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Alzheimer’s disease (AD) is the most common form of dementia worldwide, characterized by progressive memory impairment, behavioral changes, and, ultimately, loss of consciousness and death. Recently, microRNA (miRNA) dysfunction has been associated with increased production and impaired clearance of amyloid-β (Aβ) peptides, whose accumulation is one of the most well-known pathophysiological markers of this disease. In this study, we identified several miRNAs capable of targeting key proteins of the amyloidogenic pathway. The expression of one of these miRNAs, miR-31, previously found to be decreased in AD patients, was able to simultaneously reduce the levels of APP and Bace1 mRNA in the hippocampus of 17-month-old AD triple-transgenic (3xTg-AD) female mice, leading to a significant improvement of memory deficits and a reduction in anxiety and cognitive inflexibility. In addition, lentiviral-mediated miR-31 expression significantly ameliorated AD neuropathology in this model, drastically reducing Aβ deposition in both the hippocampus and subiculum. Furthermore, the increase of miR-31 levels was enough to reduce the accumulation of glutamate vesicles in the hippocampus to levels found in non-transgenic age-matched animals. Overall, our results suggest that miR-31-mediated modulation of APP and BACE1 can become a therapeutic option in the treatment of AD., Graphical Abstract

Background: Dementia is a complex pathological state that affects millions of individuals worldwide and is responsible for a huge socioeconomic burden, making it a major health concern of current times. Given the impact of dementia in both patients and caregivers, it is crucial to fully clarify the molecular mechanisms underlying dementia-associated disorders, since without this knowledge our ability to correctly diagnose and treat these diseases is severely hampered. Methods: Epigenetic mechanisms, such as miRNA-mediated post-transcriptional regulation, have been reported to play a role in dementia pathogenesis. Given their ability to bind complementary mRNA sequences, miRNAs are able to induce temporary or permanent translation repression of their mRNA targets. Results: Consequently, changes in miRNA levels may contribute to alterations in the expression of dementiarelated proteins, impacting the course of the disease. Conversely, studies have also reported that some of these proteins are able to regulate the biogenesis and transport of miRNAs, hinting at novel disease-related mechanisms that are now beginning to be explored. These findings have made miRNAs both interesting tools and promising targets in the design of novel therapeutic strategies. Moreover, the discovery of circulating miRNAs, which are released by cells of various tissues, including the brain, and travel in membrane-bound vesicles found in most biofluids, opened new possibilities concerning the usefulness of miRNAs as biomarkers of disease. Conclusion: In this context, the major aim of this review is to discuss the relevance of these small non-coding RNAs in dementia, focusing on their role as gene expression regulators, their potential as biomarkers of dementia subtype and stage, and the hypothesis of using miRNA modulation as an innovative therapeutic approach to treat dementia-related disorders.

The design and synthesis of a novel bis-furan scaffold tailored for high efficiency at inhibiting transthyretin amyloid formation is reported. In vitro results show that the discovered compounds are more efficient inhibitors of amyloid formation than tafamidis, a drug currently used in the treatment of familial amyloid polyneuropathy (FAP), despite their lower molecular weight and lipophilicity. Moreover, ex vivo experiments with the strongest inhibitor in the series, conducted in human blood plasma from normal and FAP Val30Met-transthyretin carriers, disclose remarkable affinity and selectivity profiles. The promises and challenges facing further development of this compound are discussed under the light of increasing evidence implicating transthyretin stability as a key factor not only in transthyretin amyloidoses and several associated co-morbidities, but also in Alzheimer's disease.

A novel synthetic approach to bis(indolyl)methanes has been established. Our one-pot synthetic strategy based on two consecutive hetero-Diels-Alder cycloaddition reactions of electrophilic conjugated nitrosoalkenes with indoles was extended to a range of new 1-hydroxyiminomethyl-bis(indolyl) methanes. Furthermore, a similar and broad range approach was applied to the synthesis of previously unknown 1-hydrazonomethyl-bis(indolyl)methanes. The biological evaluation of the new bis(indolyl) methanes as anti-cancer agents was investigated. (C) 2016 Elsevier Ltd. All rights reserved. Portuguese Agency for Scientific Research Fundacao para a Ciencia e a Tecnologia (FCT) [007630 UID/QUI/00313/2013] COMPETE-UE FCT Coimbra Chemistry Centre [CQC-QO-BPD-2015]

The increasing knowledge on the genetic basis of disease has allowed the development of promising gene-targeted therapies that can be applied to numerous diseases. Such genetic-based approaches involve the use of nucleic acids as therapeutic agents, either for the insertion or repair and regulation of specific genes. However, the clinical application of these large and charged molecules remains highly dependent on the development of delivery systems capable of mediating efficient cellular uptake. Since the first observations, two decades ago, that some protein-derived domains can translocate across biological membranes, a wide group of peptides called cell-penetrating peptides (CPPs) have been considered one of the most promising tools to improve non-invasive cellular delivery of therapeutic molecules. The mechanistic basis of CPP and CPP conjugate cellular uptake remains controversial. However, biophysical studies on the interactions of CPPs with membrane models have contributed to unravel the mechanisms underlying CPP membrane translocation as well as to propose relationships between those mechanisms and CPP efficiency in mediating cargo delivery. In this review, representative examples of CPPs were gathered from the most recent literature in order to emphasize the contributions of chemists, biophysicists and cell biologists towards the rational design of increasingly more efficient delivery systems. In this context, the present review aims at giving an overview of some of the most significant CPP families and their biological applications as nucleic acid delivery systems.

The reactivity of buta-2,3-dienoates towards aziridines is reported. Typically, allenoates react as the 2π-component in the [3+2] cycloaddition with azomethine ylides generated from aziridines, affording 4-methylenepyrrolidines in a site-, regio- and stereoselective fashion. However, N-cyclohexyl- or N-tert-butyl-2-benzoyl-3-phenylaziridines showed a different reactivity in the reaction with buta-2,3-dienoates. Pyrrole derivatives were obtained as single or major products resulting from a formal [3+2] cycloaddition via C–N bond cleavage of the three-membered ring heterocycle leading to functionalized pyrroles. From the reaction with allenoates bearing bulkier C-4 substituents 4-methylenepyrrolidines were also formed as minor products.

Stereoselective inverse conjugate addition of carbonucleophiles to allenoates bearing a chiral auxiliary in the ester moiety afforded optically active α,β-unsaturated carboxylic esters bearing a new stereocenter at the δ position. The rationalization of the observed selectivity was supported by semi-empirical molecular orbital calculations.

The synthesis of chiral functionalized β-amino esters via the hydride reductive amination of chiral allenes was explored. These compounds can be regarded as β-peptoids building blocks bearing a chiral side chain at the nitrogen and at the same time retaining the β-amino acid side chain. β-Enamino esters were obtained from the nucleophilic addition of α-amino esters ( l -Ala, d -Ala, l -Phe, l -Leu, l -Trp and d -Trp methyl esters) to 2,3-allenoates bearing a chiral auxiliary, which determines the stereochemistry outcome of the subsequent reduction reaction. It was also demonstrated that in the reduction of β-enamino esters derived from l -Pro and d -Pro methyl esters the chirality of the new chiral center is controlled by the α-amino ester moiety.

A highly selective two-step approach to chiral β-amino esters via the hydride reductive amination of chiral allenes is reported. β-Enamino esters were obtained from the nucleophilic addition of amines to 2,3-allenoates bearing a chiral auxiliary. The reduction of the (1R)-(−)-10-phenylsulfonylisobornyl β-enamino esters gave the corresponding β-amino esters with S configuration whereas the reduction of the (1S)-(+)-10-phenylsulfonylisobornyl β-enamino esters led to β-amino esters with R configuration. The rationalization of the observed selectivity was supported by semi-empirical molecular orbital calculations (PM3).

The synthesis and reactivity of tetrazol-5-yl-phosphorus ylides towards N-halosuccinimide/TMSN3 reagent systems was explored, opening the way to new haloazidoalkenes bearing a tetrazol-5-yl substituent. These compounds were obtained as single isomers, except in one case. X-ray crystal structures were determined for three derivatives, establishing that the non-classical Wittig reaction leads to the selective synthesis of haloazidoalkenes with (Z)-configuration. The thermolysis of the haloazidoalkenes afforded new 2-halo-2-(tetrazol-5-yl)-2H-azirines in high yields. Thus, the reported synthetic methodologies gave access to important building blocks in organic synthesis, vinyl tetrazoles and 2-halo-2-(tetrazol-5-yl)-2H-azirine derivatives.

MiRNAs are short, evolutionary conserved noncoding RNA molecules with the ability to control the magnitude of inflammation. The immunosuppressive nature of the brain is sustained by miRNA-dependent regulation of microglial cells, which become activated under neuroinflammatory conditions, such as brain injury and neurodegeneration. The pro-inflammatory and suppressive role of the most studied neuroimmune miRNAs, miR-155 and miR-146a, has been recently challenged. Although the molecular targets of these miRNAs remain unchanged across brain diseases, different kinetics of miRNA expression and degradation can produce different immune outcomes and change microglia phenotypes. Here, we discuss current knowledge regarding the implications of disruption of miRNA networks in neuroinflammation and in the pathophysiology of acute and chronic CNS diseases.

Gene delivery targeting mitochondria has the potential to transform the therapeutic landscape of mitochondrial genetic diseases. Taking advantage of the nonuniversal genetic code used by mitochondria, a plasmid DNA construct able to be specifically expressed in these organelles was designed by including a codon, which codes for an amino acid only if read by the mitochondrial ribosomes. In the present work, gemini surfactants were shown to successfully deliver plasmid DNA to mitochondria. Gemini surfactant-based DNA complexes were taken up by cells through a variety of routes, including endocytic pathways, and showed propensity for inducing membrane destabilization under acidic conditions, thus facilitating cytoplasmic release of DNA. Furthermore, the complexes interacted extensively with lipid membrane models mimicking the composition of the mitochondrial membrane, which predicts a favored interaction of the complexes with mitochondria in the intracellular environment. This work unravels new possibilities for gene therapy toward mitochondrial diseases.

A versatile and broad range approach to previously unknown bis(indolyl)methane oximes based on two consecutive hetero Diels–Alder cycloaddition reactions of electrophilic conjugated nitrosoalkenes with indoles is disclosed. The cytotoxic properties and selectivity of some adducts against several human cancer cell lines pointing to a promising role in the development of anti-tumoural drugs, in particular for leukaemia and lymphoma.

Gene knockdown has emerged as an important tool for cancer gene therapy as well as for viral infections and dominantly inherited genetic disorders. The generation of suitable siRNA delivery systems poses some challenges, namely, to avoid nuclease degradation, to surpass the cytoplasmic membrane, and to release the nucleic acids into the cytosol. Aiming at evaluating the ability of thermoresponsive block copolymers formed by units of N-isopropylacrylamide and of (3- acrylamidopropyl)trimethylammonium chloride to efficiently deliver siRNAs, an extensive study was performed with four different copolymers using a human fibrosarcoma cell line as cell model. The silencing ability and cytotoxicity of the generated copolymer-based siRNA delivery systems were found to be dependent on the cloud point of the polymer, which corresponds to the transition temperature at which the aggregation or precipitation of the polymer molecules becomes thermodynamically more favorable than their solubilization. In the present study, a system capable of delivering siRNAs efficiently, specifically and without presenting relevant cytotoxicity, even in the presence of serum, was developed. Confocal fluorescence experiments showed that the ability of the generated systems to silence the target gene is related to some extent to nucleic acid internalization, being also dependent on polymer/siRNA dissociation at 37 °C. Thus, a delicate balance between nucleic acid internalization and intracellular release must be met in order to reach an ideal knockdown efficiency. The special features and potential for manipulation of the N-isopropylacrylamide-based copolymers make them suitable materials for the design and synthesis of new and promising siRNA delivery systems. This work was supported by the Portuguese Foundation for Science and Technology and FEDER/COMPETE (research grants PTDC/QUI-BIQ/103001/2008, PTDC/DTP-FTO/ 0265/2012, and Pest-C/SAU/LA0001/2013-2014) and by the Norwegian Research Council, Project Number 190403. A.M.C., C.M.M, and A.L.C. are recipients of fellowships from the Portuguese Foundation for Science and Technology (SFRH/BD/63288/2009, SFRH/BD/79077/2011, and SFRH/BPD/46228/2008, respectively).

The successful application of gene therapy approaches is highly dependent on the efficient delivery of nucleic acids into target cells. In the present study, new peptide-based nonviral systems were developed to enhance plasmid DNA and siRNA delivery, aiming at generating appropriate gene delivery and gene silencing tools for preclinical and clinical application. For this purpose, 0 cell-penetrating peptide derived from the wild-type S4(13)-PV peptide was synthesized through the addition of a five-histidine tail to its N-terminus (H-5-S4(13)-PV), and its ability to mediate gene expression and gene silencing was evaluated and compared to that of the wild-type peptide. The histidine-enriched peptide, H-5-S4(13)-PV, proved to be generally more efficient and less toxic than the wild-type peptide in the delivery of plasmid DNA. In addition, complexes of H-5-S4(13)-PV with siRNAs, but not of S4(13)-PV, were efficiently internalized by cells and presented high knockdown activity (63%). Interestingly, systems containing the S4(13)-PV or the H-5-S4(13)-PV peptide exhibited superior biological activity when compared to those containing the reverse NLS or scrambled peptides, suggesting that both the cell-penetrating; sequence and the NLS of the S4(13)-PV peptide influence the competence Of binary and ternary complexes to accomplish nucleic acid delivery. In order to unravel the cancer therapeutic potential of formulations with the histidine-enriched peptide, their efficiency to mediate silencing of the oncogenic protein survivin was evaluated. As opposed to complexes with the wild-type peptide, H-5-S4(13)-PV complexes showed the ability to promote a high survivin knockdown at the level of both protein (44%) and mRNA (73%), in HT1080 cells.

The present work aimed at the development and application of a lipid-based nanocarrier for targeted delivery of nucleic acids to glioblastoma (GBM). For this purpose, chlorotoxin (CTX), a peptide reported to bind selectively to glioma cells while showing no affinity for non-neoplastic cells, was covalently coupled to liposomes encapsulating antisense oligonucleotides (asOs) or small interfering RNAs (siRNAs). The resulting targeted nanoparticles, designated CTX-coupled stable nucleic acid lipid particles (SNALPs), exhibited excellent features for in vivo application, namely small size (

2H-Azirine-3-carboxylates unsubstituted at C-2 act as dipolarophiles in the reaction with diazomethane giving new 4,5-dihydro-3H-pyrazole derivatives. The synthesis of a pyrimidine was also achieved via 1,3-dipolar cycloaddition of methyl 2-bromo-3-phenyl-2H-azirine-2-carboxylate with an azomethine ylide. http://www.sciencedirect.com/science/article/B6THS-493P1M1-17/1/5df1370edad7138f3cae89fadd9b5c4d

Since clinical application of conventional cancer therapies is usually limited by drug resistance and toxic side effects, combination of chemotherapeutic agents with gene therapy appears as an attractive therapeutic strategy to overcome these issues. Being selectively expressed in tumor tissues, survivin is a promising target for the development of anticancer strategies aimed at eliminating tumor cells while sparing normal tissues. In this work, we achieved substantial protein knockdown in a number of human cell lines, namely, A549, HeLa and MCF-7 cells which overexpress survivin, after treatment with anti-survivin siRNAs, which was associated with a significant reduction of cell viability, when compared to treatment with control siRNAs. Interestingly, when the survivin-silencing approach was combined with a chemotherapeutic agent, an enhancement of the therapeutic effect was achieved. Treatment with anti-survivin siRNAs resulted in high levels of caspase 3/7 activation, and an enhancement of this effect was observed when survivin silencing was combined with vinblastine. In addition, we showed that for A549 and HeLa cells survivin silencing contributes to the reversion of cell resistance to doxorubicin. Overall, we demonstrate that the combination of a survivin-directed silencing strategy with chemotherapeutic agents constitutes a valuable approach for cancer treatment.

The successful clinical application of nucleic acid-based therapeutic strategies has been limited by the poor delivery efficiency achieved by existing vectors. The development of alternative delivery systems for improved biological activity is, therefore, mandatory. Since the seminal observations two decades ago that the Tat protein, and derived peptides, can translocate across biological membranes, cell-penetrating peptides (CPPs) have been considered one of the most promising tools to improve non-invasive cellular delivery of therapeutic molecules. Despite extensive research on the use of CPPs for this purpose, the exact mechanisms underlying their cellular uptake and that of peptide conjugates remain controversial. Over the last years, our research group has been focused on the S413-PV cell-penetrating peptide, a prototype of this class of peptides that results from the combination of 13-amino-acid cell penetrating sequence derived from the Dermaseptin S4 peptide with the SV40 large T antigen nuclear localization signal. By performing an extensive biophysical and biochemical characterization of this peptide and its analogs, we have gained important insights into the mechanisms governing the interaction of CPPs with cells and their translocation across biological membranes. More recently, we have started to explore this peptide for the intracellular delivery of nucleic acids (plasmid DNA, siRNA and oligonucleotides). In this review we discuss the current knowledge of the mechanisms responsible for the cellular uptake of cell-penetrating peptides, including the S413-PV peptide, and the potential of peptide-based formulations to mediate nucleic acid delivery.

Background Cell penetrating peptides have been successfully used to mediate the intracellular delivery of a wide variety of molecules of pharmacological interest. The main aim of the present work was to evaluate the potential of the S4(13)-PV cell penetrating peptide to mediate the intracellular delivery of plasmid DNA, aiming at its use in gene therapy applications. The S4(13)-PV cell penetrating peptide is a chimeric peptide that results from the combination of a cell penetrating sequence derived from the Dermaseptin S4 peptide with the nuclear localization signal present in the Simian Virus 40 (SV40) large T antigen. Methods S413-PV cell penetrating peptide and cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane:1,2-dioleoyl-sn-glycero-3-phosphoethanolamine were complexed with pDNA at different charge ratios. Complexation of pDNA was assessed by gel electrophoresis. Luciferase assay, fluorescence microscopy and fluorescence-activated cell sorting analysis were used to evaluate reporter gene delivery to TSA and HeLa cells. Cytotoxicity of the pDNA complexes was assessed by Alamar blue assay. Results Complexes obtained through electrostatic association of the S413-PV cell penetrating peptide with plasmid DNA are able to very efficiently mediate transfection, particularly at high peptide/DNA charge ratios. Additionally, our results clearly demonstrate that, both in HeLa and TSA cells, ternary complexes, resulting from association of cationic liposomes to peptide/DNA complexes, are significantly more efficient in mediating transfection than the corresponding peptide/DNA or cationic liposome/DNA complexes. Conclusions Overall, our data highlight the potential of cell penetrating peptides for the development of improved nonviral gene delivery systems. Copyright (C) 2008 John Wiley & Sons, Ltd.

Background RNA interference provides a powerful technology for specific gene silencing. Therapeutic applications of small interfering RNA (siRNA) however require efficient vehicles for stable complexation, protection, and extra- and intracellular delivery of these nucleic acids. Here, we evaluated the potential of transferrin (Tf)-associated liposomes for siRNA complexation and gene silencing. Methods Cationic liposomes composed of DOTAP : Cholesterol associated with or without transferrin (Tf) were complexed with siRNA at different lipid/siRNA charge ratios. Complexation and protection of siRNA from enzymatic degradation was assessed with the PicoGreen intercalation assay and gel electrophoresis. Cellular internalization of these siRNA Tf-lipoplexes was detected by confocal microscopy. Luciferase assay, immunoblot and fluorescence-activated cell sorting (FACS) analysis were used to evaluate reporter gene silencing in Huh-7 hepatocarcinoma and U-373 glioma cells. c-Jun knockdown in HT-22 cells was evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Cytotoxicity of the siRNA complexes was assessed by Alamar blue, lactate dehydrogenase and MTT assays. Results Complexation of siRNA with the cationic liposomes in the presence of Tf results in the formation of stable particles and prevents serum-mediated degradation. Confocal microscopy showed fast cellular internalization of the Tf-lipoplexes via endocytosis. In the GFP glioma cells Tf-lipoplexes showed enhanced gene silencing at minimum toxicity in comparison to Tf-free lipoplexes. Targeting luciferase in the hepatocarcinoma cell line resulted in more than 70% reduction of luciferase activity, while in HT-22 cells 50% knockdown of endogenous c-Jun resulted in a significant protection from glutamate-mediated toxicity. Conclusions Cationic liposomes associated with Tf form stable siRNA lipoplexes with reduced toxicity and enhanced specific gene knockdown activity compared to conventional lipoplexes. Thus, such formulations may constitute efficient delivery systems for therapeutic siRNA applications. Copyright © 2007 John Wiley & Sons, Ltd.

The 1,3-dipolar cycloaddition of dimethyl acetylenedicarboxylate with nonstabilized azomethine ylides, generated via the decarboxylative condensation of 1,3-thiazolidine-4-carboxylic acids with aldehydes, afforded 5,7a-dihydro-1H,3H-pyrrolo[1,2-c]thiazole derivatives. 2-Substituted-1,3-thiazolidine-4-carboxylic acids led to the stereoselective formation of 5,7a-dihydro-1H,3H-pyrrolo[1,2-c]thiazoles. Quantum-chemistry calculations were carried out allowing the rationalization of the observed stereoselective formation of the anti-dipole. http://www.sciencedirect.com/science/article/B6THR-4KSSW69-5/1/f6fd75ae8164720ba1de8d3b3a44ed03

The dehalogenation of 2-halo-3-phenyl-2H-azirine-2-carboxylates is described. Using sodium borohydride and tributyltin hydride 3-phenyl-2H-azirine-2-carboxylates were obtained in moderate yields. The synthesis of a new 2-bromo-2H-azirines with a chiral auxiliary, 10-phenylsulfonylisobornyl 2-bromo-3-phenyl-2H-azirine-2-carboxylate, is reported. Its dehalogenation led to 10-phenylsulfonylisobornyl 2H-azirine-2-carboxylate as single stereoisomer together with the formation of 10-phenylsulfonylisobornyl acetate. http://www.sciencedirect.com/science/article/B6THR-482NK06-7/1/b576da25bb07c16918052295f89735c8

A phosphorus ylide bearing a 10-phenylsulfonylisoborneol unit reacted with ketenes, generated in situ from acid chlorides and triethylamine, to give allenic compounds. The reaction with methylketene led to asymmetric induction with the selective synthesis of an allene with axial chirality corresponding to an S configuration. The structure of the chiral allene 10-phenylsulfonylisobornyl penta-2,3-dienoate was determined by X-ray crystallography. Chirooptical studies of the chiral allene derivatives were done. http://www.sciencedirect.com/science/article/B6THS-495V85W-H/1/c946b9b7c8443e7586a0402c58d86df2

[alpha]-Oxophosphonium ylides react with N-chlorosuccinimide, N-bromosuccinimide and N-iodosuccinimide in the presence of azidotrimethylsilane giving the corresponding haloazidoalkenes which were completely converted to the 2-halo-2H-azirines on heating in heptane. http://www.sciencedirect.com/science/article/B6THR-43J0SY1-D/1/db29a4ddcbdab643bfdc11154aaf101f

Corrigendum to Contribution to the synthesis of chiral allenic esters q [Tetrahedron Lett. 44 (2003) 6409–6412] Teresa M. V. D. Pinho e Melo,* Ana L. Cardoso, Ant onio M. d A. Rocha Gonsalves, Richard C. Storr, Jo~ao Costa Pessoa, Jos e A. Paix~ao, Ana M. Beja and Manuela Ramos Silva Departamento de Qu imica, Universidade de Coimbra, 3004-535 Coimbra, Portugal Departamento de F isica, Universidade de Coimbra, 3004-516 Coimbra, Portugal Chemistry Department, The University of Liverpool, Liverpool L69 7ZD, UK Centro de Qu imica Estrutural, Instituto Superior T ecnico, 1049-001 Lisboa, Portugal