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Well-differentiated liposarcoma (WDLPS) is a malignant neoplasia hard to diagnose and treat. Its main molecular signature is amplification of the MDM2-containing genomic region. The MDM2 oncogene is the master regulator of p53: its overexpression enhances p53 degradation and inhibits apoptosis, leading to the tumoral phenotype. Here, we show that the MDM2 inducible promoter G-rich region folds into stable G-quadruplexes both in vitro and in vivo and it is specifically recognized by cellular helicases. Cell treatment with G-quadruplex-ligands reduces MDM2 expression and p53 degradation, thus stimulating cancer cell cycle arrest and apoptosis. Structural characterization of the MDM2 G-quadruplex revealed an extraordinarily stable, unique four-tetrad antiparallel dynamic conformation, amenable to selective targeting. These data indicate the feasibility of an out-of-the-box G-quadruplex-targeting approach to defeat WDLPS and all tumours where restoration of wild-type p53 is sought. They also point to G-quadruplex-dependent genomic instability as possible cause of MDM2 expansion and WDLPS tumorigenesis., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Naphthalene diimides showed significant anticancer activity in animal models, with therapeutic potential related to their ability to strongly interact with G-quadruplexes. Recently, a trifunctionalized naphthalene diimide, named NDI-5, was identified as the best analogue of a mini-library of novel naphthalene diimides for its high G-quadruplex binding affinity along with marked, selective anticancer activity, emerging as promising candidate drug for in vivo studies. Here we used NMR, dynamic light scattering, circular dichroism and fluorescence analyses to investigate the interactions of NDI-5 with G-quadruplexes featuring either parallel or hybrid topology. Interplay of different binding modes of NDI-5 to G-quadruplexes was observed for both parallel and hybrid topologies, with end-stacking always operative as the predominant binding event. While NDI-5 primarily targets the 5'-end quartet of the hybrid G-quadruplex model (m-tel24), the binding to a parallel G-quadruplex model (M2) occurs seemingly simultaneously at the 5'- and 3'-end quartets. With parallel G-quadruplex M2, NDI-5 formed stable complexes with 1:3 DNA:ligand binding stoichiometry. Conversely, when interacting with hybrid G-quadruplex m-tel24, NDI-5 showed multiple binding poses on a single G-quadruplex unit and/or formed different complexes comprising two or more G-quadruplex units. NDI-5 produced stabilizing effects on both G-quadruplexes, forming complexes with dissociation constants in the nM range., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Targeting of G-quadruplexes, non-canonical conformations that form in G-rich regions of nucleic acids, has been proposed as a novel therapeutic strategy toward several diseases, including cancer and infections. The unavailability of highly selective molecules targeting a G-quadruplex of choice has hampered relevant applications. Herein, we describe a novel approach, based on naphthalene diimide (NDI)-peptide nucleic acid (PNA) conjugates, taking advantage of the cooperative interaction of the NDI with the G-quadruplex structure and hybridization of the PNA with the flanking region upstream or downstream the targeted G-quadruplex. By biophysical and biomolecular assays, we show that the NDI-PNA conjugates are able to specifically recognize the G-quadruplex of choice within the HIV-1 LTR region, consisting of overlapping and therefore mutually exclusive G-quadruplexes. Additionally, the conjugates can induce and stabilize the least populated G-quadruplex at the expenses of the more stable ones. The general and straightforward design and synthesis, which readily apply to any G4 target of choice, together with both the red-fluorescent emission and the possibility to introduce cellular localization signals, make the novel conjugates available to selectively control G-quadruplex folding over a wide range of applications., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Biological transmission of arthropod-borne viruses (arboviruses) to vertebrate hosts by hematophagous insects poses a global threat because such arboviruses can result in a range of serious public health infectious diseases. Sindbis virus (SINV), the prototype Alphavirus, was used to track infections in the posterior midgut (PMG) of Aedes aegypti adult mosquitoes. Females were fed viremic blood containing a virus reporter, SINV [Thosea asigna virus-green fluorescent protein (TaV-GFP)], that leaves a fluorescent signal in infected cells. We assessed whole-mount PMGs to identify primary foci, secondary target tissues, distribution, and virus persistence. Following a viremic blood meal, PMGs were dissected and analyzed at various days of post blood-feeding. We report that virus foci indicated by GFP in midgut epithelial cells resulted in a 9.8% PMG infection and a 10.8% dissemination from these infected guts. The number of virus foci ranged from 1 to 3 per individual PMG and was more prevalent in the PMG-middle > PMG-frontal > PMG-caudal regions. SINV TaV-GFP was first observed in the PMG (primary target tissue) at 3 days post blood-feeding, was sequestered in circumscribed foci, replicated in PMG peristaltic muscles (secondary target tissue) following dissemination, and GFP was observed to persist in PMGs for 30 days postinfection.

Guanine-rich nucleic acid sequences can fold into four-stranded G-quadruplex (G4) structures. Despite growing evidence for their biological significance, considerable work still needs to be done to detail their cellular occurrence and functions. Herein, we describe an optimized core-extended naphthalene diimide (cex-NDI) to be exploited as a G4 light-up sensor. The sensing mechanism relies on the shift of the aggregate-monomer equilibrium towards the bright monomeric state upon G4 binding. In contrast with the majority of other ligands, this novel cex-NDI is able to discriminate among G4s with different topologies, with a remarkable fluorescent response for the parallel ones. We investigate this sensing by means of biophysical methods, comparing the lead compound to a non-selective analogue. We demonstrate that mitigating the affinity of the binding core for G4s results in an increased selectivity and sensitivity of the fluorescent response. This is achieved by replacing positively charged substituents with diethylene glycol (DEG) side chains. Remarkably, the limit of detection values obtained for parallel G4s are more than one order of magnitude lower than those of the parallel-selective ligand N-methyl mesoporphyrin IX (NMM). Interestingly, the classical fluorescent intercalator displacement (FID) assay failed to reveal binding of cex-NDI to G4 because of the presence a ternary complex (G4-TO-cex-NDI) revealed by electrospray-MS. Our study thus provides a rational basis to design or modify existent scaffolds to redirect the binding preference of G4 ligands.

Aedes aegypti (L.) (Diptera: Culicidae) female mosquitoes infected systemically with Sindbis virus (SINV) took longer than uninfected mosquitoes to locate and fully engorge on blood. On days 7 and 14 postexposure, blood feeding took 1.3 and 1.5 times longer in mosquitoes with a disseminated SINV infection, respectively. SINV dissemination did not affect the average weight of unfed Ae. aegypti, but did result in a 10 and 12% increase in blood imbibed compared with mosquitoes without a positive SINV dissemination and non-SINV-exposed mosquitoes, respectively. Ae. aegypti mosquitoes with a disseminated SINV infection fed an average of 4 h sooner than uninfected mosquitoes when offered a bloodmeal contained inside a DEET (N,N-diethyl-3-methylbenzamide) saturated (30%) bovine sausage casing. Together, these results indicate that behavioral changes in mosquito host-seeking, blood feeding and sensitivity to DEET occurred in mosquitoes after SINV infection and dissemination.

To determine whether a Sindbis virus (family Togaviridae, genus Alphavirus, SINV) infection in Aedes aegypti (L.) (Diptera: Culicidae) affected its response to the repellent DEET, we orally exposed Ae. aegypti to an artificial bloodmeal containing SINV or diluent and then allowed to feed on a 10% sucrose suspension containing 3% DEET. When tested seven or more days after the initial bloodmeal, although none of the diluent-exposed mosquitoes fed on the DEET-sucrose suspension, at least 60% of the SINV-exposed mosquitoes fed on the suspension. When legs from the SINV-exposed mosquitoes were tested to determine dissemination status, 89% of those that fed on the DEET-sucrose suspension contained virus. In contrast, only 34% of the nonfeeders had a disseminated infection. When offered a choice between sucrose with or without DEET, a significantly higher percentage of the SINV-exposed mosquitoes than the control mosquitoes fed on the sucrose containing 3% DEET. Together, these results indicate that mosquitoes with a disseminated SINV infection may be less responsive to DEET than uninfected mosquitoes. Therefore, repellent use may be less effective in deterring infected mosquitoes from biting than previously believed.

Differential host cell responses to the alphavirus Sindbis were observed in visceral muscles of the adult female mosquito Aedes albopictus. Following intrathoracic inoculation with SIN, muscles associated with the midgut, hindgut, and ovary resulted in clearance, persistence, and refractoriness to virus, respectively. Prominent sarcomeres characteristic of myofilaments were identified in muscles associated with these three organs by phalloidin labeling of actin, confirming these cells as muscle. The location of virus antigen mimicked the distribution of actin in both mid- and hindgut-associated muscles. Furthermore, these myofilaments remained intact following virus clearance from midgut muscles and during virus persistence in hindgut muscles. Changes in the temporal onset of virus antigen following high titer inoculum compared with standard titer inoculum was observed in anterior midgut muscles, but not in muscles associated with the posterior midgut or hindgut. Muscle bundles closely approximated the gut surface, while a wispy association was displayed at the ovary surface. Prominent ultrastructural differences were observed in the basal lamina attached to the gut compared with the ovary. Additionally, ultrastructural evidence for virus-associated pathology was observed in gut-associated muscles and gut epithelium. Visceral muscles, all composed of the same tissue type, but associated to three different organs in the insect abdomen, responded differentially to Sindbis. We speculate that variations in structure, function or physiology and ultrastructure inherent to insect host cells or organs interactions reflect the complicated milieu of the organism and contribute to differential virus phenotypic expression in muscle cells.

Virus dissemination and associated pathology were examined in Aedes albopictus after intrathoracic inoculation of Sindbis virus (SIN), the prototypic Alphavirus. At 10 days postinfection, virus RNA was detected in all three-body segments of the insect. Colocalization of virus antigen with structural pathology was observed in mosquito salivary glands and midgut-associated visceral muscles, representing yet another example of arbovirus-associated pathology in a mosquito host. SIN antigen and gross pathology were detected in lateral lobes, but not the medial lobe of salivary glands, whereas virus antigen, vacuolated cytoplasm, and myofilament misalignment were detected in the visceral muscles at the midgut exterior surface. Early in the midgut infection, virus antigen was localized in small foci on the organ surface that progressed to a grate work-like banding pattern that eventually cleared. Both the salivary glands and the midgut are essential to insect survival and reproduction. Additionally, these organs provide a pathway for virus transmission in nature. Although SIN infection may not shorten the mosquito life span, persistent coexistence could permit survival of both host and microbe as well as contribute to alterations in insect behavior.