In vivo selection of novel biotherapeutics

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The coherent dynamics of photoexcited green fluorescent proteins

The coherent dynamics of vibronic wave packets in the green fluorescent protein is reported. At room temperature the non-stationary dynamics following impulsive photoexcitation displays an oscillating optical transmissivity pattern with components at 67 fs (497 cm-1) and 59 fs (593 cm-1). Our results are complemented by ab initio calculations of the vibrational spectrum of the chromophore. This analysis shows the interplay between the dynamics of the aminoacidic structure and the electronic excitation in the primary optical events of green fluorescent proteins.Comment: accepted for publication in Physical Review Letter

Enhanced caveolae-mediated endocytosis by diagnostic ultrasound in vitro

The modulation of cellular endothelial permeability is a desirable goal for targeted delivery of labels and therapeutic macromolecules; the underlying mechanisms, however, remains poorly understood. Here, we hypothesize that a higher endothelial permeability may result as an outcome of selective enhancement of caveolar endocytosis by ultrasound (US), in the frequency and intensity range of current clinical diagnostic use. To assess the role of free radicals in this phenomenon, we exposed confluent human endothelial cells to pulsed diagnostic US for 30 min, with a mechanical index (MI) of 0.5 and 1.2, using a 1.6-MHz cardiac US scan, and endothelial cells not exposed to US were used as control. Here we show that pulsed diagnostic US with a MI of 1.2 (high mechanical index ultrasound [HMIUS]) were able to selectively enhance endothelial caveolar internalization of recombinant glutathione-S-transferase (GST)-Tat11-EGFP fusion protein (26 1 vs. 11.6 1 A.U, p < 0.001 vs. control), without disruption of plasma membrane integrity. Moreover, pulsed diagnostic US with a MI of 0.5 (low mechanical index ultrasound) did not increase caveolar endocytosis compared with control (11.4 1.2 vs. 11.6 1). Free-radical generation inhibitors, such as catalase and superoxide dismutase, reduced the HMIUS-induced caveolar internalization by a 49.29% factor; finally, HMIUS-induced caveolar endocytosis was found to be associated with a significant increase in the phosphorylation of tyr-14-caveolin1, ser1177-eNOS and Thr202/ Tyr204-ERK? compared with control. These findings show how HMIUS irradiation of human endothelial cells cause a selective enhancement of caveolar-dependent permeability, partially mediated by free radicals generation, inducing a marked increase of phosphorylation of caveolar-related proteins. Thus, the use of diagnostic US could potentially be used as an adjuvant to drive caveolar traffic of extracellular peptides by using a higher level of US energy

Identification of specific molecular structures of human immunodeficiency virus type 1 Tat relevant for its biological effects on vascular endothelial cells.

Human immunodeficiency virus type 1 (HIV-1) Tat transactivates viral genes and is released by infected cells, acting as a soluble mediator. In endothelial cells (EC), it activates a proangiogenic program by activating vascular endothelial growth factor receptor type 2 (VEGFR-2) and integrins. A structure-activity relationship study was performed by functional analysis of Tat substitution and deletion variants to define the Tat determinants necessary for EC activation. Variants were made (i) in the basic and (ii) in the cysteine-rich domains and (iii) in the C-terminal region containing the RGD sequence required for integrin recognition. Our results led to the following conclusions. (i) Besides a high-affinity binding site corresponding to VEGFR-2, EC express low-affinity binding sites. (ii) The basic and the cysteine-rich variants bind only to the low-affinity binding sites and do not promote tyrosine phosphorylation of VEGFR-2. Furthermore, they have a reduced ability to activate EC in vitro, and they lack angiogenic activity. (iii) Mutants with mutations in the C-terminal region are partially defective for in vitro biological activities and in vivo angiogenesis, but they activate VEGFR-2 as Tat wild type. In conclusion, regions encoded by the first exon of tat are necessary and sufficient for activation of VEGFR-2. However, the C-terminal region, most probably through RGD-mediated integrin engagement, is indispensable for full activation of an in vitro and in vivo angiogenic progra

Unacylated ghrelin reduces skeletal muscle reactive oxygen species generation and inflammation and prevents high-fat diet-induced hyperglycemia and whole-body insulin resistance in rodents

Excess reactive oxygen species (ROS) generation and inflammation may contribute to obesity-associated skeletal muscle insulin resistance. Ghrelin is a gastric hormone whose unacylated form (UnAG) is associated with whole-body insulin sensitivity in humans and may reduce oxidative stress in nonmuscle cells in vitro. We hypothesized that UnAG 1) lowers muscle ROS production and inflammation and enhances tissue insulin action in lean rats and 2) prevents muscle metabolic alterations and normalizes insulin resistance and hyper-glycemia in high-fat diet (HFD)-induced obesity. In 12-week-old lean rats, UnAG (4-day, twice-daily subcutaneous 200-mg injections) reduced gastrocnemius mitochondrial ROS generation and inflammatory cytokines while enhancing AKT-dependent signaling and insulinstimulated glucose uptake. In HFD-treated mice, chronic UnAG overexpression prevented obesity-associated hyperglycemia and whole-body insulin resistance (insulin tolerance test) as well as muscle oxidative stress, inflammation, and altered insulin signaling. In myotubes, UnAG consistently lowered mitochondrial ROS production and enhanced insulin signaling, whereas UnAG effects were prevented by small interfering RNA-mediated silencing of the autophagy mediator ATG5. Thus, UnAG lowers mitochondrial ROS production and inflammation while enhancing insulin action in rodent skeletal muscle. In HFD-induced obesity, these effects prevent hyperglycemia and insulin resistance. Stimulated muscle autophagy could contribute to UnAG activities. These findings support UnAG as a therapeutic strategy for obesity-associated metabolic alterations

A mouse model for adult cardiac-specific gene deletion with CRISPR/Cas9

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) 9 genomic editing has revolutionized the generation of mutant animals by simplifying the creation of null alleles in virtually any organism. However, most current approaches with this method require zygote injection, making it difficult to assess the adult, tissue-specific functions of genes that are widely expressed or which cause embryonic lethality when mutated. Here, we describe the generation of cardiac-specific Cas9 transgenic mice, which express high levels of Cas9 in the heart, but display no overt defects. In proof-of-concept experiments, we used Adeno-Associated Virus 9 (AAV9) to deliver single-guide RNA (sgRNA) that targets the Myh6 locus exclusively in cardiomyocytes. Intraperitoneal injection of postnatal cardiac-Cas9 transgenic mice with AAV9 encoding sgRNA against Myh6 resulted in robust editing of the Myh6 locus. These mice displayed severe cardiomyopathy and loss of cardiac function, with elevation of several markers of heart failure, confirming the effectiveness of this method of adult cardiac gene deletion. Mice with cardiac-specific expression of Cas9 provide a tool that will allow rapid and accurate deletion of genes following a single injection of AAV9-sgRNAs, thereby circumventing embryonic lethality. This method will be useful for disease modeling and provides a means of rapidly editing genes of interest in the heart

Maternal taurine supplementation in rats partially prevents the adverse effects of early-life protein deprivation on b-cell function and insulin sensitivity

Dietary protein restriction during pregnancy and lactation in rats impairs b-cell function and mass in neonates and leads to glucose intolerance in adult offspring. Maternal taurine (Tau) supplementation during pregnancy in rats restores b-cell function and mass in neonates, but its long-term effects are unclear. The prevention of postnatal catch-up growth has been suggested to improve glucose tolerance in adult offspring of low-protein (LP)-fed mothers. The objective of this study was to examine the relative contribution of b-cell dysfunction and insulin resistance to impaired glucose tolerance in 130-day-old rat offspring of LP-fed mothers and the effects of maternal Tau supplementation on b-cell function and insulin resistance in these offspring. Pregnant rats were fed i) control, ii) LP, and iii) LPCTau diets during gestation and lactation. Offspring were given a control diet following weaning. A fourth group consisting of offspring of LP-fed mothers, maintained on a LP diet following weaning, was also studied (LP-all life). Insulin sensitivity in the offspring of LP-fed mothers was reduced in females but not in males. In both genders, LP exposure decreased b-cell function. Tau supplementation improved insulin sensitivity in females and b-cell function in males. The LP-all life diet improved b-cell function in males. We conclude that i) maternal Tau supplementation has persistent effects on improving glucose metabolism (b-cell function and insulin sensitivity) in adult rat offspring of LP-fed mothers and ii) increasing the amount of protein in the diet of offspring adapted to a LP diet after weaning may impair glucose metabolism (b-cell function) in a gender-specific manner.Fil: Tang, Christine. University Of Toronto; Canadá;Fil: Marchand, K.elly. University of Western Ontario. Lawson Health Research Institute; Canadá;Fil: Lam, Loretta. University Of Toronto; Canadá;Fil: Lux, Victoria Adela R.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Thyssen, Sandra M.. University of Western Ontario. Lawson Health Research Institute; Canadá;Fil: Guo, June. University Of Toronto; Canadá;Fil: Giacca, A.dria. University Of Toronto; Canadá;Fil: Arany, Edith. University of Western Ontario. Lawson Health Research Institute; Canadá

HIV-1 Tat, apoptosis and the mitochondria: a tubulin link?

The Tat protein of HIV-1 is a powerful activator of viral gene expression. Besides this essential function at the HIV-1 promoter, the protein also exerts a remarkable number of other biological activities, among which the induction of cellular apoptosis. Two papers now published in Retrovirology provide possible molecular mechanisms for the pro-apoptotic effect of Tat, which involve the cell's microtubular network and the mitochondrial pathway of apoptosis

Virus-mediated gene transfer to induce therapeutic angiogenesis: Where do we stand?

The potential to induce therapeutic angiogenesis through gene transfer has engendered much excitement as a possible treatment for tissue ischemia. After 10 years of clinical experimentation, however, it now appears clear that several crucial issues are still to be resolved prior to achieving clinical success. These include the understanding of whether functional blood vessels might arise as a result of the delivery of a single angiogenic factor or require more complex cytokine combinations, the identification of the proper timing of therapeutic gene expression and, most notably, the development of more efficacious gene delivery tools. Viral vectors based on the adeno-associated virus (AAV) appear particularly suitable to address the last requirement, since they display a specific tropism for skeletal muscle cells and cardiomyocytes, and drive expression of the therapeutic genes in these cells for indefinite periods of time. In this review, I discuss the current applications of gene therapy for cardiovascular disorders, with particular attention to the possible improvements in the technologies involved in virus-mediated gene transfer