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The Inhibition of NADPH Oxidase 1/Protein Disulfide-Isomerase Increases BIM in Pancreatic CancerPancreatic cancer requires elevated protein synthesis. To maintain endoplasmic reticulum (ER) homeostasis, chaperones are overexpressed. Persistent ER stress activates the unfolded protein response (UPR), which can become overwhelmed and lead to cell death. Reactive oxygen species (ROS) from NADPH oxidases (Noxs) can regulate ER homeostasis. In particular, the chaperone protein disulfide-isomerase (PDI) colocalizes with Nox1 in the ER to regulate its activity. Because Nox1 is expressed in the stroma of pancreatic cancer, I studied the extent to which Nox1-derived ROS help establish an adaptive ER homeostasis via PDI in pancreatic cancer cells. I generated a pancreatic cancer mouse model lacking the Nox1 gene in the whole body (Nox1-null KPC mice). Nox1-competent KPC mice were used as a control. The induction of Cre recombination was done by administration of tamoxifen for five consecutive days. After 2 months of tamoxifen induction, the pancreas was harvested. Western blotting was carried out to evaluate PDI, UPR activation, ER stress and cell death. The Nox1-null KPC mice were viable. The lack of Nox1 reduced PDI, C/EBP homologous protein (CHOP) in KPC mice. The UPR sensor IRE1α and the growth arrest and DNA damage-inducible 34 (GADD34) were absent in Nox1-competent KPC mice, but they were recovered in Nox1- null KPC mice. In Nox1-null KPC mice, the apoptotic protein BIM was increased. I concluded that in pancreatic cancer, the lack of Nox1 decreased PDI, which led to an accumulation of unfolded proteins and activation of UPR system. GADD34 recovery increased protein synthesis, worsening the scenario. Persistent ER stress overwhelmed the UPR system, which caused BIM-induced cell death.
Identification and Characterization of Force Sensitive Domains using an In Vivo Dorosphila Synthetic Biology PlatformThe Notch protein is a family of highly conserved transmembrane signaling proteins that are found in all metazoan life. This protein family plays many roles in developmental and regulatory pathways in these organisms such as the development of the human vertebral column and the Drosophila adult wings. The Drosophila Notch protein has been the focus of recent research as this protein has shown to be dependent on a unique signaling mechanism that relies on the force being applied to the receptor by the endocytosis of a bound ligand for a cleavage event to occur. This protein has been shown to only signal in the presence of this endocytosis force which allows for cleavage at the S2 and S3 sites on the protein which allows for a transcription factor to be released from the membrane and enter the nucleus. In synthetic biology, this protein has found a role in creating customizable cell-cell signaling platforms known as Synthetic Notch or Syn-Notch. As demonstrated in previous research, Notch proteins contain highly modifiable ligand binding and transcription factor domains that can be interchanged with other ligand binding and transcription factor domains from non-Notch proteins without affecting the protein signaling. In addition to the ligand-binding and transcription factor, the Notch protein contains a Negative Regulatory Region (NRR) which functions as a force sensor domain and allows for the force signaling activation seen in the native Notch proteins. This region of the Notch protein is often conserved in Syn-Notch systems as a means of controlling the signaling of Syn-Notch proteins used in precision medicine. The unique force-sensing function of this domain has raised questions regarding the characteristics of this domain that allow for it to act in a force-sensing manner and if this domain is as interchangeable as its other components. In this study, domains from other non-Drosophila Notch proteins and non-Notch protein domains were evaluated in an in vivo Drosophila platform for the ability to function as a force sensor domain. In this screen, several unique force sensor domains were identified as having the ability to recapitulate the force sensing characteristic of the canonical Notch NRR. While a common thread between these domains in terms of defining characteristics remains elusive, this study was able to demonstrate the feasibility of this screening method to identify force-sensitive domains and identify 4 domains that exhibit this characteristic. The identification of these domains which are force sensitive not only allows for the characteristics to function as force-sensitive domains but also provides alternative force sensors with differing force sensitivities when compared to the canonical Notch NRR for use in Syn-Notch systems. The development of these receptors will not only contribute to the field of synthetic biology as means to create synthetic platforms for use in future research, but these receptors will allow for the development of even more precise treatments using these synthetic receptor systems in clinical therapies.
Prevalence of Chronic Medical Conditions Among the 1990-1991 Gulf War Female VeteransBackground: The care of the large number of women joining the U.S. military is receiving increased attention. Currently, 56% of all women veterans alive served during the 1990–1991 Gulf War (GW). Veterans of the GW have reported poorer health outcomes than their counterparts who did not deploy because of the potential health risks associated with military service during the GW, and the possible use of chemical and biologic warfare agents. Military service and deployment affect women differently than men, however gaps exist in the research of common chronic conditions among veterans due to the lack of gender-specific results from published studies. Purpose: The specific aims of this study were 1) to examine and compare the prevalence of chronic medical conditions (CMCs) among deployed GW female veterans and GW era female veterans that did not deploy; 2) to examine the prevalence of exposure to environmental toxicants among deployed GW female veterans and compare the prevalence of CMCs between exposed and unexposed females; and 3) to examine and compare the prevalence of CMCs among deployed male and deployed female veterans of the GW. Methods: Data from three longitudinal epidemiologic studies of the health of the 1990-1991 U.S. GW veterans were analyzed for this study. Cross tabs and logistic regression were done to calculate the prevalence and prevalence ratios (PRs) of seventeen self-reported CMCs among the study population. PRs were also used for assessing the relationship between self-reported exposure to environmental toxicants and the prevalence of CMCs among deployed female veterans. Results: The most prevalent CMCs reported by both deployed and non-deployed female veterans were headaches and high blood cholesterol. Compared to non-deployed females, significantly higher proportions of deployed females reported migraine headaches, osteoarthritis, and skin problems. In addition, the prevalence of headaches was almost three times higher for deployed female veterans, compared to deployed male veterans. Anthrax vaccine, oil well fires, pyridostigmine bromide, petroleum combustion products and chemical weapons were the toxicants with the highest proportions of reported exposure among deployed female veterans. Compared to unexposed females, significantly higher proportions of exposed females reported migraine headaches, headaches (other than migraines), high blood cholesterol, hypertension, acid reflux/GERD, irritable bowel syndrome, colon polyps, thyroid disease, osteoarthritis, tinnitus, asthma, chronic lung disease, and skin problems. Conclusions: Compared to their counterparts, higher prevalence of some CMCs were observed among deployed female veterans of the 1990–1991 GW, especially among those that reported toxicant exposures. This information about CMCs and exposures is useful for guiding medical assessments and policy development affecting this group of female veterans.
The Perioperative Synergy IndexThe presence of synergy within perioperative services has a significant bearing on a hospital’s viability and positive patient outcomes. Synergy is a positive emergence that arises as a value-added return on investment when all contributing factors and personnel work toward common goals. The purposes of this research were to develop a perioperative synergy model and to create an index to evaluate perioperative synergy. Research questions were: (a) What are the antecedents of synergy in the context of perioperative services? (b) What research is currently being conducted on high functioning perioperative services across perioperative disciplines? (c) What are the items necessary to ensure face and content validity of a perioperative synergy index? (d) How should the items be worded to ensure clarity, readability, and usability of a perioperative synergy index? (e) What are the underlying constructs that constitute perioperative services synergy? and (f) What are the psychometric properties of the perioperative synergy index? A three-phase survey methodology was adopted. For Phase 1, a directed content analysis of 20 years of research literature on high performing ORs was conducted using nine domains of a theoretical perioperative model. The analysis yielded 221 items that were presented to a panel of perioperative experts who rated each item’s relevance to perioperative success on a 4-point scale. Expert agreement on content was evaluated using a content validity index. In Phase 2, experts judged the wording and clarity of each item and of the instructions. Inter-rater agreement on clarity of items and instructions was evaluated using Gwet’s AC1 coefficient. For Phase 3, perioperative managers across the nation were surveyed and asked to rate levels of agreement with the items on a 6-point Likert scale as each item pertained to their hospital. Confirmatory factor analyses were completed on the responses, resulting in a model consisting of nine constructs and 53 indicators. Construct, convergent, and discriminant validity were assessed. Reliability was evaluated using composite reliability, inter-item correlations, and average inter-item correlations. Factor scores were employed to calculate a perioperative synergy index coefficient that ranged from 0 to 1, with higher values indicative of higher functioning perioperative services at the hospitals involved in the study. Managers can use the index to identify areas in need of improvement and can use the model to guide process improvements. Following further refinement, the index may serve as a new way to evaluate performance and standardize protocols across healthcare systems.
Twist1 Evokes Matrix Metalloproteinase 9 and Collagen IV Synthesis in Activated Pancreatic Stellate CellsBackground: Pancreatic cells are embedded in an extracellular matrix (ECM) and are also surrounded by a thin sheet of specialized extracellular matrix known as basal lamina. In healthy pancreas, pancreatic stellate cells (PaSCs) are responsible for the synthesis of basal lamina, which is mainly composed of collagen IV and laminin. In chronic pancreatitis (CP), PaSCs are responsible for the synthesis of fibrotic tissue, which is mainly composed of fibronectin and collagen I. Reactive oxygen species (ROS), which are predominant inflammatory mediators in CP, evoke the formation of fibrotic tissue by PaSCs. One of the sources of ROS is NADPH oxidase (Nox) enzymes. In particular, Nox1 has been associated with both fibrogenesis in a CP mouse model and an up-regulation of the transcription factor Twist1 and matrix metalloproteinase (MMP) 9 in CP-activated PaSCs. I sought to determine the functional relationship between Twist1 and MMP-9, as well as other PaSC-produced proteins. Therefore, my aim was to assess the extent to which Twist1 up-regulates MMP-9 and other PaSC-produced proteins. Methods: To overexpress Twist1, I infected activated PaSCs from Nox1-null mice with retroviruses expressing either mouse Twist1 or the empty backbone. I compared the relative expression of MMP-9 and other PaSC-produced proteins using quantitative PCR. To examine whether MMP-9 expression is regulated by Twist1 at the transcriptional level, I carried out a dual-luciferase reporter assay using a pGL2 Luciferase Reporter Vector carrying the human MMP-9 (pGL2-hMMP-9) promoter. I co-transfected HEK293 cells with human Twist1 in pCMV6 vector, or the empty backbone (negative control) along with human pGL2-hMMP-9 promoter vector or pGL3-control vector using Lipofectamine 3000. As a positive control, I co-transfected HEK293 cells with human NF-ĸB1 in pFUW-tetO vector along with the pGL2-hMMP-9 promoter. After 48 h, I performed the dual-luciferase reporter gene assay. Results: I found that the up-regulation of Twist1 in culture-activated PaSCs from Nox1-null mice increased MMP-9 mRNA level, but it did not modify the expression of PaSC-produced proteins linked to fibrosis [e.g., collagen I, fibronectin, α-smooth muscle actin, transforming growth factor-β (TGF- β), and interleukin-6 (IL-6)]. Therefore, I studied the expression of collagen IV, a component of basal lamina and found that the expression of Twist1 in activated PaSCs from Nox1-null mice increased collagen IV at the mRNA level. I also found that Twist1 increased the expression of MMP-9 at the transcriptional level in a NF-ĸB dependent manner using a dual-luciferase assay. Conclusion: Twist1 in PaSCs induced the expression of MMP-9 and collagen IV, at the transcriptional level. NF-ĸB was required for the transcriptional up-regulation of MMP-9 by Twist1. The expression of other PaSC-produced proteins, including collagen I, fibronectin, IL-6, α-smooth muscle actin, and TGF-β, were not affected. Since Twist1 in activated PaSCs was responsible for the synthesis and remodeling of the basal lamina in healthy pancreas, I concluded that the overexpression of Twist1 using a retrovirus approach was not sufficient to change the phenotype of PaSC from a basal lamina “maker” to a fibrotic tissue “maker.”