A Kinesin Driven Enzyme Linked Immunosorbant Assay (ELISA) for Ultra Low Protein Detection Applications

<p>Gene Ontology bar charts for three different criteria: biological process (A), cell localization (B) and molecular function (C). The list of differentially expressed genes after RSV diet in neocortex were classified by Gene Ontology (GO) and drawn using Panther (<a href="https://www.pantherdb.org/" target="_blank">www.pantherdb.org/</a>)</p

Micelle-like Nanoparticles as Carriers for DNA and siRNA

Gene therapy represents a potential efficient approach of disease prevention and therapy. However, due to their poor <i>in vivo</i> stability, gene molecules need to be associated with delivery systems to overcome extracellular and intracellular barriers and allow access to the site of action. Cationic polymeric nanoparticles are popular carriers for small interfering RNA (siRNA) and DNA-based therapeutics for which efficient and safe delivery are important factors that need to be optimized. Micelle-like nanoparticles (MNP) (half micelles, half polymeric nanoparticles) can overcome some of the disadvantages of such cationic carriers by unifying in one single carrier the best of both delivery systems. In this review, we will discuss how the unique properties of MNP including self-assembly, condensation and protection of nucleic acids, improved cell association and gene transfection, and low toxicity may contribute to the successful application of siRNA- and DNA-based therapeutics into the clinic. Recent developments of MNP involving the addition of stimulus-sensitive functions to respond specifically to pathological or externally applied “triggers” (e.g., temperature, pH or enzymatic catalysis, light, or magnetic fields) will be discussed. Finally, we will overview the use of MNP as two-in-one carriers for the simultaneous delivery of different agents (small molecules, imaging agents) and nucleic acid combinations

Image_2_Neuronal Calcium and cAMP Cross-Talk Mediated by Cannabinoid CB1 Receptor and EF-Hand Calcium Sensor Interactions.TIF

<p>Endocannabinoids are important players in neural development and function. They act via receptors, whose activation inhibits cAMP production. The aim of the paper was to look for calcium- and cAMP-signaling cross-talk mediated by cannabinoid CB₁ receptors (CB₁R) and to assess the relevance of EF-hand CaM-like calcium sensors in this regard. Using a heterologous expression system, we demonstrated that CB₁R interacts with calneuron-1 and NCS1 but not with caldendrin. Furthermore, interaction motives were identified in both calcium binding proteins and the receptor, and we showed that the first two sensors competed for binding to the receptor in a Ca²⁺-dependent manner. Assays in neuronal primary cultures showed that, CB₁R-NCS1 complexes predominate at basal Ca²⁺ levels, whereas in the presence of ionomycin, a calcium ionophore, CB₁R-calneuron-1 complexes were more abundant. Signaling assays following forskolin-induced intracellular cAMP levels showed in mouse striatal neurons that binding of CB₁R to NCS1 is required for CB₁R-mediated signaling, while the binding of CB₁R to calneuron-1 completely blocked G_i-mediated signaling in response to a selective receptor agonist, arachidonyl-2-chloroethylamide. Calcium levels and interaction with calcium sensors may even lead to apparent Gs coupling after CB₁R agonist challenge.</p

Image_3_Neuronal Calcium and cAMP Cross-Talk Mediated by Cannabinoid CB1 Receptor and EF-Hand Calcium Sensor Interactions.TIFF

<p>Endocannabinoids are important players in neural development and function. They act via receptors, whose activation inhibits cAMP production. The aim of the paper was to look for calcium- and cAMP-signaling cross-talk mediated by cannabinoid CB₁ receptors (CB₁R) and to assess the relevance of EF-hand CaM-like calcium sensors in this regard. Using a heterologous expression system, we demonstrated that CB₁R interacts with calneuron-1 and NCS1 but not with caldendrin. Furthermore, interaction motives were identified in both calcium binding proteins and the receptor, and we showed that the first two sensors competed for binding to the receptor in a Ca²⁺-dependent manner. Assays in neuronal primary cultures showed that, CB₁R-NCS1 complexes predominate at basal Ca²⁺ levels, whereas in the presence of ionomycin, a calcium ionophore, CB₁R-calneuron-1 complexes were more abundant. Signaling assays following forskolin-induced intracellular cAMP levels showed in mouse striatal neurons that binding of CB₁R to NCS1 is required for CB₁R-mediated signaling, while the binding of CB₁R to calneuron-1 completely blocked G_i-mediated signaling in response to a selective receptor agonist, arachidonyl-2-chloroethylamide. Calcium levels and interaction with calcium sensors may even lead to apparent Gs coupling after CB₁R agonist challenge.</p

Network analysis using STRING.

<p>The list of differentially expressed genes in neocortex after RSV diet was subjected to Network analysis using the STRING software as described in Methods. Square interaction score was set at medium confidence (0.4; scores range from highest, 0.9, to low, 0.15, confidence). Other selected parameters were: average node degree: 1.12, local clustering coefficient: 0.32 and active interaction sources including: text mining, experiments, databases, co-expression, neighborhood, gene fusion and co-occurrence. Moreover, no restrictions were forced in the number of interactions to show. Overexpressed genes are shown within red rectangles. The colors of the edges represent the different types of protein associations, either from known or predicted interactions: from curated databases (blue), experimentally determined (magenta), text mining (green) and co-expression (black).</p

Gene expression evaluated by reverse transcription, quantitative real-time PCR (RT-qPCR) in control and in 100 μM RSV-treated C6 glioma cells.

<p>mRNA samples isolated from cells incubated for 24 h were analyzed by RT-qPCR using specific probes for <i>Atp1b2</i>, <i>Vamp2</i>, <i>Rab2a</i>, and <i>Dnm1</i>. β-actin mRNA was used as control. Data are mean ± SEM of four independent experiments. * p < 0.05 according to Student’s t test.</p

Venn diagram constructed with lists of differentially expressed genes corresponding to the three tissues (neocortex, heart and muscle) as identified using the GEO2R tool.

<p>The lists of genes were first curated as indicated in Material and Methods.</p

Number of differentially expressed genes detected by GeneSpring depending on p-value and absolute fold change (FC) values.

<p>Analysis of data, from control- and RSV-enriched diet both in quintuplicates, were performed using moderated T-test, the multiple testing correction of Benjamini-Hochberg and asymptotic p-value (adjusted) computation.</p

Selected differentially expressed genes.

<p>Selected differentially expressed genes.</p

Adjuvant nanoparticle anti-IL6 siRNA suppresses thermal ablation-induced hepatocyte proliferation in the untreated, distant hepatic lobe.

<p>Adjuvant MNP anti-IL6 siRNA given 15 minutes after hepatic thermal ablation <b>(C)</b> in C57Bl mice (n = 5–6 animals/arm) suppressed hepatocyte proliferation in the distant, untreated liver lobe (with CDC47 staining, mean ± standard deviation) compared to hepatic thermal ablation alone <b>(A, D,</b> p<0.01). Hepatic thermal ablation combined with MNP scrambled siRNA was not significantly different from either thermal ablation alone or ablation combined with MNP anti-IL6 siRNA <b>(B, D)</b>.</p