Short interfering RNA delivered by a hybrid nanoparticle targeting VEGF: Biodistribution and anti-tumor effect

BackgroundThe use of RNA interference (iRNA) therapy has proved to be an interesting target therapy for the cancer treatment; however, siRNAs are unstable and quickly eliminated from the bloodstream. To face these barriers, the use of biocompatible and efficient nanocarriers emerges as an alternative to improve the success application of iRNA to the cancer, including breast cancer.ResultsA hybrid nanocarrier composed of calcium phosphate as the inorganic phase and a block copolymer containing polyanions as organic phase, named HNPs, was developed to deliver VEGF siRNA into metastatic breast cancer in mice. The particles presented a rounded shape by TEM images with average size measured by DLS suitable and biocompatible for biomedical applications. The XPS and EDS spectra confirmed the hybrid composition of the nanoparticles. Moreover, after intravenous administration, the particles accumulated mainly in the tumor site and kidneys, which demonstrates the tumor targeting accumulation through the Enhanced Permeability and Retention Effect (EPR). A significant decrease in size of the tumors treated with the nanoparticles containing siVEGF (HNPs-siVEGF) was observed and the reduction was related to enhanced tumor accumulation of siRNA as well as in vivo VEGF silencing at gene and protein levels.ConclusionThe hybrid system prepared was successful in promoting the RNAi effect in vivo with very low toxicity.General significanceThis study shows the valuable development of a hybrid nanoparticle carrying VEGF siRNA, as well as their tumor targeting, accumulation and reduction in mice triple-negative breast cancer.     

Chromosome 10 in the tomato plant carries clusters of genes responsible for field resistance/defence to Phytophthora infestans

The main objective of the present study was to reanalyse tomato expression data that was previously submitted to the Tomato Expression Database to dissect the resistance/defence genomic and metabolic responses of tomato to Phytophthora infestans under field conditions. Overrepresented gene sets belonging to chromosome 10 were identified using the Gene Set Enrichment Analysis, and we found that these genes tend to be located towards the end of the chromosome 10. An analysis of syntenic regions between Arabidopsis thaliana chromosomes and the tomato chromosome 10 allowed us to identify conserved regions in the two genomes. In addition to allowing for the identification of tomato candidate genes participating in resistance/defence in the field, this approach allowed us to investigate the relationships of the candidate genes with chromosomal position and participation in metabolic functions, thus offering more insight into the phenomena occurring during the infection process.     

Using Multidetector Computed Tomography in a Swine Model to Assess the Effects of Sublingual Nitroglycerin and Intravenous Adenosine on Epicardial Coronary Arteries

This study examines the effects of intravenous infusion of adenosine and sublingual nitroglycerin on coronary angiograms obtained by current-generation multidetector computed tomography. We assessed coronary vasodilation at baseline and after intravenous adenosine (140 µg/kg/min) or sublingual nitroglycerin spray (800 µg) in 7 female swine (weight, 40.9 ± 1.4 kg) by using electrocardiogram-gated coronary angiography with a 64-detector scanner (rotation time, 400 ms; 120kV; 400 mA) and intravenous contrast (300 mg/mL iohexol, 4.5 mL/s, 2 mL/kg). Cross-sectional areas of segments in the left anterior descending, circumflex, and right coronary arteries were evaluated in oblique orthogonal views. Images were acquired at an average heart rate of 73 ± 11 beats per minute. Changes in aortic pressure were not significant with nitroglycerin but decreased (approximately 10%) with adenosine. Of the 76 segments analyzed (baseline range, 2 to 39 mm²), 1 distal segment could not be assessed after adenosine. Segment cross-sectional area increased by 11.3% with nitroglycerin but decreased by 9.6% during adenosine infusion. The results of the present study are consistent with the practice of using sublingual nitroglycerin to enhance visualization of epicardial vessels and suggest that intravenous adenosine may hinder coronary artery visualization. This study is the first repeated-measures electrocardiogram-gated CT evaluation to use the same imaging technology to assess changes in coronary cross-sectional area before and after treatment with a vasodilator. The nitroglycerin-associated changes in our swine model were modest in comparison with previously reported human studies.Abbreviations: LAD, left anterior descending artery; MDCT, multidetector computed tomographyRecent advances in multidetector computed tomography (MDCT) for coronary angiography, their increased availability, and the pending release of a new generation of CT scanners contribute to this methodology''s potential for revolutionizing the early diagnosis and functional assessment of coronary artery disease.^15,16,25 However, the benefits of methodologic advances do not diminish the need to validate and assess the safety, efficacy, and costs of technology.¹⁵ In this regard, the present study uses an animal model with coronary anatomy analogous to that of humans^11,43 to assess the effects of sublingual nitroglycerin and intravenous adenosine on coronary epicardial vessel visualization by using baseline imaging as a control. The radiation exposure associated with CT procedures precludes human studies that involve repeated measures on the same subjects.Sublingual nitroglycerin is hypothesized to enhance the visualization of epicardial coronary vessels due to its vasodilatory properties.^8-10 Nitroglycerin acts as a nitric oxide generator to induce relaxation of vascular smooth muscle independent of endothelial function.^1,18,20 Current evidence supporting improved coronary visualization with sublingual nitroglycerin is derived from clinical cross-sectional studies that compare results in different groups of patients with heterogeneous coronary lesions.^5,6,16,27,44 A recent survey reported that more than 80% of facilities in the United States routinely use nitroglycerin in cardiac MDCT angiography.²³In contrast to the action of nitroglycerin on epicardial vascular smooth muscle, intravenous adenosine frequently is used as a pharmacologic stress to affect vasodilatation within the coronary microcirculation.²⁰ Although current-generation CT scanners have limited capability to assess myocardial tissue perfusion, research efforts from advanced imaging centers suggest that future-generation CT scanners soon will permit myocardial perfusion imaging.^13,14,29 No studies to date have assessed epicardial vessel changes by MDCT with intravenous doses of adenosine appropriate for myocardial perfusion imaging.The objective of this study was to provide an animal model for MDCT studies that permits repeated measures of epicardial vessels without the limitations of cross-sectional designs or the complications of heterogeneity of vascular lesions within clinical populations. This study used routes of administration of nitroglycerin and adenosine that are consistent with clinical practice.     

Discovery of Phytophthora infestans Genes Expressed in Planta through Mining of cDNA Libraries

Background

Phytophthora infestans (Mont.) de Bary causes late blight of potato and tomato, and has a broad host range within the Solanaceae family. Most studies of the Phytophthora – Solanum pathosystem have focused on gene expression in the host and have not analyzed pathogen gene expression in planta.

Methodology/Principal Findings

We describe in detail an in silico approach to mine ESTs from inoculated host plants deposited in a database in order to identify particular pathogen sequences associated with disease. We identified candidate effector genes through mining of 22,795 ESTs corresponding to P. infestans cDNA libraries in compatible and incompatible interactions with hosts from the Solanaceae family.

Conclusions/Significance

We annotated genes of P. infestans expressed in planta associated with late blight using different approaches and assigned putative functions to 373 out of the 501 sequences found in the P. infestans genome draft, including putative secreted proteins, domains associated with pathogenicity and poorly characterized proteins ideal for further experimental studies. Our study provides a methodology for analyzing cDNA libraries and provides an understanding of the plant – oomycete pathosystems that is independent of the host, condition, or type of sample by identifying genes of the pathogen expressed in planta.     

Presence and expression of the mating type locus in Paracoccidioides brasiliensis isolates

Paracoccidioides brasiliensis has been classified in the phylum Ascomycota, order Onygenales, family Ajellomycetaceae, even in the absence of a known sexual cycle or mating system. The objective of this work was to determine the presence of the mating type locus in 71 P. brasiliensis isolates from various sources. A PCR assay using specific primers for the MAT 1 gene was developed and applied for the detection of such genes. Two heterothallic groups (MAT1-1 or MAT1-2) were recognized and, in some isolates, gene expression was confirmed indicating the existence of a basal gene expression. The distribution of two mating type loci in the studied population suggested that sexual reproduction might occur in P. brasiliensis. This finding points towards the possibility of applying a more precise definition of the concept of biological species to P. brasiliensis. Further studies should be conducted to confirm the sexual capacity of this fungus and its implications among phylogenetic species and geographical distribution.