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.     

Effect of Glomus mosseae on accumulation efficiency,hazard index and antioxidant defense mechanisms in tomato under metal(loid) Stress

In the present study, the phytoremedation potential along with growth, physiological and biochemical response of tomato (Solanum lycopersicum) was assessed under heavy metal(loid) (HM) and arbuscular mycorrhizal fungus (AMF) amendment. Effect of AMF on uptake and accumulation of metal(loid)s was assessed and accumulation characteristics were expressed in terms of bioabsorption coefficient (BAC), bioconcentration factor (BCF), translocation factor (TLF) and transfer factor (TF). Results showed that AMF-inoculated plants showed not only a better growth, chlorophyll content, strengthened non-enzymatic and enzymatic defense mechanism, but also accumulated higher concentration of metal(loid)s. The correlation between biochemical and physiological parameters was significant at 0.01 level. A significant difference (p ≤ 0.001) in antioxidant enzyme activity was found on increasing metal(loid) dose and application of AMF. The accumulation of Cd and Pb in edible part exceeded the chronic reference dose stated by USEPA. The target hazard quotient (THQ) was >1 for Cd and Pb, whereas <1 for As. The study shows that tomato has good potential as Cd and Pb phytoremediator, hence must not be consumed when grown on Cd or Pb contaminated sites.     

Substrate-energy metabolism and metabolic risk factors for cardiovascular disease in relation to fetal growth and adult body composition

The effect of fetal programming on intermediary metabolism is uncertain. Therefore, we examined whether fetal programming affects oxidative and nonoxidative macronutrient metabolism and the prevalence of the metabolic syndrome in adult life. Healthy older men, aged 64-72 years, with either a lower birth weight (LBW, or=75th %ile; n = 13) had measurements of 1) net oxidative metabolism using indirect calorimetry before and for 6 h after a mixed meal (3,720 kJ) and 2) postprandial oxidation of exogenous [13C]palmitic acid. Body composition was measured using dual-energy X-ray absorptiometry. After adjustment for current weight and height, the LBW group had a lower resting energy expenditure (REE) in the preprandial (4.01 vs. 4.54 kJ/min, P = 0.015) and postprandial state (4.60 vs. 5.20 kJ/min, P = 0.004), and less fat-free mass than the HBW group. The BW category was a significant, independent, and better predictor of REE than weight plus height. There were no significant differences between groups in net oxidative and nonoxidative macronutrient (protein, fat, carbohydrate) metabolism (or of exogenous [13C]palmitate) or in the prevalence of the metabolic syndrome, which was present almost twice as commonly in the LBW than in the HBW group. The study suggests that fetal programming affects both pre- and postprandial EE in older life by mechanisms that are at least partly related to the mass of the fat-free body. BW was found to be a significant predictor of REE that was independent of adult weight plus height.     

N-Acetyl-L-Leucine Accelerates Vestibular Compensation after Unilateral Labyrinthectomy by Action in the Cerebellum and Thalamus

An acute unilateral vestibular lesion leads to a vestibular tone imbalance with nystagmus, head roll tilt and postural imbalance. These deficits gradually decrease over days to weeks due to central vestibular compensation (VC). This study investigated the effects of i.v. N-acetyl-DL-leucine, N-acetyl-L-leucine and N-acetyl-D-leucine on VC using behavioural testing and serial [¹⁸F]-Fluoro-desoxyglucose ([¹⁸F]-FDG)-μPET in a rat model of unilateral chemical labyrinthectomy (UL). Vestibular behavioural testing included measurements of nystagmus, head roll tilt and postural imbalance as well as sequential whole-brain [¹⁸F]-FDG-μPET was done before and on days 1,3,7 and 15 after UL. A significant reduction of postural imbalance scores was identified on day 7 in the N-acetyl-DL-leucine (p < 0.03) and the N-acetyl-L-leucine groups (p < 0.01), compared to the sham treatment group, but not in the N-acetyl-D-leucine group (comparison for applied dose of 24 mg i.v. per rat, equivalent to 60 mg/kg body weight, in each group). The course of postural compensation in the DL- and L-group was accelerated by about 6 days relative to controls. The effect of N-acetyl-L-leucine on postural compensation depended on the dose: in contrast to 60 mg/kg, doses of 15 mg/kg and 3.75 mg/kg had no significant effect. N-acetyl-L-leucine did not change the compensation of nystagmus or head roll tilt at any dose. Measurements of the regional cerebral glucose metabolism (rCGM) by means of μPET revealed that only N-acetyl-L-leucine but not N-acetyl-D-leucine caused a significant increase of rCGM in the vestibulocerebellum and a decrease in the posterolateral thalamus and subthalamic region on days 3 and 7. A similar pattern was found when comparing the effect of N-acetyl-L-leucine on rCGM in an UL-group and a sham UL-group without vestibular damage. In conclusion, N-acetyl-L-leucine improves compensation of postural symptoms after UL in a dose-dependent and specific manner, most likely by activating the vestibulocerebellum and deactivating the posterolateral thalamus.     

Lamin A/C Depletion Enhances DNA Damage-Induced Stalled Replication Fork Arrest

The human LMNA gene encodes the essential nuclear envelope proteins lamin A and C (lamin A/C). Mutations in LMNA result in altered nuclear morphology, but how this impacts the mechanisms that maintain genomic stability is unclear. Here, we report that lamin A/C-deficient cells have a normal response to ionizing radiation but are sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with ICL agents (cisplatin, camptothecin, and mitomycin), lamin A/C-deficient cells displayed normal γ-H2AX focus formation but a higher frequency of cells with delayed γ-H2AX removal, decreased recruitment of the FANCD2 repair factor, and a higher frequency of chromosome aberrations. Similarly, following hydroxyurea-induced replication stress, lamin A/C-deficient cells had an increased frequency of cells with delayed disappearance of γ-H2AX foci and defective repair factor recruitment (Mre11, CtIP, Rad51, RPA, and FANCD2). Replicative stress also resulted in a higher frequency of chromosomal aberrations as well as defective replication restart. Taken together, the data can be interpreted to suggest that lamin A/C has a role in the restart of stalled replication forks, a prerequisite for initiation of DNA damage repair by the homologous recombination pathway, which is intact in lamin A/C-deficient cells. We propose that lamin A/C is required for maintaining genomic stability following replication fork stalling, induced by either ICL damage or replicative stress, in order to facilitate fork regression prior to DNA damage repair.     

Real-time analysis of membrane permeabilizing effects of oleanane saponins

Saponins are a group of plant and marine derived glycosides with numerous biological functions. Two important characteristics of certain plant saponins are their ability to enhance cytotoxicity of type I ribosome inactivating proteins and stimulation of the immune system. The main objective of the present study was to investigate in real-time the permeabilizing effects of saponins on cell membrane. A set of oleanane saponins (glycyrrhizinic acid, Gypsophila, Saponaria and Quillaja saponins) and a steroid saponin (digitonin) were tested. The effects of these saponins on lysosomal membranes and hemolysis, along with their charge were also studied. Real-time monitoring of cell membrane permeabilization facilitated a highly sensitive analysis of the cellular kinetics. Saponins showed variable permeabilizing effects on cellular and lysosomal membranes at concentrations from 6 μM and hemolysis from 3 μM. Further, the results suggest that charge of the saponin may be relevant for permeabilizing effects of oleanane saponins.     

Animal Viruses Probe Dataset (AVPDS) for Microarray-Based Diagnosis and Identification of Viruses

AVPDS (Animal Viruses Probe dataset) is a dataset of virus-specific and conserve oligonucleotides for identification and diagnosis of viruses infecting animals. The current dataset contain 20,619 virus specific probes for 833 viruses and their subtypes and 3,988 conserved probes for 146 viral genera. Dataset of virus specific probe has been divided into two fields namely virus name and probe sequence. Similarly conserved probes for virus genera table have genus, and subgroup within genus name and probe sequence. The subgroup within genus is artificially divided subgroups with no taxonomic significance and contains probes which identifies viruses in that specific subgroup of the genus. Using this dataset we have successfully diagnosed the first case of Newcastle disease virus in sheep and reported a mixed infection of Bovine viral diarrhea and Bovine herpesvirus in cattle. These dataset also contains probes which cross reacts across species experimentally though computationally they meet specifications. These probes have been marked. We hope that this dataset will be useful in microarray-based detection of viruses. The dataset can be accessed through the link https://dl.dropboxusercontent.com/u/94060831/avpds/HOME.html.