Development of Novel Biodegradable Polymeric Nanoparticles-in-Microsphere Formulation for Local Plasmid DNA Delivery in the Gastrointestinal Tract

There is a critical need for development of novel delivery systems to facilitate the translation of nucleic acid-based macromolecules into clinically-viable therapies. The aim of this investigation was to develop and evaluate a novel nanoparticles-in-microsphere oral system (NiMOS) for gene delivery and transfection in specific regions of the gastrointestinal (GI) tract. Plasmid DNA, encoding for the enhanced green fluorescent protein (EGFP-N1), was encapsulated in type B gelatin nanoparticles. NiMOS were prepared by further protecting the DNA-loaded nanoparticles in a poly(epsilon-caprolactone) (PCL) matrix to form microspheres of less than 5.0 μm in diameter. In order to evaluate the biodistribution following oral administration, radiolabeled (¹¹¹In-labeled) gelatin nanoparticles and NiMOS were administered orally to fasted Balb/C mice. The results of biodistribution studies showed that, while gelatin nanoparticles traversed through the GI tract fairly quickly with more than 54% of the administered dose per gram localizing in the large intestine at the end of 2 h, NiMOS resided in the stomach and small intestine for relatively longer duration. Following oral administration of EGFP-N1 plasmid DNA at 100 μg dose in the control and test formulations, the quantitative and qualitative results presented in this study provide the necessary evidence for transfection potential of NiMOS upon oral administration. After 5 days post-administration, transgene expression in the small and large intestine of mice was observed. Based on these results, NiMOS show significant potential as novel gene delivery vehicle for therapeutic and vaccination purposes.     

Cadmium Remediation by Arbuscular Mycorrhizal Fungus–Colonized Celery Plants Supplemented with Ethylenediaminetetraacetic Acid

A pot trial using Glomus mosseae along with EDTA (ethylenediaminetetraacetic acid) was conducted for the phytoextraction of cadmium (Cd) by celery (Apium graveolens Linn.) plants from soil artificially contaminated with Cd under glass house conditions. The experiment is a 2 × 2 × 4 factorial design with two levels of G. mosseae inoculations (G. mosseae inoculated and uninoculated), two EDTA concentrations (without and with 2.5 mmol kg^?1 soil EDTA) and four Cd concentrations (0, 5, 10, and 20 mg kg^?1 soil). The results indicate the formation of an effective symbiosis between G. mosseae and celery in the contaminated soil. However, an increase in Cd input level and EDTA addition showed strong phytotoxic effect on celery plants and G. mosseae, as a considerable decrease in the frequency of root colonization and spore density was noticed. However, the plants were able to withstand the stressed condition due to the benefits provided by G. mosseae through increased P accumulation, chlorophyll content, and plant growth, resulting in an increase in Cd accumulation, which was good enough for the phytoextraction purpose. Thus, celery plants inoculated with G. mosseae and later supplemented with EDTA could be an effective and potentially suitable practice for the remediation of Cd-contaminated sites.     

Integrating image quality in 2nu-SVM biometric match score fusion

This paper proposes an intelligent 2nu-support vector machine based match score fusion algorithm to improve the performance of face and iris recognition by integrating the quality of images. The proposed algorithm applies redundant discrete wavelet transform to evaluate the underlying linear and non-linear features present in the image. A composite quality score is computed to determine the extent of smoothness, sharpness, noise, and other pertinent features present in each subband of the image. The match score and the corresponding quality score of an image are fused using 2nu-support vector machine to improve the verification performance. The proposed algorithm is experimentally validated using the FERET face database and the CASIA iris database. The verification performance and statistical evaluation show that the proposed algorithm outperforms existing fusion algorithms.     

N-linked (N-) Glycoproteomics of Urimary Exosomes*

Epithelial cells lining the urinary tract secrete urinary exosomes (40–100 nm) that can be targeted to specific cells modulating their functionality. One potential targeting mechanism is adhesion between vesicle surface glycoproteins and target cells. This makes the glycopeptide analysis of exosomes important. Exosomes reflect the physiological state of the parent cells; therefore, they are a good source of biomarkers for urological and other diseases. Moreover, the urine collection is easy and noninvasive and urinary exosomes give information about renal and systemic organ systems. Accordingly, multiple studies on proteomic characterization of urinary exosomes in health and disease have been published. However, no systematic analysis of their glycoproteomic profile has been carried out to date, whereas a conserved glycan signature has been found for exosomes from urine and other sources including T cell lines and human milk.Here, we have enriched and identified the N-glycopeptides from these vesicles. These enriched N-glycopeptides were solved for their peptide sequence, glycan composition, structure, and glycosylation site using collision-induced dissociation MS/MS (CID-tandem MS) data interpreted by a publicly available software GlycopeptideId. Released glycans from the same sample was also analyzed with MALDI-MS.We have identified the N-glycoproteome of urinary exosomes. In total 126 N-glycopeptides from 51 N-glycosylation sites belonging to 37 glycoproteins were found in our results. The peptide sequences of these N-glycopeptides were identified unambiguously and their glycan composition (for 125 N-glycopeptides) and structures (for 87 N-glycopeptides) were proposed. A corresponding glycomic analysis with released N-glycans was also performed. We identified 66 unique nonmodified N-glycan compositions and in addition 13 sulfated/phosphorylated glycans were also found. This is the first systematic analysis of N-glycoproteome of urinary exosomes.Urine is a combination of plasma filtrate and the secretion profile of cells lining the urino-genital tract. This secretion profile, in addition to proteins and metabolites, also contains exosomes and larger microvesicles that have glycoproteins on their surface. In healthy individuals, ∼70% of the urinary proteome originates from kidneys and the rest represents plasma filtered by glomeruli (1). Proteins present in urine are a collection of proteins secreted by a number of tissues, which changes in disease states (2). Therefore, the urinary proteome may serve as a rich source of biomarkers for urogenital and systemic diseases, which have been reviewed previously (3). Moreover, urine collection is a noninvasive procedure, which makes it an ideal candidate for discovery of novel biomarkers. Only a few large scale studies on urinary proteome and glycoproteome have been published (4, 5). However, most of them have not focused on the glycopeptide characterization.Microvesicles, including exosomes are secreted by many cell types and involved in functions including antigen-presentation, cell-to-cell communication, and immunomodulation (6). These are specialized compartments of cells and they mirror the physiological state of cells secreting them while also providing information about the environment into which they are secreted. For instance, the immunosuppressive and pro-angiogenic environment of cancer may be mediated partially by exosomes (6). Exosomes and other types of microvesicles are abundantly found in urine and thought to be mainly secreted by epithelial cells lining the urinary system (7). These vesicles contain DNA, RNA, and proteins.Glycosylation is an important post-translational modification of proteins and lipids and appears to play many roles, e.g. in cell adhesion, cell-to-cell communication, and immune response (8). Glycosylation is also very important for targeting of proteins to various compartments of the cells. Accordingly, glycans of glycoproteins have important roles in protein sorting to membrane microdomains and furthermore in influencing their intracellular trafficking (9). Microvesicles have a glycan signature that is distinct from the parent cell, suggesting that they originate from specialized membrane microdomains implying a role of glycosylation in microvesicle protein sorting (10, 11). Changes in N-glycans of exosomes from expressed prostatic secretions correlate with disease severity (12). HIV-1 particles were found to have a glycome (the comprehensive glycan profile of a protein, cell, or tissue) largely shared with microvesicles, which is taken to imply that the virus hijacks the glycomachinery of infected cells and uses it systematically to infect additional cells or to deceive the normal immunodefence (13). Thus, the specific glycoproteins of exosomes may be of major impact in targeting exosomes to distinct cells and tissues.Exosome uptake in various cell types has been shown to occur through the mechanisms of clathrin-mediated endocytosis, phagocytosis, and micropinocytosis (14, 15). The uptake of exosomes by dendritic cells and macrophages has been shown to be inhibited by mannose, N-acetylglucosamine, and lactose residues, respectively. This uptake is mediated by a C-type lectin in dendritic cells and galectin-5 in macrophages (14, 15). All this points toward a system in which the exosome glycosylation pattern is kept specific by the cells secreting them to suit the target cell makeup and uptake pathways, and further downstream functions. Taken together, these findings suggest that better understanding of surface glycosylation patterns as well as the glycomics and glycoproteomics of exosomes might help in establishing the specificity of exosome uptake by target cells and activated downstream pathways. This information about exosome uptake might be utilized in therapeutics involving exosomes. Glycome and glycoproteome of urinary microvesicles will provide information not only about the functional state of constituent proteins, but it will also highlight the similarities and differences among proteins that are specifically targeted to exosomes.An N-glycopeptide analysis using collision induced dissociation (CID)-Tandem MS has been reported previously for different sample types (16, 17). This approach provides information about the composition of glycan, partial structure, glycosylation site, and peptide sequence from the same molecule compared with approaches where released glycans or peptides of N-glycopeptides are analyzed separately.We have published an algorithm for an automated analysis of N-glycopeptides (18, 19). A public, web-based software with some changes to the original was developed and named as GlycopeptideId (www.appliednumerics.com, GlycopeptideID version 28–02-28 0.91 beta). The major change was to analyze glycan structures against a database and not with an iterative de novo glycan structure analysis. The proposed structures were further manually validated by the presence of diagnostic ions, when they were available for the given structure.This software was utilized in this study and a comprehensive glycopeptide characterization of urinary exosomal glycoproteins was carried out. We report here the glycan structure determination of urinary exosomal glycoproteins. We have characterized 126 N-glycopeptides representing 51 N-glycosylation sites that belong to 37 glycoproteins. Additionally, glycomic analysis of released N-glycans was also performed and 66 unique modified and 13 sulfated and/or phosphorylated glycans were found. A third of total glycan compositions were common to both the analysis, whereas approximately a third were unique to both the analysis.     

Insights towards sulfonamide drug specificity in α-carbonic anhydrases

Carbonic anhydrases (CAs, EC 4.2.1.1) are a group of metalloenzymes that play important roles in carbon metabolism, pH regulation, CO₂ fixation in plants, ion transport etc., and are found in all eukaryotic and many microbial organisms. This family of enzymes catalyzes the interconversion of CO₂ and HCO₃^?. There are at least 16 different CA isoforms in the alpha structural class (α-CAs) that have been isolated in higher vertebrates, with CA isoform II (CA II) being ubiquitously abundant in all human cell types. CA inhibition has been exploited clinically for decades for various classes of diuretics and anti-glaucoma treatment. The characterization of the overexpression of CA isoform IX (CA IX) in certain tumors has raised interest in CA IX as a diagnostic marker and drug target for aggressive cancers and therefore the development of CA IX specific inhibitors. An important goal in the field of CA is to identify, rationalize, and design potential compounds that will preferentially inhibit CA IX over all other isoforms of CA. The variations in the active sites between isoforms of CA are subtle and this causes non-specific CA inhibition which leads to various side effects. In the case of CA IX inhibition, CA II along with other isoforms of CA provide off-target binding sites which is undesirable for cancer treatment. The focus of this article is on CA IX inhibition and two different structural approaches to CA isoform specific drug designing: tail approach and fragment addition approach.     

Real time monitoring of the cell viability during treatment with tumor-targeted toxins and saponins using impedance measurement

This work describes the application of an impedance-based measurement for the real time evaluation of targeted tumor therapies in cell culture (HeLa cells). We used a treatment procedure that is well established in cells and mice. Therein, tumor cells are treated with a combination of an epidermal growth factor-based targeted toxin named SE and particular plant glycosides called saponins. In the present study HeLa cells were seeded in different numbers onto interdigitated electrode structures integrated into the bottom of a 96 well plate. The cells were treated with SE in the presence and absence of the saponin SpnS-1 (isolated from Saponaria officinalis roots). The impedance was directly correlated with the viability of the cells. As expected from known end point measurements, a concentration dependent enhancement of toxicity was observed; however, with the impedance measurement we were for the first time able to trace the temporal changes of cell death during the combination treatment. This substantially added to the understanding of initial cellular mechanisms in the augmentation of the toxicity of targeted toxins by saponins and indicated the superiority of real time monitoring over end point assays. The method is less labor intensive and label-free with ease of monitoring the effects at each time point.     

Effects of Friedreich's ataxia GAA repeats on DNA replication in mammalian cells

Friedreich's ataxia (FRDA) is a common hereditary degenerative neuro-muscular disorder caused by expansions of the (GAA)n repeat in the first intron of the frataxin gene. The expanded repeats from parents frequently undergo further significant length changes as they are passed on to progeny. Expanded repeats also show an age-dependent instability in somatic cells, albeit on a smaller scale than during intergenerational transmissions. Here we studied the effects of (GAA)n repeats of varying lengths and orientations on the episomal DNA replication in mammalian cells. We have recently shown that the very first round of the transfected DNA replication occurs in the lack of the mature chromatin, does not depend on the episomal replication origin and initiates at multiple single-stranded regions of plasmid DNA. We now found that expanded GAA repeats severely block this first replication round post plasmid transfection, while the subsequent replication cycles are only mildly affected. The fact that GAA repeats affect various replication modes in a different way might shed light on their differential expansions characteristic for FRDA.     

Intravenous Delivery of Targeted Liposomes to Amyloid-β Pathology in APP/PSEN1 Transgenic Mice

Extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles constitute the major neuropathological hallmarks of Alzheimer’s disease (AD). It is now apparent that parenchymal Aβ plaque deposition precedes behavioral signs of disease by several years. The development of agents that can target these plaques may be useful as diagnostic or therapeutic tools. In this study, we synthesized an Aβ-targeted lipid conjugate, incorporated it in stealth liposomal nanoparticles and tested their ability to bind amyloid plaque deposits in an AD mouse model. The results show that the particles maintain binding profiles to synthetic Aβ aggregates comparable to the free ligand, and selectively bind Aβ plaque deposits in brain tissue sections of an AD mouse model (APP/PSEN1 transgenic mice) with high efficiency. When administered intravenously, these long circulating nanoparticles appear to cross the blood-brain barrier and bind to Aβ plaque deposits, labeling parenchymal amyloid deposits and vascular amyloid characteristic of cerebral amyloid angiopathy.