RVG29-Functionalized Lipid Nanoparticles for Quercetin Brain Delivery and Alzheimers Disease

Purpose: Lipid nanoparticles (SLN and NLC) were functionalized with the RVG29 peptide in order to target the brain and increase the neuronal uptake through the nicotinic acetylcholine receptors. These nanosystems were loaded with quercetin to take advantage of its neuroprotective properties mainly for Alzheimer's disease. Methods: The functionalization of nanoparticles with RVG29 peptide was confirmed by NMR and FTIR. Their morphology was assessed by transmission electron microscopy and nanoparticles size, polydispersity and zeta potential were determined by dynamic light scattering. The in vitro validation tests were conducted in hCMEC/D3 cells, a human blood-brain barrier model and thioflavin T binding assay was conducted to assess the process of amyloid-beta peptide fibrillation typical of Alzheimer's disease. Results: RVG29-nanoparticles displayed spherical morphology and size below 250 nm, which is compatible with brain applications. Zeta potential values were between −20 and −25 mV. Quercetin entrapment efficiency was generally higher than 80% and NLC nanoparticles were able to encapsulate up to 90%. The LDH assay showed that there is no cytotoxicity in hCMEC/D3 cell line and RVG29-nanoparticles clearly increased in 1.5-fold the permeability across the in vitro model of blood-brain barrier after 4 h of incubation compared with non-functionalized nanoparticles. Finally, this nanosystem was capable of inhibiting amyloid-beta aggregation in thioflavin T binding assay, suggesting its great potential for neuroprotection. Conclusions: RVG29-nanoparticles that simultaneously target the blood-brain barrier and induce neurons protection against amyloid-beta fibrillation proved to be an efficient way of quercetin delivery and a promising strategy for future approaches in Alzheimer's disease. [Figure not available: see fulltext.]. (c) 2020, Springer Science+Business Media, LLC, part of Springer Nature

Chronic stress targets adult neurogenesis preferentially in the suprapyramidal blade of the rat dorsal dentate gyrus

First Online: 29 August 2017The continuous generation of new neurons and glial cells in the adult hippocampal dentate gyrus (DG) represents an important form of adult neuroplasticity, involved in normal brain function and behavior but also associated with the etiopathogenesis and treatment of psychiatric disorders. Despite the large number of studies addressing cell genesis along the septotemporal axis, data on the anatomical gradients of cytogenesis along the DG transverse axis is scarce, especially after exposure to stress. As such, in this study we characterized both basal proliferation and survival of adult-born neural cells along the transverse axis of the rat dorsal DG, and after stress exposure. In basal conditions, both proliferating cells and newborn neurons and glial cells were preferentially located at the subgranular zone and suprapyramidal blade. Exposure to chronic stress induced an overall decrease in the generation of adult-born neural cells and, more specifically, produced a regional-specific decrease in the survival of adult-born neurons at the suprapyramidal blade. No particular region-specific alterations were observed on surviving adult-born glial cells. This work reveals, for the first time, a distinct survival profile of adult-born neural cells, neurons and glial cells, among the transverse axis of the DG, in both basal and stress conditions. Our results unveil that adult-born neurons are preferentially located in the suprapyramidal blade and suggest a regional-specific impact of chronic stress in this blade with potential repercussions for its functional significance.NDA, PP, AMP, ARMS, MM and LP received fellowships from the Portuguese Foundation for Science and Technology (FCT). This work was funded by FCT (IF/01079/2014). This article has been developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). This work has been funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038.info:eu-repo/semantics/publishedVersio

Targeting p53 for melanoma treatment: counteracting tumour proliferation, dissemination and therapeutic resistance

Melanoma is the deadliest form of skin cancer, primarily due to its high metastatic propensity and therapeutic resistance in advanced stages. The frequent inactivation of the p53 tumour suppressor protein in melanomagenesis may predict promising outcomes for p53 activators in melanoma therapy. Herein, we aimed to investigate the antitumor potential of the p53-activating agent SLMP53-2 against melanoma. Two- and three-dimensional cell cultures and xenograft mouse models were used to unveil the antitumor activity and the underlying molecular mechanism of SLMP53-2 in melanoma. SLMP53-2 inhibited the growth of human melanoma cells in a p53-dependent manner through induction of cell cycle arrest and apoptosis. Notably, SLMP53-2 induced p53 stabilization by disrupting the p53–MDM2 interaction, enhancing p53 transcriptional activity. It also promoted the expression of p53-regulated microRNAs (miRNAs), including miR-145 and miR-23a. Moreover, it displayed anti-invasive and antimigratory properties in melanoma cells by inhibiting the epithelial-to-mesenchymal transition (EMT), angiogenesis and extracellular lactate production. Importantly, SLMP53-2 did not induce resistance in melanoma cells. Additionally, it synergized with vemurafenib, dacarbazine and cisplatin, and resensitized vemurafenib-resistant cells. SLMP53-2 also exhibited antitumor activity in human melanoma xenograft mouse models by repressing cell proliferation and EMT while stimulating apoptosis. This work discloses the p53-activating agent SLMP53-2 which has promising therapeutic potential in advanced melanoma, either as a single agent or in combination therapy. By targeting p53, SLMP53-2 may counteract major features of melanoma aggressiveness.This work received financial support from PT national funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) through LAQV/REQUIMTE (UID/QUI/50006/2020), iMed.ULisboa (UIDB/04138/2020), and PTDC/QUIQOR/29664/2017, PTDC/MEC-ONC/32018/2017. We thank FCT for the fellowships SFRH/BD/ 128673/2017 (J. Loureiro), 2020.04613.BD (J. Calheiros), PD/BD/143126/2019 (V. Barcherini)

Beyond new neurons in the adult hippocampus: imipramine acts as a pro-astrogliogenic factor and rescues cognitive impairments induced by stress exposure

Depression is a prevalent, socially burdensome disease. Different studies have demonstrated the important role of astrocytes in the pathophysiology of depression as modulators of neurotransmission and neurovascular coupling. This is evidenced by astrocyte impairments observed in brains of depressed patients and the appearance of depressive-like behaviors upon astrocytic dysfunctions in animal models. However, little is known about the importance of de novo generated astrocytes in the mammalian brain and in particular its possible involvement in the precipitation of depression and in the therapeutic actions of current antidepressants (ADs). Therefore, we studied the modulation of astrocytes and adult astrogliogenesis in the hippocampal dentate gyrus (DG) of rats exposed to an unpredictable chronic mild stress (uCMS) protocol, untreated and treated for two weeks with antidepressants—fluoxetine and imipramine. Our results show that adult astrogliogenesis in the DG is modulated by stress and imipramine. This study reveals that distinct classes of ADs impact differently in the astrogliogenic process, showing different cellular mechanisms relevant to the recovery from behavioral deficits induced by chronic stress exposure. As such, in addition to those resident, the newborn astrocytes in the hippocampal DG might also be promising therapeutic targets for future therapies in the neuropsychiatric field.ARMS: ELC, NDA, PP, AMP, JSC, MM, AJR, JFO, and L.P. received fellowships from the Portuguese Foundation for Science and Technology (FCT) (IF/00328/2015 to J.F.O.; 2020.02855.CEECIND to LP). This work was funded by FCT (IF/01079/2014, PTDC/MED-NEU/31417/2017 Grant to JFO), BIAL Foundation Grants (037/18 to J.F.O. and 427/14 to L.P.), “la Caixa” Foundation Health Research Grant (LCF/PR/HR21/52410024) and Nature Research Award for Driving Global Impact—2019 Brain Sciences (to L.P.). This was also co-funded by the Life and Health Sciences Research Institute (ICVS), and by FEDER, through the Competitiveness Internationalization Operational Program (POCI), and by National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020. Moreover, this work has been funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122; by National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020; “la Caixa” Foundation (ID 100010434 to A.J.R.), under the agreement LCF/PR/HR20/52400020; and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 101003187 to A.J.R.)

Yicathins B and C and analogues: total synthesis, lipophilicity and biological activities

Natural products had always be an important source of new hits and leads in drug discovery. The marine environment has been regarded as a significant souce of novel and exquisite bioactive compounds. Yicathins B and C are two marine derived xanthones that have shown antibacterial and antifungal activities. Herein, the total synthesis of these yicathins is reported for the first time as well as six novel analogues. As marine natural products tend to bear very lipophilic scaffolds, the lipophilicity of yicathins and its analogues was evaluated using the classical octanol:water system and a biomimetic model based system. As the xanthonic nucleus is a â privileged structureâ , other biological activities were evaluated, namely antitumor and anti-inflammatory activities. An interesting anti-inflammatory activity was identified for yicathins analogues that paves the way for the design of dual activity (anti-infective and anti-inflammatory) marine inspired xanthones derivatives.This work was supported through national funds provided by FCT/MCTES - Foundation for Science and Technology from the Minister of Science, Technology and Higher Education (PIDDAC) and European Regional Development Fund (ERDF) through the COMPETE - Programa Operacional Factores de Competitividade (POFC) programme, under the projects PTDC/MAR-BIO/4694/2014 (reference POCI-01-0145-FEDER-016790; Project 3599 - Promover a ProducAo Cientifica e Desenvolvimento Tecnologico e a ConstituicAo de Redes Tematicas (3599-PPCDT)) and PTDC/SAU-PUB/28736/2017 (reference POCI-01-0145-FEDER- 028736) in the framework of the programme PT2020. D. R. P. L. is grateful for research grant PTDC/MAR-BIO/4694/2014-BI-2017-003. J. X. S. thanks the FCT PhD Programmes and Programa Operacional Capital Humano (POCH), specifically the BiotechHealth Programme (Doctoral Programme on Cellular and Molecular Biotechnology Applied to Health Sciences), reference PD/00016/2012; through the FCT and POCH for PhD grants (SFRH/BD/98105/2013 and SFRH/BD/116167/2016). The authors would like to thank Sara Cravo and Gisela Adriano for the technical support, the Centro de Apoio Cientifico e Tecnoloxico a Investigation (C.A.C.T.I., University of Vigo, Pontevedra, Spain) for HRMS analysis, the Centro de Materiais da Universidade do Porto (CEMUP, Porto, Portugal) for HRMS, and the Departamento de Quimica da Universidade de Aveiro (Portuguese NMR network) for the NMR analysis

SLMP53-2 Restores Wild-Type-Like Function to Mutant p53 through Hsp70: Promising Activity in Hepatocellular Carcinoma.

Half of human cancers harbor TP53 mutations that render p53 inactive as a tumor suppressor. In these cancers, reactivation of mutant p53 (mutp53) through restoration of wild-type-like function constitutes a valuable anticancer therapeutic strategy. In order to search for mutp53 reactivators, a small library of tryptophanol-derived oxazoloisoindolinones was synthesized and the potential of these compounds as mutp53 reactivators and anticancer agents was investigated in human tumor cells and xenograft mouse models. By analysis of their anti-proliferative effect on a panel of p53-null NCI-H1299 tumor cells ectopically expressing highly prevalent mutp53, the compound SLMP53-2 was selected based on its potential reactivation of multiple structural mutp53. In mutp53-Y220C-expressing hepatocellular carcinoma (HCC) cells, SLMP53-2-induced growth inhibition was mediated by cell cycle arrest, apoptosis, and endoplasmic reticulum stress response. In these cells, SLMP53-2 restored wild-type-like conformation and DNA-binding ability of mutp53-Y220C by enhancing its interaction with the heat shock protein 70 (Hsp70), leading to the reestablishment of p53 transcriptional activity. Additionally, SLMP53-2 displayed synergistic effect with sorafenib, the only approved therapy for advanced HCC. Notably, it exhibited potent antitumor activity in human HCC xenograft mouse models with a favorable toxicological profile. Collectively, SLMP53-2 is a new mutp53-targeting agent with promising antitumor activity, particularly against HCC

Can parasites adapt to pollutants? A multigenerational experiment with a Daphnia x Metschnikowia model system exposed to the fungicide tebuconazole

There is increasing evidence about negative effects of fungicides on non-target organisms, including parasitic species, which are key elements in food webs. Previous experiments showed that environmentally relevant concentrations of fungicide tebuconazole are toxic to the microparasite Metschnikowia bicuspidata, a yeast species that infects the planktonic crustacean Daphnia spp. However, due to their short-term nature, this and other experimental studies were not able to test if parasites could potentially adapt to these contaminants. Here, we tested if M. bicuspidata parasite can adapt to tebuconazole selective pressure. Infected D. magna lineages were reared under control conditions (no tebuconazole) and environmentally realistic tebuconazole concentrations, for four generations, and their performance was compared in a follow-up reciprocal assay. Additionally, we assessed whether the observed effects were transient (phenotypic) or permanent (genetic), by reassessing parasite fitness after the removal of selective pressure. Parasite fitness was negatively affected throughout the multigenerational exposure to the fungicide: prevalence of infection and spore load decreased, whereas host longevity increased, in comparison to control (naive) parasite lineages. In a follow-up reciprocal assay, tebuconazole-conditioned (TEB) lineages performed worse than naive parasite lineages, both in treatments without and with tebuconazole, confirming the cumulative negative effect of tebuconazole. The underperformance of TEB lineages was rapidly reversed after removing the influence of the selective pressure (tebuconazole), demonstrating that the costs of prolonged exposure to tebuconazole were phenotypic and transient. The microparasitic yeast M. bicuspidata did not reveal potential for rapid evolution to an anthropogenic selective pressure; instead, the long-term exposure to tebuconazole was hazardous to this non-target species. These findings highlight the potential environmental risks of azole fungicides on non-target parasit- This work was supported by the European Regional Development Fund (programmes COMPETE2020 and PT2020) and by National Funds (Portuguese Science Foundation - FCT, I.P.), through the strategic programmes UID/AMB/50017/2013 and UID/BIA/04050/2019 (POCI-01-0145-FEDER-007569), as well as by the research projects FunG-Eye (POCI-01-0145-FEDER-029505) and EcoAgriFood (NORTE-01-0145-FEDER-000009). Part of the work presented here was developed during the PhD of Ana P. Cuco, who was supported by FCT (PhD grant SFRH/BD/81661/2011). JW was supported by Beethoven Life-1 grant from the German Science Foundation and Polish National Science Center (WO 1587/9-1). Nelson Abrantes is the recipient of an individual postdoctoral research contract (CEECIND/01653/2017)

White matter microstructural perturbations after total sleep deprivation in depression

BackgroundTotal sleep deprivation (TSD) transiently reverses depressive symptoms in a majority of patients with depression. How TSD modulates diffusion tensor imaging (DTI) measures of white matter (WM) microstructure, which may be linked with TSD’s rapid antidepressant effects, remains uncharacterized.MethodsPatients with depression (N = 48, mean age = 33, 26 women) completed diffusion-weighted imaging and Hamilton Depression Rating (HDRS) and rumination scales before and after >24 h of TSD. Healthy controls (HC) (N = 53, 23 women) completed the same assessments at baseline, and after receiving TSD in a subset of HCs (N = 15). Tract based spatial statistics (TBSS) investigated voxelwise changes in fractional anisotropy (FA) across major WM pathways pre-to-post TSD in patients and HCs and between patients and HCs at baseline. Post hoc analyses tested for TSD effects for other diffusion metrics, and the relationships between change in diffusion measures with change in mood and rumination symptoms.ResultsSignificant improvements in mood and rumination occurred in patients with depression (both p < 0.001), but not in HCs following TSD. Patients showed significant (p < 0.05, corrected) decreases in FA values in multiple WM tracts, including the body of the corpus callosum and anterior corona radiata post-TSD. Significant voxel-level changes in FA were not observed in HCs who received TSD (p > 0.05). However, differential effects of TSD between HCs and patients were found in the superior corona radiata, frontal WM and the posterior thalamic radiation (p < 0.05, corrected). A significant (p < 0.05) association between change in FA and axial diffusivity within the right superior corona radiata and improvement in rumination was found post-TSD in patients.ConclusionTotal sleep deprivation leads to rapid microstructural changes in WM pathways in patients with depression that are distinct from WM changes associated with TSD observed in HCs. WM tracts including the superior corona radiata and posterior thalamic radiation could be potential biomarkers of the rapid therapeutic effects of TSD. Changes in superior corona radiata FA, in particular, may relate to improvements in maladaptive rumination

Impact of Continuous Axenic Cultivation in Leishmania infantum Virulence

Experimental infections with visceral Leishmania spp. are frequently performed referring to stationary parasite cultures that are comprised of a mixture of metacyclic and non-metacyclic parasites often with little regard to time of culture and metacyclic purification. This may lead to misleading or irreproducible experimental data. It is known that the maintenance of Leishmania spp. in vitro results in a progressive loss of virulence that can be reverted by passage in a mammalian host. In the present study, we aimed to characterize the loss of virulence in culture comparing the in vitro and in vivo infection and immunological profile of L. infantum stationary promastigotes submitted to successive periods of in vitro cultivation. To evaluate the effect of axenic in vitro culture in parasite virulence, we submitted L. infantum promastigotes to 4, 21 or 31 successive in vitro passages. Our results demonstrated a rapid and significant loss of parasite virulence when parasites are sustained in axenic culture. Strikingly, the parasite capacity to modulate macrophage activation decreased significantly with the augmentation of the number of in vitro passages. We validated these in vitro observations using an experimental murine model of infection. A significant correlation was found between higher parasite burdens and lower number of in vitro passages in infected Balb/c mice. Furthermore, we have demonstrated that the virulence deficit caused by successive in vitro passages results from an inadequate capacity to differentiate into amastigote forms. In conclusion, our data demonstrated that the use of parasites with distinct periods of axenic in vitro culture induce distinct infection rates and immunological responses and correlated this phenotype with a rapid loss of promastigote differentiation capacity. These results highlight the need for a standard operating protocol (SOP) when studying Leishmania species

Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity

A growing interest in asparagine (Asn) metabolism has currently been observed in cancer and infection fields. Asparagine synthetase (AS) is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A, and the type B, which preferably uses glutamine. Absent in humans and present in trypanosomatids, AS-A was worthy of exploring as a potential drug target candidate. Appealingly, it was reported that AS-A was essential in Leishmania donovani, making it a promising drug target. In the work herein we demonstrate that Leishmania infantum AS-A, similarly to Trypanosoma spp. and L. donovani, is able to use both ammonia and glutamine as nitrogen donors. Moreover, we have successfully generated LiASA null mutants by targeted gene replacement in L. infantum, and these parasites do not display any significant growth or infectivity defect. Indeed, a severe impairment of in vitro growth was only observed when null mutants were cultured in asparagine limiting conditions. Altogether our results demonstrate that despite being important under asparagine limitation, LiAS-A is not essential for parasite survival, growth or infectivity in normal in vitro and in vivo conditions. Therefore we exclude AS-A as a suitable drug target against L. infantum parasites