to dissect amyotrophic lateral sclerosis (ALS), a neurodegenerative/neuroinflammatory disease that is heterogeneous in its clinical presentation and is multigenic, multifactorial and non-cell autonomous. These models include both vertebrates and invertebrates such as yeast, worms, flies, zebrafish, mice, rats, guinea pigs, dogs and, more recently, non-human primates. Despite their obvious differences and peculiarities, only the concurrent and comparative analysis of these various systems will allow the untangling of the causes and mechanisms of ALS for finally obtaining new efficacious therapeutics. However, harnessing these powerful organisms poses numerous challenges. In this context, we present here an updated and comprehensive review of how eukaryotic unicellular and multicellular organisms that reproduce a few of the main clinical features of the disease have helped in ALS research to dissect the pathological pathways of the disease insurgence and progression. We describe common features as well as discrepancies among these models, highlighting new insights and emerging roles for experimental organisms in ALS.

Functional Inactivation of Drosophila GCK Orthologs Causes Genomic Instability and Oxidative Stress in a Fly Model of MODY-2.

Maturity-onset diabetes of the young (MODY) type 2 is caused by heterozygous inactivating mutations in the gene encoding glucokinase (GCK), a pivotal enzyme for glucose homeostasis. In the pancreas GCK regulates insulin secretion, while in the liver it promotes glucose utilization and storage. We showed that silencing the Drosophila GCK orthologs Hex-A and Hex-C results in a MODY-2-like hyperglycemia. Targeted knock-down revealed that Hex-A is expressed in insulin producing cells (IPCs) whereas Hex-C is specifically expressed in the fat body. We showed that Hex-A is essential for insulin secretion and it is required for Hex-C expression. Reduced levels of either Hex-A or Hex-C resulted in chromosome aberrations (CABs), together with an increased production of advanced glycation end-products (AGEs) and reactive oxygen species (ROS). This result suggests that CABs, in GCK depleted cells, are likely due to hyperglycemia, which produces oxidative stress through AGE metabolism. In agreement with this hypothesis, treating GCK-depleted larvae with the antioxidant vitamin B6 rescued CABs, whereas the treatment with a B6 inhibitor enhanced genomic instability. Although MODY-2 rarely produces complications, our data revealed the possibility that MODY-2 impacts genome integrity.

Optimal Design of CNT-Nanocomposite Nonlinear Shells.

Carbon nanotube/polymer nanocomposite plate- and shell-like structures will be the next generation lightweight structures in advanced applications due to the superior multifunctional properties combined with lightness. Here material optimization of carbon nanotube/polymer nanocomposite beams and shells is tackled via ad hoc nonlinear finite element schemes so as to control the loss of stability and overall nonlinear response. Three types of optimizations are considered: variable through-the-thickness volume fraction of random carbon nanotubes (CNTs) distributions, variable volume fraction of randomly oriented CNTs within the mid-surface, aligned CNTs with variable orientation with respect to the mid-surface. The collapse load, which includes both limit points and deformation thresholds, is chosen as the objective/cost function. An efficient computation of the cost function is carried out using the Koiter reduced order model obtained starting from an isogeometric solid-shell model to accurately describe the point-wise material distribution. The sensitivity to geometrical imperfections is also investigated. The optimization is carried out making use of the Global Convergent Method of Moving Asymptotes. The extensive numerical analyses show that varying the volume fraction distribution as well as the CNTs orientation can lead to significantly enhanced performances towards the loss of elastic stability making these lightweight structures more stable. The most striking result is that for curved shells, the unstable postbuckling response of the baseline material can be turned into a globally stable response maintaining the same amount of nanostructural reinforcement but simply tailoring strategically its distribution.

P2X7 Receptor Agonist 2'(3')-O-(4-Benzoylbenzoyl)ATP Differently Modulates Cell Viability and Corticostriatal Synaptic Transmission in Experimental Models of Huntington's Disease.

Huntington's disease (HD) is a life-threatening neurodegenerative disorder. Altered levels and functions of the purinergic ionotropic P2X7 receptors (P2X7Rs) have been found in animal and cellular models of HD, suggesting their possible role in the pathogenesis of the disease; accordingly, the therapeutic potential of P2X7R antagonists in HD has been proposed. Here we further investigated the effects of P2X7R ligands in in vitro and ex vivo HD experimental models. In ST14A/Q120 rat striatal cells, we found a reduction of P2X7R expression; however, the P2X7R agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate (BzATP) induced cellular death, and this effect was fully reversed by the antagonist periodate-oxidized adenosine 5'-triphosphate (OxATP). Moreover, in corticostriatal slices from symptomatic R6/2 mice, BzATP reduced the synaptic transmission to a larger extent than in wild-type (WT) mice. Such an effect was accompanied by a concomitant increase of the paired-pulse ratio, suggesting a presynaptic inhibitory action. This was confirmed to be the case, since while the effects of BzATP were unaffected by the P2X7R antagonist OxATP, they were blocked by the adenosine A1 receptor (A1R) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), suggesting possible BzATP hydrolysis to 2'(3')-O-(4-benzoylbenzoyl)adenosine (Bz-adenosine) and consequent activation of A1Rs as a mechanism. Taken together, these data point out that 1) P2X7R expression and activity are confirmed to be altered in the presence of HD mutation; 2) in some experimental settings, such an abnormal functioning can be ascribed to presynaptic A1Rs activation.