Distinct 5′ UTRs regulate XIAP expression under normal growth conditions and during cellular stress

X-chromosome linked inhibitor of apoptosis, XIAP, is cellular caspase inhibitor and a key regulator of apoptosis. We and others have previously shown that XIAP expression is regulated primarily at the level of protein synthesis; the 5′ untranslated region (UTR) of XIAP mRNA contains an Internal Ribosome Entry Site (IRES) that supports cap-independent expression of XIAP protein during conditions of pathophysiological stress, such as serum deprivation or gamma irradiation. Here, we show that XIAP is encoded by two distinct mRNAs that differ in their 5′ UTRs. We further show that the dominant, shorter, 5′ UTR promotes a basal level of XIAP expression under normal growth conditions. In contrast, the less abundant longer 5′ UTR contains an IRES and supports cap-independent translation during stress. Our data suggest that the combination of alternate regulatory regions and distinct translational initiation modes is critical in maintaining XIAP levels in response to cellular stress and may represent a general mechanism of cellular adaptation

Framework, principles and recommendations for utilising participatory methodologies in the co-creation and evaluation of public health interventions

Background: Due to the chronic disease burden on society, there is a need for preventive public health interventions to stimulate society towards a healthier lifestyle. To deal with the complex variability between individual lifestyles and settings, collaborating with end-users to develop interventions tailored to their unique circumstances has been suggested as a potential way to improve effectiveness and adherence. Co-creation of public health interventions using participatory methodologies has shown promise but lacks a framework to make this process systematic. The aim of this paper was to identify and set key principles and recommendations for systematically applying participatory methodologies to co-create and evaluate public health interventions. Methods: These principles and recommendations were derived using an iterative reflection process, combining key learning from published literature in addition to critical reflection on three case studies conducted by research groups in three European institutions, all of whom have expertise in co-creating public health interventions using different participatory methodologies. Results: Key principles and recommendations for using participatory methodologies in public health intervention co-creation are presented for the stages of: Planning (framing the aim of the study and identifying the appropriate sampling strategy); Conducting (defining the procedure, in addition to manifesting ownership); Evaluating (the process and the effectiveness) and Reporting (providing guidelines to report the findings). Three scaling models are proposed to demonstrate how to scale locally developed interventions to a population level. Conclusions: These recommendations aim to facilitate public health intervention co-creation and evaluation utilising participatory methodologies by ensuring the process is systematic and reproducible

Differential contribution of the m7G-cap to the 5′ end-dependent translation initiation of mammalian mRNAs

Many mammalian mRNAs possess long 5′ UTRs with numerous stem-loop structures. For some of them, the presence of Internal Ribosome Entry Sites (IRESes) was suggested to explain their significant activity, especially when cap-dependent translation is compromised. To test this hypothesis, we have compared the translation initiation efficiencies of some cellular 5′ UTRs reported to have IRES-activity with those lacking IRES-elements in RNA-transfected cells and cell-free systems. Unlike viral IRESes, the tested 5′ UTRs with so-called ‘cellular IRESes’ demonstrate only background activities when placed in the intercistronic position of dicistronic RNAs. In contrast, they are very active in the monocistronic context and the cap is indispensable for their activities. Surprisingly, in cultured cells or cytoplasmic extracts both the level of stimulation with the cap and the overall translation activity do not correlate with the cumulative energy of the secondary structure of the tested 5′ UTRs. The cap positive effect is still observed under profound inhibition of translation with eIF4E-BP1 but its magnitude varies for individual 5′ UTRs irrespective of the cumulative energy of their secondary structures. Thus, it is not mandatory to invoke the IRES hypothesis, at least for some mRNAs, to explain their preferential translation when eIF4E is partially inactivated

Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin

Variation in cellular gene expression levels has been shown to be inherited. Expression is controlled at transcriptional and post-transcriptional levels. Internal ribosome entry sites (IRES) are used by viruses to bypass inhibition of cap-dependent translation, and by eukaryotic cells to control translation under conditions when protein synthesis is inhibited. We aimed at identifying genomic determinants of variability in IRES-mediated translation of viral [Encephalomyocarditis virus (EMCV)] and cellular IRES [X-linked inhibitor-of-apoptosis (XIAP) and c-myc]. Bicistronic lentiviral constructs expressing two fluorescent reporters were used to transduce laboratory and B lymphoblastoid cell lines [15 CEPH pedigrees (n = 205) and 50 unrelated individuals]. IRES efficiency varied according to cell type and among individuals. Control of IRES activity has a significant genetic component (h2 of 0.47 and 0.36 for EMCV and XIAP, respectively). Quantitative linkage analysis identified a suggestive locus (LOD 2.35) on chromosome 18q21.2, and genome-wide association analysis revealed of a cluster of SNPs on chromosome 3, intronic to the FHIT gene, marginally associated (P = 5.9E-7) with XIAP IRES function. This study illustrates the in vitro generation of intermediate phenotypes by using cell lines for the evaluation of genetic determinants of control of elements such as IRES

Ribosomal scanning on the 5′-untranslated region of the human immunodeficiency virus RNA genome

Translation initiation on most eukaryotic mRNAs occurs via a cap-dependent scanning mechanism and its efficiency is modulated by their 5′-untranslated regions (5′-UTR). The human immunodeficiency virus type 1 (HIV-1) 5′-UTR contains a stable TAR hairpin directly at its 5′-end, which possibly masks the cap structure. In addition, the 5′-UTR is relatively long and contains several stable RNA structures that are essential for viral replication. These characteristics may interfere with ribosomal scanning and suggest that translation is initiated via internal entry of ribosomes. Literature on the HIV-1 5′-UTR-driven translation initiation mechanism is controversial. Both scanning and internal initiation have been shown to occur in various experimental systems. To gain further insight in the translation initiation process, we determined which part of the 5′-UTR is scanned. To do so, we introduced upstream AUGs at various positions across the 5′-UTR and determined the effect on expression of a downstream reporter gene that was placed under control of the gag start codon. This strategy allowed us to determine the window of ribosomal scanning on the HIV-1 5′-UTR

Extensive Translatome Remodeling during ER Stress Response in Mammalian Cells

In this work we have described the translatome of two mammalian cell lines, NIH3T3 and Jurkat, by scoring the relative polysome association of ∼10,000 mRNA under normal and ER stress conditions. We have found that translation efficiencies of mRNA correlated poorly with transcript abundance, although a general tendency was observed so that the highest translation efficiencies were found in abundant mRNA. Despite the differences found between mouse (NIH3T3) and human (Jurkat) cells, both cell types share a common translatome composed by ∼800–900 mRNA that encode proteins involved in basic cellular functions. Upon stress, an extensive remodeling in translatomes was observed so that translation of ∼50% of mRNA was inhibited in both cell types, this effect being more dramatic for those mRNA that accounted for most of the cell translation. Interestingly, we found two subsets comprising 1000–1500 mRNA whose translation resisted or was induced by stress. Translation arrest resistant class includes many mRNA encoding aminoacyl tRNA synthetases, ATPases and enzymes involved in DNA replication and stress response such as BiP. This class of mRNA is characterized by high translation rates in both control and stress conditions. Translation inducible class includes mRNA whose translation was relieved after stress, showing a high enrichment in early response transcription factors of bZIP and zinc finger C2H2 classes. Unlike yeast, a general coordination between changes in translation and transcription upon stress (potentiation) was not observed in mammalian cells. Among the different features of mRNA analyzed, we found a relevant association of translation efficiency with the presence of upstream ATG in the 5′UTR and with the length of coding sequence of mRNA, and a looser association with other parameters such as the length and the G+C content of 5′UTR. A model for translatome remodeling during the acute phase of stress response in mammalian cells is proposed

Confronting crises: Fundamental leadership attributes for managing natural disasters

This evidence-based research dissertation examined the situational complexity that leaders in networked service organizations (NSOs) face when preparing for and responding to the impact of natural disasters. The purpose of the study was to identify the fundamental competencies needed by those leaders to execute responses that are most effective and lessen the impact of the crises. Crises scenarios are notably pressure-filled, and require rapid decision-making to bring about effective responses. Natural disasters, frequently unpredictable, typically have a significant and dire impact on communities, as well as on the organizational structures and systems that serve these communities during the natural disasters. The inherent ambiguity and uncertainty in dynamic and fluid situations require specific leadership skills for efficient operations and responses. Specifically, this dissertation examined the characteristics and abilities of leaders who positively influenced organizational effectiveness when preparing for the potential impact of and response to natural disasters. A systematic review of the literature found that leaders of NSOs must understand the internal and external environments, must adapt to them, and must communicate broadly with stakeholders. Leaders must exhibit constant and continuous environmental awareness and understanding in order to decode and analyze these inherently stress-filled situations. In addition, NSO leaders who effectively managed the cyclical nature of the crises were better able to assess, interpret, and synthesize ambiguous information related to crises, and provide a more effective organizational response. A realist synthesis and configurative analysis of the primary research studies used as evidence showed that leaders must possess the fundamental competencies of decision-making, environmental awareness, adaptability and information exchange, and must further demonstrate those capabilities throughout each stage of the disaster life cycle — response, recovery, mitigation, and preparation. Additionally, competent leaders facilitated the process of organizational learning throughout the disaster life cycle in order to positively influence the next stage of the cycle — with the goal of being better prepared to reduce further the impact of the next natural disaster.Running head: LEADER ATTRIBUTES FOR MANAGING NATURAL DISASTERS Confronting Crises: Fundamental Leadership Attributes for Managing Natural Disasters by Eric Baranick A Thesis Submitted to the Graduate Faculty of University of Maryland University College in Partial Fulfillment of The Requirements for the Degree of Doctor of Management Dissertation Committee: Dr. Deborah Wharff and Dr. Eric Dent Date Submitted: December 31, 2014 LEADER ATTRIBUTES FOR MANAGING NATURAL DISASTERS 2 Dedication I am indebted to many friends and my family for their understanding and patience during the past three and a half years as this journey concludes. The insight from my subject matter experts (SMEs) was invaluable and much appreciated. The support from the University of Maryland University College Doctor of Management Program and each professor was continuous and I am grateful to them all – Dr. Bijlani, Dr. Breckon, Dr. Shepard, Dr. Watts, Dr. Winters, Dr. Witz and last, but not least, my dissertation advisors Dr. Wharff and Dr. Dent and teaching assistant Dr. Kearney, for pushing me academically and challenging me to make the most of this endeavor. Furthermore, I am indebted to my cohort members, in particular Margaret, Tom, and Jim, to whom I also direct my sincere congratulations. I am grateful to my parents, who encouraged me and provided advice and support throughout this journey as I tackled the many frustrations, and to my sister, who used her wittiness to encourage me to go forward. Finally, a great deal of gratitude goes to my wife, Maria, and our three children, Erica, Amanda, and William who have sacrificed family vacations, weekend outings and a general sense of my presence over the past three and a half years, as I immersed myself in research and writing this dissertation. This would not have been possible without their love and support, and I am forever grateful. LEADER ATTRIBUTES FOR MANAGING NATURAL DISASTERS 3 Abstract This evidence-based research dissertation examined the situational complexity that leaders in networked service organizations (NSOs) face when preparing for and responding to the impact of natural disasters. The purpose of the study was to identify the fundamental competencies needed by those leaders to execute responses that are most effective and lessen the impact of the crises. Crises scenarios are notably pressure-filled, and require rapid decision-making to bring about effective responses. Natural disasters, frequently unpredictable, typically have a significant and dire impact on communities, as well as on the organizational structures and systems that serve these communities during the natural disasters. The inherent ambiguity and uncertainty in dynamic and fluid situations require specific leadership skills for efficient operations and responses. Specifically, this dissertation examined the characteristics and abilities of leaders who positively influenced organizational effectiveness when preparing for the potential impact of and response to natural disasters. A systematic review of the literature found that leaders of NSOs must understand the internal and external environments, must adapt to them, and must communicate broadly with stakeholders. Leaders must exhibit constant and continuous environmental awareness and understanding in order to decode and analyze these inherently stress-filled situations. In addition, NSO leaders who effectively managed the cyclical nature of the crises were better able to assess, interpret, and synthesize ambiguous information related to crises, and provide a more effective organizational response. A realist synthesis and configurative analysis of the primary research studies used as evidence showed that leaders must possess the fundamental competencies of decision-making, environmental awareness, adaptability and information exchange, and must further demonstrate those capabilities throughout each stage of the disaster life cycle – response, LEADER ATTRIBUTES FOR MANAGING NATURAL DISASTERS 4 recovery, mitigation, and preparation. Additionally, competent leaders facilitated the process of organizational learning throughout the disaster life cycle in order to positively influence the next stage of the cycle – with the goal of being better prepared to reduce further the impact of the next natural disaster. Keywords: Networked service organization, natural disasters, leadership competencies, agility, environmental awareness, organizational learning LEADER ATTRIBUTES FOR MANAGING NATURAL DISASTERS 5 Table of Contents Abstract .................................................................................................................................. 3 Table of Contents .................................................................................................................... 5 List of Figures ......................................................................................................................... 8 Chapter One: Introduction and Overview of Relevant Literature .......................................... 9 Statement and Significance of the Problem ............................................................................ 9 Importance to Management .................................................................................................. 13 Research Question and Propositions ..................................................................................... 19 Propositions .......................................................................................................................... 20 Definitions of Key Terms within the Literature ................................................................... 20 Organization of the Dissertation ........................................................................................... 21 Chapter Two: Methodology .................................................................................................. 23 Introduction ........................................................................................................................... 23 Evidence-Based Movement .................................................................................................. 23 Systematic Reviews .............................................................................................................. 26 Identification of Evidence ..................................................................................................... 28 Quality Appraisal of Evidence .............................................................................................. 31 Data Analysis and Realist Synthesis ..................................................................................... 33 Advisory Panel of Subject Matter Experts (SMEs) .............................................................. 35 Summary ............................................................................................................................... 36 Chapter Three: Systematic Review of the Literature ............................................................ 37 Theoretical Framework ......................................................................................................... 38 Complex Adaptive Systems Theory ..................................................................................... 38 Complex Leadership Theory ................................................................................................ 39 LEADER ATTRIBUTES FOR MANAGING NATURAL DISASTERS 6 Organizational Learning Theory ........................................................................................... 41 Critical Factors in Organizational Preparedness and Response to Crises ............................ 44 Critical Factors in Leader Preparedness and Response to Crises ......................................... 53 Relationship between Leaders’ Competencies and Effective NSOs .................................... 66 Summary ............................................................................................................................... 80 Chapter 4: Analysis and Discussion .................................................................................... 82 Introduction ........................................................................................................................... 82 Finding One: Environmental awareness underpinned effective NSO leader decision-making. ................................................................................................................................ 87 Finding Two: Leader flexibility, agility, and adaptability to contextual situations positively influence organizational response to crises. ......................................................................... 90 Finding Three: Leader facilitated organizational learning reduced ambiguity and uncertainty. ........................................................................................................................... 93 Alternative Perspective ......................................................................................................... 95 Summary ............................................................................................................................... 97 Chapter 5: Conceptual Framework ...................................................................................... 99 Thematic Issue One: Complex adaptive systems theory provides an explanation for organizations demanding environmental awareness in crises scenarios. ........................... 103 Thematic Issue Two: Complex leadership theory clarifies how flexibility, adaptability, and agile decision-making improve NSO preparedness and response to natural disasters. ...... 105 Thematic Issue Three: Organizational learning is a critical component of how NSOs are able to prepare for and respond to crises. ........................................................................... 106 Summary ............................................................................................................................. 108 Chapter 6: Conclusions, Implications and Trends ............................................................. 109 LEADER ATTRIBUTES FOR MANAGING NATURAL DISASTERS 7 Introduction ......................................................................................................................... 109 Overall Conclusions ............................................................................................................ 109 Implications for Management/Practitioner ......................................................................... 112 Implications of Emerging Trends ....................................................................................... 113 Limitations and Areas for Future Research ........................................................................ 115 Summary ............................................................................................................................. 116 References ........................................................................................................................... 118 Appendix A: Subject Matter Expert Evaluation & Feedback Form ................................... 131 Appendix B: Literature Summary Table ........................................................................... 144 Appendix C: Affirmative responses to network traits ........................................................ 148 LEADER ATTRIBUTES FOR MANAGING NATURAL DISASTERS 8 List of Figures Figure 1. Disaster management cycle. .................................................................................. 18 Figure 2. Process for narrowing the studies. ........................................................................ 30 Figure 3. Managerial leadership behaviors. .......................................................................... 55 Figure 4. Organizational agility dimensions and correlations. ............................................. 57 Figure 5. Generic leadership traits and response rate.. ......................................................... 59 Figure 6. Descriptive statistics and correlations. .................................................................. 64 Figure 7. Effective leader behaviors.. .................................................................................. 73 Figure 8. Definitions of leadership by for-profit and public organizations.. ....................... 75 Figure 9. Resilience descriptors and statistics.. ................................................................... 78 Figure 10. Articles by year of publication. ........................................................................... 85 Figure 11. Disaster life cycle.. .............................................................................................. 87 Figure 12. Conceptual framework ..................................................................................... 101 Figure 13. Propositions and related leadership competencies. .......................................... 102 LEADER ATTRIBUTES FOR MANAGING NATURAL DISASTERS 9 Chapter One: Introduction and Overview of Relevant Literature Statement and Significance of the Problem This dissertation examined the leadership competencies required to prepare for and respond to the impact of potential natural disaster crises managed by NSOs (networked service organizations). The effects of natural disasters are chaotic and indiscriminate, however, this dissertation assessed empirical evidence to determine how to lessen their impact on an organization and the affected population through focused preparation and adequate response. Between 1971 and 1995, disasters caused unparalleled damage affecting over a hundred million people on average per year (Twigg & Steiner, 2002). The 2009 International Federation of the Red Cross and Red Crescent (IFRC) annual World Disasters Report noted a 62% increase in the number of disaster-related deaths between 1989-1998 and 1999-2008. The impact of natural disasters was further exacerbated by the rise in the number of people affected by disasters over the same ten-year period, rising from 2.130 billion to 2.695 billion, respectively (IFRC, 2009, p. 180). This 26.5% increase was far greater than the world’s population growth rate, which the World Bank estimated to be 1.2% per year for the past decade (World Bank, 2012) and highlights the exponential increase the impact of natural disasters is having on humanity and the service organizations that respond accordingly. The increasing frequency of disasters and increasing number of people affected highlight the importance of well-prepared and competent leaders of NSOs to effectively and efficiently adapt to the a changing environment. However, some recent examples highlight that leaders are not always prepared, and a less than adequate response ensues. Farazmand (2007) argued that following Hurricane Katrina, both federal and local government agencies were unprepared to respond to a disaster of this scope. This lack of LEADER ATTRIBUTES FOR MANAGING NATURAL DISASTERS 10 preparation resulted in a management crisis and a breakdown in response activities at various levels. Analyzing the same event, Comfort (2007) found that those in leadership roles were unable to comprehend the existing hazards and vulnerabilities in the impacted area, thus contributing to the perceptions of a poor response by those directly affected, and the public at large. Additionally, similar events in the United States, including the 1992 Hurricane Andrew and the 1994 Northridge Earthquake, demonstrated the broad impact disasters have on community infrastructure requiring extensive response and recovery, both economic and organizational, to meet the needs of the affected population (Harrald, 2006). Crises by their very nature are complex events and, for a variety of reasons, leaders of NSOs sometimes find it overwhelming to prepare for them. Van Wart and Kapucu (2011) concluded the limited frequency of disasters contributed to the lack of preparedness and poor response when 35,000 people in Europe fell victim to an unparalleled heat wave in 2003. Hypothetically, leaders may be able to prepare for unanticipated events, but planning for every possible disaster is financially and operationally not feasible (Van Wart & Kapucu, 2011). This dissertation evaluated actions organizations may take to improve disaster preparation and response, by focusing specifically on leadership competencies. There are potentially additional actions to be taken by other members of the organization that could influence effectiveness and efficiency; however, examining the leadership competencies that facilitated organizational response and preparedness to crises, specifically within NSOs, bounded the scope of this dissertation. Leadership devoid of requisite competencies and organizations lacking systems and structures have the potential to influence negatively the organizational response to disasters. Following the 2004 Indian Ocean tsunami, the lack of coordination and information sharing by responding organizations led to the impression of poor response efforts by those in the LEADER ATTRIBUTES FOR MANAGING NATURAL DISASTERS 11 affected community (Harrald, 2006). Stakeholder perceptions must be incorporated into the decision-making by NSO leadership during response and planning scenarios. Pearson and Mitroff (1993) underscored how leader awareness of public perception of the response efforts could positively, or conversely, negatively influence the outcome (p.55). Likewise, the ability to adapt while planning for the impact and outcome following a disaster could improve organizational response and have a positive effect on community perception. Boin and t’Hart (2003) found that following the 1992 transportation crisis in Amsterdam (airplane crash), the promises by government leaders to respond were initially well received. However, unforeseen problems relating to the long-term provisions of public services to those affected by the event during the response led to a significant backlash against the Dutch government, further illustrating that leaders must be able to confront a broad spectrum of crises, and a range of contingencies and competencies are required for an effective organizational response. Organizational effectiveness is a construct that requires extensive examination of the goals and other contextual outcome-based variables that provide insight into how organizations measure their success. Scholars have focused on a variety of methods for determining effectiveness, to include goal attainment, reputational measures, and resource dimensions (Lecy, Schmitz, & Swedlund, 2012, p. 439). One aspect of NSO goal attainment and organizational effectiveness was achieved when the networked organizational structure improved the ability of each individual organization to be better prepared and respond more efficiently than the individual organizations acting alone (McGuire & Silvia, 2009, p. 37). Scholars have debated the actual metric of the specific dimension indicating NSO success. More recently, however, there has been a movement toward examining a multidimensional approach for determining networked organizational LEADER ATTRIBUTES FOR MANAGING NATURAL DISASTERS 12 effectiveness. These four dimensions most prominent in the li

Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical applications

This work was supported by the Medical Research Council.Background Many biomedical applications require the expression or production of therapeutic hetero-multimeric proteins/protein complexes: in most cases only accomplished by co-ordinated co-expression within the same cell. Foot-and-mouth disease virus 2A (F2A) and ‘2A-like’ sequences are now widely used for this purpose. Since 2A mediates a co-translational ‘cleavage’ at its own C-terminus, sequences encoding multiple proteins (linked via 2As) can be concatenated into a single ORF: a single transgene. It has been shown that in some cases, however, the cleavage efficiency of shorter versions of F2A may be inhibited by the C-terminus of certain gene sequences immediately upstream of F2A. This paper describes further work to optimise F2A for co-expression strategies. Results We have inserted F2A of various lengths in between GFP and CherryFP ‘reporter’ proteins (in reciprocal or tandem arrangements). The co-expression of these proteins and cleavage efficiencies of F2As of various lengths were studied by in vitro coupled transcription and translation in rabbit reticulocyte lysates, western blotting of HeLa cell lysates and fluorescence microscopy. Conclusions Optimal and suboptimal lengths of F2A sequences were identified as a result of detailed ‘fine-tuning’ of the F2A sequence. Based on our data and the model according to which 2A activity is a product of its interaction with the exit tunnel of the ribosome, we suggest the length of the F2A sequence which is not ‘sensitive’ to the C-terminus of the upstream protein that can be successfully used for co-expression of two proteins for biomedical applications.Publisher PDFPeer reviewe