Enhancing Neuronal Cell Uptake of Therapeutic Nucleic Acids with Tetrahedral DNA Nanostructures.

The successful translation of therapeutic nucleic acids (NAs) for the treatment of neurological disorders depends on their safe and efficient delivery to neural cells, in particular neurons. DNA nanostructures can be a promising NAs delivery vehicle. Nonetheless, the potential of DNA nanostructures for neuronal cell delivery of therapeutic NAs is unexplored. Here, tetrahedral DNA nanostructures (TDN) as siRNA delivery scaffolds to neuronal cells, exploring the influence of functionalization with two different reported neuronal targeting ligands: C4-3 RNA aptamer and Tet1 peptide are investigated. Nanostructures are characterized in vitro, as well as in silico using molecular dynamic simulations to better understand the overall TDN structural stability. Enhancement of neuronal cell uptake of TDN functionalized with the C4-3 Aptamer (TDN-Apt), not only in neuronal cell lines but also in primary neuronal cell cultures is demonstrated. Additionally, TDN and TDN-Apt nanostructures carrying siRNA are shown to promote silencing in a process aided by chloroquine-induced endosomal disruption. This work presents a thorough workflow for the structural and functional characterization of the proposed TDN as a nano-scaffold for neuronal delivery of therapeutic NAs and for targeting ligands evaluation, contributing to the future development of new neuronal drug delivery systems based on DNA nanostructures.

Impact of Conventional and Potential New Metal-Based Drugs on Lipid Metabolism in Osteosarcoma MG-63 Cells.

This work investigated the mechanisms of action of conventional drugs, cisplatin and oxaliplatin, and the potentially less deleterious drug Pd2Spermine (Spm) and its Pt(II) analog, against osteosarcoma MG-63 cells, using nuclear-magnetic-resonance metabolomics of the cellular lipidome. The Pt(II) chelates induced different responses, namely regarding polyunsaturated-fatty-acids (increased upon cisplatin), suggesting that cisplatin-treated cells have higher membrane fluidity/permeability, thus facilitating cell entry and justifying higher cytotoxicity. Both conventional drugs significantly increased triglyceride levels, while Pt2Spm maintained control levels; this may reflect enhanced apoptotic behavior for conventional drugs, but not for Pt2Spm. Compared to Pt2Spm, the more cytotoxic Pd2Spm (IC50 comparable to cisplatin) induced a distinct phospholipids profile, possibly reflecting enhanced de novo biosynthesis to modulate membrane fluidity and drug-accessibility to cells, similarly to cisplatin. However, Pd2Spm differed from cisplatin in that cells had equivalent (low) levels of triglycerides as Pt2Spm, suggesting the absence/low extent of apoptosis. Our results suggest that Pd2Spm acts on MG-63 cells mainly through adaptation of cell membrane fluidity, whereas cisplatin seems to couple a similar effect with typical signs of apoptosis. These results were discussed in articulation with reported polar metabolome adaptations, building on the insight of these drugs' mechanisms, and particularly of Pd2Spm as a possible cisplatin substitute.

Dengue Virus Capsid Protein Facilitates Genome Compaction and Packaging.

Dengue virus (DENV) is a single-stranded (+)-sense RNA virus that infects humans and mosquitoes, posing a significant health risk in tropical and subtropical regions. Mature virions are composed of an icosahedral shell of envelope (E) and membrane (M) proteins circumscribing a lipid bilayer, which in turn contains a complex of the approximately 11 kb genomic RNA with capsid (C) proteins. Whereas the structure of the envelope is clearly defined, the structure of the packaged genome in complex with C proteins remains elusive. Here, we investigated the interactions of C proteins with viral RNA, in solution and inside mature virions, via footprinting and cross-linking experiments. We demonstrated that C protein interaction with DENV genomes saturates at an RNA:C protein ratio below 1:250. Moreover, we also showed that the length of the RNA genome interaction sites varies, in a multimodal distribution, consistent with the C protein binding to each RNA site mostly in singlets or pairs (and, in some instances, higher numbers). We showed that interaction sites are preferentially sites with low base pairing, as previously measured by 2'-acetylation analyzed by primer extension (SHAPE) reactivity indicating structuredness. We found a clear association pattern emerged: RNA-C protein binding sites are strongly associated with long-range RNA-RNA interaction sites, particularly inside virions. This, in turn, explains the need for C protein in viral genome packaging: the protein has a chief role in coordinating these key interactions, promoting proper packaging of viral RNA. Such sites are, thus, highly consequential for viral assembly, and, as such, may be targeted in future drug development strategies against these and related viruses.

Purple Urine Bag Syndrome: A Peculiar Presentation of a Urinary Tract Infection.

Purple urine bag syndrome (PUBS) is a peculiar phenomenon and corresponds to the appearance of purplish-colored urine. It is associated with urinary tract infections occurring mainly in debilitated elderly women with constipation and long-term indwelling urinary catheters. We share a case involving PUBS in an 87-year-old female patient, explore the pathophysiology, and discuss potential management options for this uncommon syndrome.

Engineering a Novel Self-Assembled Multi-siRNA Nanocaged Architecture with Controlled Enzyme-Mediated siRNA Release.

The RNA interference (RNAi) chemical and structural design space has evolved since its original definitions. Although this has led to the development of RNAi molecules that are starting to address the issues of silencing efficiency and delivery to target organs and cells, there is an on-going interest to improve upon their properties to attain wider therapeutic applicability. Taking advantage of the flexibility given by DNA and RNA structural and chemical properties, we here investigated unconventional RNAi encoding structures, designated by caged-siRNA structures (CsiRNAs), to explore novel features that could translate into advantageous properties for cellular delivery and intracellular activity. Using the principles of controlled nucleic acid self-assembly, branched DNA-RNA hybrid intermediates were formed, ultimately leading to the assembly of a "closed" structure encompassing multiple RNAi units. The RNAi active regions are further triggered by an encoded RNAse H-mediated release mechanism, while the overall structure possesses easily addressable anchors for hybridization-based functionalization with active biological moieties. We confirmed the production of correct structures and demonstrated that the encoded RNAi sequences maintain gene silencing activity even within this novel unconventional nanoarchitecture, aided by the intracellularly triggered RNAse H release mechanism. With this design, functionalization is easily achieved with no negative effects on the silencing activity, warranting further development of these novel molecular structures as a multi-RNAi platform for therapeutic delivery.

Advances in Microfluidics for the Implementation of Liquid Biopsy in Clinical Routine.

In recent years, we have seen major advances in the field of liquid biopsy and its implementation in the clinic, mainly driven by breakthrough developments in the area of molecular biology. New developments have seen an integration of microfluidics and also biosensors in liquid biopsy systems, bringing advantages in terms of cost, sensitivity and automation. Without a doubt, the next decade will bring the clinical validation and approval of these combined solutions, which is expected to be crucial for the wide implementation of liquid biopsy systems in clinical routine.

Response of Osteosarcoma Cell Metabolism to Platinum and Palladium Chelates as Potential New Drugs.

This paper reports the first metabolomics study of the impact of new chelates Pt2Spm and Pd2Spm (Spm = Spermine) on human osteosarcoma cellular metabolism, compared to the conventional platinum drugs cisplatin and oxaliplatin, in order to investigate the effects of different metal centers and ligands. Nuclear Magnetic Resonance metabolomics was used to identify meaningful metabolite variations in polar cell extracts collected during exposure to each of the four chelates. Cisplatin and oxaliplatin induced similar metabolic fingerprints of changing metabolite levels (affecting many amino acids, organic acids, nucleotides, choline compounds and other compounds), thus suggesting similar mechanisms of action. For these platinum drugs, a consistent uptake of amino acids is noted, along with an increase in nucleotides and derivatives, namely involved in glycosylation pathways. The Spm chelates elicit a markedly distinct metabolic signature, where inverse features are observed particularly for amino acids and nucleotides. Furthermore, Pd2Spm prompts a weaker response from osteosarcoma cells as compared to its platinum analogue, which is interesting as the palladium chelate exhibits higher cytotoxicity. Putative suggestions are discussed as to the affected cellular pathways and the origins of the distinct responses. This work demonstrates the value of untargeted metabolomics in measuring the response of cancer cells to either conventional or potential new drugs, seeking further understanding (or possible markers) of drug performance at the molecular level.

Metabolic Aspects of Palladium(II) Potential Anti-Cancer Drugs.

This mini-review reports on the existing knowledge of the metabolic effects of palladium [Pd(II)] complexes with potential anticancer activity, on cell lines and murine models. Most studies have addressed mononuclear Pd(II) complexes, although increasing interest has been noted in bidentate complexes, as polynuclear structures. In addition, the majority of records have reported in vitro studies on cancer cell lines, some including the impact on healthy cells, as potentially informative in relation to side effects. Generally, these studies address metabolic effects related to the mechanisms of induced cell death and antioxidant defense, often involving the measurement of gene and protein expression patterns, and evaluation of the levels of reactive oxygen species or specific metabolites, such as ATP and glutathione, in relation to mitochondrial respiration and antioxidant mechanisms. An important tendency is noted toward the use of more untargeted approaches, such as the use of omic sciences e.g., proteomics and metabolomics. In the discussion section of this mini-review, the developments carried out so far are summarized and suggestions of possible future developments are advanced, aiming at recognizing that metabolites and metabolic pathways make up an important part of cell response and adaptation to therapeutic agents, their further study potentially contributing valuably for a more complete understanding of processes such as biotoxicity or development of drug resistance.

Structural and Functional Properties of the Capsid Protein of Dengue and Related Flavivirus.

Dengue, West Nile and Zika, closely related viruses of the Flaviviridae family, are an increasing global threat, due to the expansion of their mosquito vectors. They present a very similar viral particle with an outer lipid bilayer containing two viral proteins and, within it, the nucleocapsid core. This core is composed by the viral RNA complexed with multiple copies of the capsid protein, a crucial structural protein that mediates not only viral assembly, but also encapsidation, by interacting with host lipid systems. The capsid is a homodimeric protein that contains a disordered N-terminal region, an intermediate flexible fold section and a very stable conserved fold region. Since a better understanding of its structure can give light into its biological activity, here, first, we compared and analyzed relevant mosquito-borne Flavivirus capsid protein sequences and their predicted structures. Then, we studied the alternative conformations enabled by the N-terminal region. Finally, using dengue virus capsid protein as main model, we correlated the protein size, thermal stability and function with its structure/dynamics features. The findings suggest that the capsid protein interaction with host lipid systems leads to minor allosteric changes that may modulate the specific binding of the protein to the viral RNA. Such mechanism can be targeted in future drug development strategies, namely by using improved versions of pep14-23, a dengue virus capsid protein peptide inhibitor, previously developed by us. Such knowledge can yield promising advances against Zika, dengue and closely related Flavivirus.

West Nile Virus Capsid Protein Interacts With Biologically Relevant Host Lipid Systems.

West Nile and dengue viruses are closely related flaviviruses, originating mosquito-borne viral infections for which there are no effective and specific treatments. Their capsid proteins sequence and structure are particularly similar, forming highly superimposable α-helical homodimers. Measuring protein-ligand interactions at the single-molecule level yields detailed information of biological and biomedical relevance. In this work, such an approach was successfully applied on the characterization of the West Nile virus capsid protein interaction with host lipid systems, namely intracellular lipid droplets (an essential step for dengue virus replication) and blood plasma lipoproteins. Dynamic light scattering measurements show that West Nile virus capsid protein binds very low-density lipoproteins, but not low-density lipoproteins, and this interaction is dependent of potassium ions. Zeta potential experiments show that the interaction with lipid droplets is also dependent of potassium ions as well as surface proteins. The forces involved on the binding of the capsid protein with lipid droplets and lipoproteins were determined using atomic force microscopy-based force spectroscopy, proving that these interactions are K+-dependent rather than a general dependence of ionic strength. The capsid protein interaction with host lipid systems may be targeted in future therapeutic strategies against different flaviviruses. The biophysical and nanotechnology approaches employed in this study may be applied to characterize the interactions of other important proteins from different viruses, in order to understand their life cycles, as well as to find new strategies to inhibit them.

Methods for Lipid Droplet Biophysical Characterization in Flaviviridae Infections.

Lipid droplets (LDs) are intracellular organelles for neutral lipid storage, originated from the endoplasmic reticulum. They play an essential role in lipid metabolism and cellular homeostasis. In fact, LDs are complex organelles, involved in many more cellular processes than those initially proposed. They have been extensively studied in the context of LD-associated pathologies. In particular, LDs have emerged as critical for virus replication and assembly. Viruses from the Flaviviridae family, namely dengue virus (DENV), hepatitis C virus (HCV), West Nile virus (WNV), and Zika virus (ZIKV), interact with LDs to usurp the host lipid metabolism for their own viral replication and pathogenesis. In general, during Flaviviridae infections it is observed an increasing number of host intracellular LDs. Several viral proteins interact with LDs during different steps of the viral life cycle. The HCV core protein and DENV capsid protein, extensively interact with LDs to regulate their replication and assembly. Detailed studies of LDs in viral infections may contribute for the development of possible inhibitors of key steps of viral replication. Here, we reviewed different techniques that can be used to characterize LDs isolated from infected or non-infected cells. Microscopy studies have been commonly used to observe LDs accumulation and localization in infected cell cultures. Fluorescent dyes, which may affect LDs directly, are widely used to probe LDs but there are also approaches that do not require the use of fluorescence, namely stimulated Raman scattering, electron and atomic force microscopy-based approaches. These three are powerful techniques to characterize LDs morphology. Raman scattering microscopy allows studying LDs in a single cell. Electron and atomic force microscopies enable a better characterization of LDs in terms of structure and interaction with other organelles. Other biophysical techniques, such as dynamic light scattering and zeta potential are also excellent to characterize LDs in terms of size in a simple and fast way and test possible LDs interaction with viral proteins. These methodologies are reviewed in detail, in the context of viral studies.

Impact of the Pd2Spermine Chelate on Osteosarcoma Metabolism: An NMR Metabolomics Study.

A metabolomics study of Pd2Spermine(Spm) on osteosarcoma MG-63 and osteoblastic HOb cells is presented to assess the impact of the potential palladium drug on cell metabolism compared with cisplatin (cDDP). Despite its higher cytotoxicity, Pd2Spm induced lower (and reversible) metabolic impact on MG-63 cells and the absence of apoptosis; conversely, it induced significant deviations in osteoblastic amino acid metabolism. However, when in combination with doxorubicin and methotrexate, Pd2Spm induced strong metabolic deviations on lipids, choline compounds, amino acids, nucleotides, and compounds related to antioxidative mechanisms (e.g., glutathione, inositol, hypoxanthine), similarly to the cDDP cocktail. Synergetic effects included triggering of lipid biosynthesis by Pd2Spm in the presence of doxorubicin (and reinforced by methotrexate) and changes in the glycosylation substrate uridine diphosphate acetylgalactosamine and methionine and serine metabolisms. This work provides promising results related to the impact of Pd2Spm on osteosarcoma cellular metabolism, particularly in drug combination protocols. Lipid metabolism, glycosylation, and amino acid metabolisms emerge as relevant features for targeted studies to further understand a potential anticancer mechanism of combined Pd2Spm.

Prediction of Gestational Diabetes through NMR Metabolomics of Maternal Blood.

Metabolic biomarkers of pre- and postdiagnosis gestational diabetes mellitus (GDM) were sought, using nuclear magnetic resonance (NMR) metabolomics of maternal plasma and corresponding lipid extracts. Metabolite differences between controls and disease were identified through multivariate analysis of variable selected (1)H NMR spectra. For postdiagnosis GDM, partial least squares regression identified metabolites with higher dependence on normal gestational age evolution. Variable selection of NMR spectra produced good classification models for both pre- and postdiagnostic GDM. Prediagnosis GDM was accompanied by cholesterol increase and minor increases in lipoproteins (plasma), fatty acids, and triglycerides (extracts). Small metabolite changes comprised variations in glucose (up regulated), amino acids, betaine, urea, creatine, and metabolites related to gut microflora. Most changes were enhanced upon GDM diagnosis, in addition to newly observed changes in low-Mw compounds. GDM prediction seems possible exploiting multivariate profile changes rather than a set of univariate changes. Postdiagnosis GDM is successfully classified using a 26-resonance plasma biomarker. Plasma and extracts display comparable classification performance, the former enabling direct and more rapid analysis. Results and putative biochemical hypotheses require further confirmation in larger cohorts of distinct ethnicities.

Insulin-like growth factor I receptor pathway inhibition by ADW742, alone or in combination with imatinib, doxorubicin, or vincristine, is a novel therapeutic approach in Ewing tumor.

PURPOSE: Ewing tumor cell survival and proliferation depends on several autocrine loops. Targeting these loops is a promising therapeutic approach. We recently showed the cytostatic role of imatinib, an inhibitor of the SCF-KIT loop, on Ewing tumor cells, and in this study, we intend to analyze the inhibition of the insulin-like growth factor I receptor (IGF1R) loop. EXPERIMENTAL DESIGN: We analyzed IGF1R blockade by ADW742, a small molecule specific for this receptor, alone and in combination with imatinib, vincristine, and doxorubicin on Ewing tumor cell lines. We studied the effect on proliferation, apoptosis, cell cycle, pathway phosphorylation, soft-agar growth, motility, and vascular endothelial growth factor expression levels. RESULTS: Treatment with ADW742 induced down-regulation of IGF1R/AKT/mammalian target of rapamycin (mTOR) phosphorylation, which was deeper in cell lines having higher IGF1R activation levels. Treatment also induced dose-dependent inhibition of cell proliferation (IC50 = 0.55-1.4 micromol/L), inducing a G1 phase blockage and apoptosis. Addition of imatinib to ADW742 synergistically augmented these effects and was especially effective in inhibiting AKT/mTOR phosphorylation and reducing vascular endothelial growth factor expression in cell lines having high IGF1R activation levels. Combination with usual chemotherapeutic agents vincristine and doxorubicin showed synergistic interactions. CONCLUSIONS: Inhibition of Ewing tumor cell proliferation by ADW742 is mediated through blockade of IGF1R signaling. Combination of ADW742 with imatinib, vincristine, and doxorubicin induces a significant reduction of tumor cell growth, mainly by the increase in apoptosis with a pattern depending on IGF1R activation levels. This study supports a potential role for ADW742 in the treatment of Ewing tumor and AKT/mTOR as a possible surrogate marker of response to therapy.