Intracellular delivery of an antisense oligonucleotide via endocytosis of a G protein-coupled receptor

Gastrin-releasing peptide receptor (GRPR), a member of the G protein-coupled receptor superfamily, has been utilized for receptor-mediated targeting of imaging and therapeutic agents; here we extend its use to oligonucleotide delivery. A splice-shifting antisense oligonucleotide was conjugated to a bombesin (BBN) peptide, and its intracellular delivery was tested in GRPR expressing PC3 cells stably transfected with a luciferase gene interrupted by an abnormally spliced intron. The BBN-conjugate produced significantly higher luciferase expression compared to unmodified oligonucleotide, and this increase was reversed by excess BBN peptide. Kinetic studies revealed a combination of saturable, receptor-mediated endocytosis and non-saturable pinocytosis for uptake of the conjugate. The Km value for saturable uptake was similar to the EC50 value for the pharmacological response, indicating that receptor-mediated endocytosis was a primary contributor to the response. Use of pharmacological and molecular inhibitors of endocytosis showed that the conjugate utilized a clathrin-, actin- and dynamin-dependent pathway to enter PC3 cells. The BBN-conjugate partially localized in endomembrane vesicles that were associated with Rab7 or Rab9, demonstrating that it was transported to late endosomes and the trans-golgi network. These observations suggest that the BBN-oligonucleotide conjugate enters cells via a process of GRPR mediated endocytosis followed by trafficking to deep endomembrane compartments

Body Mass Index (BMI) assessment among Macau students: age group differences and weight management strategies

There is evidence that rapid weight gain during the first year of life is associated with being overweight later in life. Therefore, overweight tendencies need to be detected at an appropriate age, and suitable strategies need to be implemented for weight management to achieve optimal long-term health. The objective of this study was to investigate comparisons in BMI status and associated categories in male and female students over ten years in two phases, including 2008-2013 and 2009-2014. Weight and height data were collected to obtain BMI (Body Mass Index) over ten years in two phases. The first phase occurred from 2008 to 2013, and the second phase occurred from 2009 to 2014 in a population of 10846 school children (Males: 6970, 64.3%, and females: 3875, 35.7%) in Macau. Their ages ranged from 6 years old in 2008 to 11 years old in 2013. The same age range was observed in the second phase, i.e., 2009-2014. Statistical analyses included descriptive statistics, such as the mean, standard deviation, t-tests to determine gender differences (year-wise) and a Chi square test for independence to determine the relationship between BMI (Underweight, Standard, Overweight and Obese) and age groups. In the first phase (2008-2013), the findings indicated a higher BMI level among the male students than the female students across all age groups (2008 t = 5.24, 2009 t = 88.25, 2010 t = 11.32, 2011 t = 17.45, 2012 t = 19.70 and 2013 t = 19.92). In the second phase (2009-2014), a higher BMI level was found among the male students than the female students across all age groups (2009 t = 2.68, 2010 t = 2.886, 2011 t = 3.076, 2013 t = 4.228, and 2014 t = 2.405). The results of the two phases combined (2008 to 2014 and 2009 to 2014) revealed that male students in 2008 had a higher BMI level than their counterparts in 2009 in the two age categories (8 years t = 3.025 and 11 years t = 3.377). Female students in the second phase (2009-2014) showed a higher BMI level than their male counterparts (9 years, t = 3.151). The results indicate the need to have focused strategies and structured interventions for male students at the critical age range of 8 to 9 years old. The results of this study also imply the need for the delivery of suitable school intervention activities at the appropriate time. Specifically, the prevention of weight gain should start early in life to encourage the development of healthier behaviours and habits throughout childhood and later ages

Mapping the contribution of β3-containing GABA(A )receptors to volatile and intravenous general anesthetic actions

BACKGROUND: Agents belonging to diverse chemical classes are used clinically as general anesthetics. The molecular targets mediating their actions are however still only poorly defined. Both chemical diversity and substantial differences in the clinical actions of general anesthetics suggest that general anesthetic agents may have distinct pharmacological targets. It was demonstrated previously that the immobilizing action of etomidate and propofol is completely, and the immobilizing action of isoflurane partly mediated, by β3-containing GABA(A )receptors. This was determined by using the β3(N265M) mice, which carry a point mutation known to decrease the actions of general anesthetics at recombinant GABA(A )receptors. In this communication, we analyzed the contribution of β3-containing GABA(A )receptors to the pharmacological actions of isoflurane, etomidate and propofol by means of β3(N265M) mice. RESULTS: Isoflurane decreased core body temperature and heart rate to a smaller degree in β3(N265M) mice than in wild type mice, indicating a minor but significant role of β3-containing GABA(A )receptors in these actions. Prolonged time intervals in the ECG and increased heart rate variability were indistinguishable between genotypes, suggesting no involvement of β3-containing GABA(A )receptors. The anterograde amnesic action of propofol was indistinguishable in β3(N265M) and wild type mice, suggesting that it is independent of β3-containing GABA(A )receptors. The increase of heart rate variability and prolongation of ECG intervals by etomidate and propofol were also less pronounced in β3(N265M) mice than in wild type mice, pointing to a limited involvement of β3-containing GABA(A )receptors in these actions. The lack of etomidate- and propofol-induced immobilization in β3(N265M) mice was also observed in congenic 129X1/SvJ and C57BL/6J backgrounds, indicating that this phenotype is stable across different backgrounds. CONCLUSION: Our results provide evidence for a defined role of β3-containing GABA(A )receptors in mediating some, but not all, of the actions of general anesthetics, and confirm the multisite model of general anesthetic action. This pharmacological separation of anesthetic endpoints also suggests that subtype-selective substances with an improved side-effect profile may be developed

A Quantitative Study of the Mechanisms behind Thymic Atrophy in Gαi2-Deficient Mice during Colitis Development

Mice deficient for the G protein subunit Gαi2 spontaneously develop colitis, a chronic inflammatory disease associated with dysregulated T cell responses. We and others have previously demonstrated a thymic involution in these mice and an aberrant thymocyte dynamics. The Gαi2−/− mice have a dramatically reduced fraction of double positive thymocytes and an increased fraction of single positive (SP) thymocytes. In this study, we quantify a number of critical parameters in order to narrow down the underlying mechanisms that cause the dynamical changes of the thymocyte development in the Gαi2−/− mice. Our data suggest that the increased fraction of SP thymocytes results only from a decreased number of DP thymocytes, since the number of SP thymocytes in the Gαi2−/− mice is comparable to the control littermates. By measuring the frequency of T cell receptor excision circles (TRECs) in the thymocytes, we demonstrate that the number of cell divisions the Gαi2−/− SP thymocytes undergo is comparable to SP thymocytes from control littermates. In addition, our data show that the mature SP CD4+ and CD8+ thymocytes divide to the same extent before they egress from the thymus. By estimating the number of peripheral TREC+ T lymphocytes and their death rate, we could calculate the daily egression of thymocytes. Gαi2−/− mice with no/mild and moderate colitis were found to have a slower export rate in comparison to the control littermates. The quantitative measurements in this study suggest a number of dynamical changes in the thymocyte development during the progression of colitis

Regulation of mammary gland branching morphogenesis by the extracellular matrix and its remodeling enzymes.

A considerable body of research indicates that mammary gland branching morphogenesis is dependent, in part, on the extracellular matrix (ECM), ECM-receptors, such as integrins and other ECM receptors, and ECM-degrading enzymes, including matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). There is some evidence that these ECM cues affect one or more of the following processes: cell survival, polarity, proliferation, differentiation, adhesion, and migration. Both three-dimensional culture models and genetic manipulations of the mouse mammary gland have been used to study the signaling pathways that affect these processes. However, the precise mechanisms of ECM-directed mammary morphogenesis are not well understood. Mammary morphogenesis involves epithelial 'invasion' of adipose tissue, a process akin to invasion by breast cancer cells, although the former is a highly regulated developmental process. How these morphogenic pathways are integrated in the normal gland and how they become dysregulated and subverted in the progression of breast cancer also remain largely unanswered questions

Measuring the perception of quality physical education in Latin American professionals

Um plano instável para o desenvolvimento de currículos e questões de apoio na educac¸ão física (PE) criou confusão entre os profissionais. O objetivo desta pesquisa é investigar os fatores percebidos como importantes no desenvolvimento da educac¸ão física de qualidade (QPE) por profissionais de países da América Latina (AL). Um questionário composto por 24 itens com base no QPE foi respondido por 468 profissionais coletados em 6 cidades da América Latina. Uma análise fatorial exploratória dos 24 itens usando extrac¸ão ML e rotac¸ão obliminar direta foram aplicados, e os 17 itens retidos foram agrupados em uma soluc¸ão de três fatores denominada Elementos de Desenvolvimento e Suporte para QPE na Escola (DSFQPE) ( = 0,935), Valor essencial do QPE (CVPE) ( = 0,890) e Arranjo Curricular das Atividades Físicas (CAPA) ( = 0,850). Os itens retidos indicaram propriedades excelentes e o referencial básico percebido pelos profissionais de EF em países da América Latina como importante na investigac¸ão do PEQ. © 2018 Colegio ´ Brasileiro de Ciencias ˆ do Esporte. Publicado por Elsevier Editora Ltda. Este e´ um artigo Open Access sob uma licenc¸a CC BY-NC-N

Antimicrobial Peptides and Skin: A Paradigm of Translational Medicine

Antimicrobial peptides (AMPs) are small, cationic, amphiphilic peptides with broad-spectrum microbicidal activity against both bacteria and fungi. In mammals, AMPs form the first line of host defense against infections and generally play an important role as effector agents of the innate immune system. The AMP era was born more than 6 decades ago when the first cationic cyclic peptide antibiotics, namely polymyxins and tyrothricin, found their way into clinical use. Due to the good clinical experience in the treatment of, for example, infections of mucus membranes as well as the subsequent understanding of mode of action, AMPs are now considered for treatment of inflammatory skin diseases and for improving healing of infected wounds. Based on the preclinical findings, including pathobiochemistry and molecular medicine, targeted therapy strategies are developed and first results indicate that AMPs influence processes of diseased skin. Importantly, in contrast to other antibiotics, AMPs do not seem to propagate the development of antibiotic-resistant micro-organisms. Therefore, AMPs should be tested in clinical trials for their efficacy and tolerability in inflammatory skin diseases and chronic wounds. Apart from possible fields of application, these peptides appear suited as an example of the paradigm of translational medicine for skin diseases which is today seen as a `two-way road' - from bench to bedside and backwards from bedside to bench. Copyright (c) 2012 S. Karger AG, Base

Prevalence and prognosis of myocardial scar in patients with known or suspected coronary artery disease and normal wall motion

<p>Abstract</p> <p>Background</p> <p>Some patients may have normal wall motion after myocardial infarction. The aim of this study was to determine the prevalence and prognosis of patients with myocardial scar in the absence of abnormal wall motion. We studied patients with suspected or known coronary artery disease (CAD) who were referred for cardiovascular magnetic resonance (CMR) for the assessment of global and regional cardiac function and late gadolinium enhancement (LGE) and had normal left ventricular wall motion. Prognostic value was determined by the occurrence of hard endpoints (cardiac death and nonfatal myocardial infarction) and major adverse cardiac events (MACE) which also included hospitalization due to unstable angina or heart failure or life threatening ventricular arrhythmia.</p> <p>Results</p> <p>A total 1148 patients (70.3%) were studied. LGE was detected in 104 patients (9.1%). Prevalence of LGE increased in patients with increased left ventricular mass. Average follow-up time was 955 ± 542 days. LGE was the strongest predictor for hard endpoints and MACE.</p> <p>Conclusion</p> <p>LGE was detected in 9.1% of patients with suspected or known CAD and normal wall motion. LGE was the strongest predictor of significant cardiac events.</p

Use of radiolabelled choline as a pharmacodynamic marker for the signal transduction inhibitor geldanamycin

There is an urgent need to develop non-invasive pharmacodynamic endpoints for the evaluation of new molecular therapeutics that inhibit signal transduction. We hypothesised that, when labelled appropriately, changes in choline kinetics could be used to assess geldanamycin pharmacodynamics, which involves inhibition of the HSP90 molecular chaperone→Raf1→Mitogenic Extracellular Kinase→Extracellular Signal-Regulated Kinase 1 and 2 signal transduction pathway. Towards identifying a potential pharmacodynamic marker response, we have studied radiolabelled choline metabolism in HT29 human colon carcinoma cells following treatment with geldanamycin. We studied the effects of geldanamycin, on net cellular accumulation of (methyl-14C)choline and (methyl-14C)phosphocholine production. In parallel experiments, the effects of geldanamycin on extracellular signal-regulated kinase 1 and 2 phosphorylation and cell viability were also assessed. Additional validation studies were carried out with the mitogenic extracellular kinase inhibitor U0126 as a positive control; a cyclin-dependent kinase-2 inhibitor roscovitine and the phosphatidylinositol 3-kinase inhibitor LY294002 as negative controls. Hemicholinium-3, an inhibitor of choline transport and choline kinase activity was included as an additional control. In exponentially growing HT29 cells, geldanamycin inhibited extracellular signal-regulated kinase 1 and 2 phosphorylation in a concentration- and time-dependent manner. These changes were associated with a reduction in (methyl-14C)choline uptake, (methyl-14C) phosphocholine production and cell viability. Brief exposure to U0126, suppressed phosphocholine production to the same extent as Hemicholinium-3. In contrast to geldanamycin and U0126, which act upstream of extracellular signal-regulated kinase 1 and 2, roscovitine and LY294002 failed to suppress phosphocholine production. Our results suggest that when labelled with carbon-11 isotope, (methyl-11C)choline may be a useful pharmacodynamic marker for the non-invasive evaluation of geldanamycin analogues