Transmissible gastroenteritis virus: Identification of M protein-binding peptide ligands with antiviral and diagnostic potential

The membrane (M) protein is one of the major structural proteins of coronavirus particles. In this study, the M protein of transmissible gastroenteritis virus (TGEV) was used to biopan a 12-mer phage display random peptide library. Three phages expressing TGEV-M-binding peptides were identified and characterized in more depth. A phage-based immunosorbent assay (phage-ELISA) capable of differentiating TGEV from other coronaviruses was developed using one phage, phTGEV-M7, as antigen. When the phage-ELISA was compared to conventional antibody-based ELISA for detecting infections, phage-ELISA exhibited greater sensitivity. A chemically synthesized, TGEV-M7 peptide (pepTGEV-M7; HALTPIKYIPPG) was evaluated for antiviral activity. Plaque-reduction assays revealed that pepTGEV-M7 was able to prevent TGEV infection in vitro (p \u3c 0.01) following pretreatment of the virus with the peptide. Indirect immunofluorescence and real-time RT-PCR confirmed the inhibitory effects of the peptide. These results indicate that pepTGEV-M7 might be utilized for virus-specific diagnostics and treatment

Transmissible gastroenteritis virus: Identification of M protein-binding peptide ligands with antiviral and diagnostic potential

The membrane (M) protein is one of the major structural proteins of coronavirus particles. In this study, the M protein of transmissible gastroenteritis virus (TGEV) was used to biopan a 12-mer phage display random peptide library. Three phages expressing TGEV-M-binding peptides were identified and characterized in more depth. A phage-based immunosorbent assay (phage-ELISA) capable of differentiating TGEV from other coronaviruses was developed using one phage, phTGEV-M7, as antigen. When the phage-ELISA was compared to conventional antibody-based ELISA for detecting infections, phage-ELISA exhibited greater sensitivity. A chemically synthesized, TGEV-M7 peptide (pepTGEV-M7; HALTPIKYIPPG) was evaluated for antiviral activity. Plaque-reduction assays revealed that pepTGEV-M7 was able to prevent TGEV infection in vitro (p \u3c 0.01) following pretreatment of the virus with the peptide. Indirect immunofluorescence and real-time RT-PCR confirmed the inhibitory effects of the peptide. These results indicate that pepTGEV-M7 might be utilized for virus-specific diagnostics and treatment

Development of novel breast cancer-targeted spect imaging peptides by phage display

"May 2014."Dissertation Supervisor: Dr. Susan L. Deutscher.Includes vita.Malignancy of the breast is the leading cause of cancer among women and the second leading cause of cancer-related death. Despite implementation of mammograms and breast exams, there still exists a need to detect highly aggressive cancer at its earliest stage. Development of targeted molecules, such as peptides, by phage display allows for specific imaging of a targeted antigen. The hypothesis of this dissertation was that phage display could be used select novel peptides for breast cancer imaging. In vivo phage display selection is a strategy that allows for selection of peptides based on their function in the environment in which they will be used. The work here demonstrated that in vivo phage display successfully was able to select a peptide with improved pharmacokinetics and tumor targeting in comparison to a previous generation. Additionally, the selection yielded a peptide that mimicked a putative tumor vasculature. associated protein termed EGFL6. EGFL6 was determined to be expressed in breast cancer and may serve as the basis for future investigations. Finally, a peptide was discovered that bound to and imaged BT-474 human breast cancer tumors in mice. This cell line is of importance because it represents resistance susceptible breast cancer, a major cause of death among women. These results demonstrate the utility of phage display and highlight the novel targeting agents that can be obtained by the technology.Includes bibliographical references (pages 137-151)

The role of AMPK in the regulation of nitric oxide synthesis by perivascular adipose tissue

Nitric oxide (NO) is a key signalling molecule in the cardiovascular, genitourinary, respiratory, nervous, and gastrointestinal systems. NO is released by the endothelium of blood vessels and inhibits vascular smooth muscle cell (VSMC) migration and proliferation, modulates vascular tone and maintains cardiovascular homeostasis. Several clinical studies have shown that a reduction of NO bioavailability has a significant role in the development of endothelial dysfunction and other cardiovascular diseases. Perivascular adipose tissue (PVAT) is the outer layer of connective tissue that surrounds most systemic blood vessels, adjacent to the adventitia. Adipose tissue provides a protective layer of tissue surrounding most of the body organs and, in the case of PVAT, regulates vascular function by production of many autocrine and paracrine molecules including adiponectin, prostacyclin and angiotensin 1–7. AMP-activated protein kinase (AMPK) plays a major role in sensing cellular energy status and signalling this information back to the mitochondria to modulate their function according to the energy demands of the cell. Previous work in our laboratory has demonstrated that the presence of PVAT enhances vascular relaxation to cromakalim in endothelium-intact thoracic aortic rings from wild type (WT), but not in mice lacking AMPKα1. Compared to previous studies which examined the role of AMPK in regulation of NO release from the endothelium, the current research has focused on the mechanisms by which AMPK regulates NO production by PVAT and what effect this has on vascular tone. In this project experiments were conducted using wild type (WT) and global AMPKα1 knockout (KO) mice. Thoracic and abdominal aortic PVAT was utilised to measure the difference in NO production between fat depots. The anti-contractile effect of PVAT from WT and KO aortic rings was studied using wire myography. Furthermore, immunoprecipitation was used to measure the Cav-1/eNOS coupling and immunofluorescence was used to study Cav-1/eNOS colocalization in 3T3-L1 adipocytes. The results showed that there was a significant decrease in NO production in conditioned media derived from both the thoracic and abdominal PVAT from KO mice compared to WT mice, although the quantities of NO generated by the abdominal aortic PVAT was much lower. Overall, eNOS activity in WT thoracic PVAT was significantly increased compared to WT abdominal PVAT suggesting that changes in eNOS activity could account for the difference in NO production. Furthermore, in abdominal PVAT, considerably more caveolin-1 (Cav-1), a negative regulator of eNOS, was detected in both WT and KO mice compared with thoracic PVAT, which may also limit NO production by abdominal PVAT. Cav-1 was detected in eNOS immunoprecipitates and levels of Cav-1/eNOS association were increased in abdominal PVAT relative to thoracic PVAT of WT mice and the total Cav-1 levels in abdominal PVAT lysates was increased. Intriguingly, Cav-1/eNOS association was significantly increased in thoracic PVAT from KO mice compared with WT mice, which might account for the reduction in NO production in KO thoracic PVAT compared with WT. In 3T3-L1 adipocyte adipogenesis, eNOS levels and NO production were significantly reduced. Incubation of 3T3-L1 preadipocytes or adipocytes with methyl-β-cyclodextrin (MβCD), which disrupts Cav-1 and therefore leading to a noticeable reduction in Cav-1/eNOS colocalization and a significant increase in NO production both in preadipocytes and adipocytes compared with non-treated cells. Incubation of 3T3-L1 adipocytes with a mutant cell–permeable scaffolding domain peptide of Cav-1 (CAV-AP) reduced Cav-1/eNOS colocalization compared with control. In functional experiments, CAV-AP reduced PE-induced contraction in both WT and KO thoracic aortic rings lacking endothelium. Overall, the results presented in this thesis demonstrate that AMPKα1 has an important role in regulating NO production by PVAT, likely through regulating Cav-1/eNOS binding. Manipulating Cav-1/eNOS binding in adipocytes using scaffolding mutant peptides without interfering with the other biological effects of endogenous Cav-1 may have beneficial effects in restoration of NO production under conditions or diseases associated with impaired NO bioavailability