Evolutionarily conserved role of calcineurin in phosphodegron-dependent degradation of phosphodiesterase 4D.

Calcineurin is a widely expressed and highly conserved Ser/Thr phosphatase. Calcineurin is inhibited by the immunosuppressant drug cyclosporine A (CsA) or tacrolimus (FK506). The critical role of CsA/FK506 as an immunosuppressant following transplantation surgery provides a strong incentive to understand the phosphatase calcineurin. Here we uncover a novel regulatory pathway for cyclic AMP (cAMP) signaling by the phosphatase calcineurin which is also evolutionarily conserved in Caenorhabditis elegans. We found that calcineurin binds directly to and inhibits the proteosomal degradation of cAMP-hydrolyzing phosphodiesterase 4D (PDE4D). We show that ubiquitin conjugation and proteosomal degradation of PDE4D are controlled by a cullin 1-containing E(3) ubiquitin ligase complex upon dual phosphorylation by casein kinase 1 (CK1) and glycogen synthase kinase 3beta (GSK3beta) in a phosphodegron motif. Our findings identify a novel signaling process governing G-protein-coupled cAMP signal transduction-opposing actions of the phosphatase calcineurin and the CK1/GSK3beta protein kinases on the phosphodegron-dependent degradation of PDE4D. This novel signaling system also provides unique functional insights into the complications elicited by CsA in transplant patients.

Family Stability and Childhood Behavioral Outcomes: A Critical Review of the Literature

As the definition of what is considered a family changes in our society, the family unit itself continues to undergo changes. These changes can sometimes lead to decreased stability within the family unit. One of the greatest challenges facing those researching this phenomenon is the lack of consistency within the existing body of research surrounding what familial instability actually is (the definition). This critical review of the literature examines the current body of literature in order to identify what is known about family stability and its impact on adolescent behavior, as well as what gaps currently exist. This review focuses on definitions of family stability, current factors surrounding the stability of the family unit, and addresses the implications that the current body of literature presents.

RNA polymerase II large subunit degradation induced by ecteinascidin 743: Molecular characterization and subsequent rational investigation of antitumor mechanisms in cancers with clinical response

Ecteinascidin 743 (Et-743), which is a novel DNA minor groove alkylator with a unique spectrum of antitumor activity, is currently being evaluated in phase II/III clinical trials. Although the precise molecular mechanisms responsible for the observed antitumor activity are poorly understood, recent data suggests that post-translational modifications of RNA polymerase II Large Subunit (RNAPII LS) may play a central role in the cellular response to this promising anticancer agent. The stalling of an actively transcribing RNAPII LS at Et-743-DNA adducts is the initial cellular signal for transcription-coupled nucleotide excision repair (TC-NER). In this manner, Et-743 poisons TC-NER and produces DNA single strand breaks. Et-743 also inhibits the transcription and RNAPII LS-mediated expression of selected genes. Because the poisoning of TC-NER and transcription inhibition are critical components of the molecular response to Et-743 treatment, we have investigated if changes in RNAPII LS contribute to the disruption of these two cellular pathways. In addition, we have studied changes in RNAPII LS in two tumors for which clinical responses were reported in phase I/II clinical trials: renal cell carcinoma and Ewing's sarcoma. Our results demonstrate that Et-743 induces degradation of the RNAPII LS that is dependent on active transcription, a functional 26S proteasome, and requires functional TC-NER, but not global genome repair. Additionally, we have provided the first experimental data indicating that degradation of RNAPII LS might lead to the inhibition of activated gene transcription. A set of studies performed in isogenic renal carcinoma cells deficient in von Hippel-Lindau protein, which is a ubiquitin-E3-ligase for RNAPII LS, confirmed the central role of RNAPII LS degradation in the sensitivity to Et-743. Finally, we have shown that RNAPII LS is also degraded in Ewing's sarcoma tumors following Et-743 treatment and provide data to suggest that this event plays a role in decreased expression of the Ewing's sarcoma oncoprotein, EWS-Fli1. Altogether, these data implicate degradation of RNAPII LS as a critical event following Et-743 exposure and suggest that the clinical activity observed in renal carcinoma and Ewing's sarcoma may be mediated by disruption of molecular pathways requiring a fully functional RNAPII LS. ^

Federally regulated institutional review boards: The aspects of a "model" IRB---Is the current system in need of a tune up or complete overhaul?

Institutional Review Boards (IRBs) are the primary gatekeepers for the protection of ethical standards of federally regulated research on human subjects in this country. This paper focuses on what general, broad measures that may be instituted or enhanced to exemplify a "model IRB". This is done by examining the current regulatory standards of federally regulated IRBs, not private or commercial boards, and how many of those standards have been found either inadequate or not generally understood or followed. The analysis includes suggestions on how to bring about changes in order to make the IRB process more efficient, less subject to litigation, and create standardized educational protocols for members. The paper also considers how to include better oversight for multi-center research, increased centralization of IRBs, utilization of Data Safety Monitoring Boards when necessary, payment for research protocol review, voluntary accreditation, and the institution of evaluation/quality assurance programs. ^ This is a policy study utilizing secondary analysis of publicly available data. Therefore, the research for this paper focuses on scholarly medical/legal journals, web information from the Department of Health and Human Services, Federal Drug Administration, and the Office of the Inspector General, Accreditation Programs, law review articles, and current regulations applicable to the relevant portions of the paper. ^ Two issues are found to be consistently cited by the literature as major concerns. One is a need for basic, standardized educational requirements across all IRBs and its members, and secondly, much stricter and more informed management of continuing research. There is no federally regulated formal education system currently in place for IRB members, except for certain NIH-based trials. Also, IRBs are not keeping up with research once a study has begun, and although regulated to do so, it does not appear to be a great priority. This is the area most in danger of increased litigation. Other issues such as voluntary accreditation and outcomes evaluation are slowing gaining steam as the processes are becoming more available and more sought after, such as JCAHO accrediting of hospitals. ^ Adopting the principles discussed in this paper should promote better use of a local IRBs time, money, and expertise for protecting the vulnerable population in their care. Without further improvements to the system, there is concern that private and commercial IRBs will attempt to create a monopoly on much of the clinical research in the future as they are not as heavily regulated and can therefore offer companies quicker and more convenient reviews. IRBs need to consider the advantages of charging for their unique and important services as a cost of doing business. More importantly, there must be a minimum standard of education for all IRB members in the area of the ethical standards of human research and a greater emphasis placed on the follow-up of ongoing research as this is the most critical time for study participants and may soon lead to the largest area for litigation. Additionally, there should be a centralized IRB for multi-site trials or a study website with important information affecting the trial in real time. There needs to be development of standards and metrics to assess the performance of the IRBs for quality assurance and outcome evaluations. The boards should not be content to run the business of human subjects' research without determining how well that function is actually being carried out. It is important that federally regulated IRBs provide excellence in human research and promote those values most important to the public at large.^

Critical indicators associated with negative performance in a selectively manned unit

Introduction. Selectively manned units have a long, international history, both military and civilian. Some examples include SWAT teams, firefighters, the FBI, the DEA, the CIA, and military Special Operations. These special duty operators are individuals who perform a highly skilled and dangerous job in a unique environment. A significant amount of money is spent by the Department of Defense (DoD) and other federal agencies to recruit, select, train, equip and support these operators. When a critical incident or significant life event occurs, that jeopardizes an operator's performance; there can be heavy losses in terms of training, time, money, and potentially, lives. In order to limit the number of critical incidents, selection processes have been developed over time to “select out” those individuals most likely to perform below desired performance standards under pressure or stress and to "select in" those with the "right stuff". This study is part of a larger program evaluation to assess markers that identify whether a person will fail under the stresses in a selectively manned unit. The primary question of the study is whether there are indicators in the selection process that signify potential negative performance at a later date. ^ Methods. The population being studied included applicants to a selectively manned DoD organization between 1993 and 2001 as part of a unit assessment and selection process (A&S). Approximately 1900 A&S records were included in the analysis. Over this nine year period, seventy-two individuals were determined to have had a critical incident. A critical incident can come in the form of problems with the law, personal, behavioral or family problems, integrity issues, and skills deficit. Of the seventy-two individuals, fifty-four of these had full assessment data and subsequent supervisor performance ratings which assessed how an individual performed while on the job. This group was compared across a variety of variables including demographics and psychometric testing with a group of 178 individuals who did not have a critical incident and had been determined to be good performers with positive ratings by their supervisors.^ Results. In approximately 2004, an online pre-screen survey was developed in the hopes of preselecting out those individuals with items that would potentially make them ineligible for selection to this organization. This survey has aided the organization to increase its selection rates and save resources in the process. (Patterson, Howard Smith, & Fisher, Unit Assessment and Selection Project, 2008) When the same prescreen was used on the critical incident individuals, it was found that over 60% of the individuals would have been flagged as unacceptable. This would have saved the organization valuable resources and heartache.^ There were some subtle demographic differences between the two groups (i.e. those with critical incidents were almost twice as likely to be divorced compared with the positive performers). Upon comparison of Psychometric testing several items were noted to be different. The two groups were similar when their IQ levels were compared using the Multidimensional Aptitude Battery (MAB). When looking at the Minnesota Multiphasic Personality Inventory (MMPI), there appeared to be a difference on the MMPI Social Introversion; the Critical Incidence group scored somewhat higher. When analysis was done, the number of MMPI Critical Items between the two groups was similar as well. When scores on the NEO Personality Inventory (NEO) were compared, the critical incident individuals tended to score higher on Openness and on its subscales (Ideas, Actions, and Feelings). There was a positive correlation between Total Neuroticism T Score and number of MMPI critical items.^ Conclusions. This study shows that the current pre-screening process is working and would have saved the organization significant resources. ^ If one was to develop a profile of a candidate who potentially could suffer a critical incident and subsequently jeopardize the unit, mission and the safety of the public they would look like the following: either divorced or never married, score high on the MMPI in Social Introversion, score low on MMPI with an "excessive" amount of MMPI critical items; and finally scores high on the NEO Openness and subscales Ideas, Feelings, and Actions.^ Based on the results gleaned from the analysis in this study there seems to be several factors, within psychometric testing, that when taken together, will aid the evaluators in selecting only the highest quality operators in order to save resources and to help protect the public from unfortunate critical incidents which may adversely affect our health and safety.^

A critical assessment of the National Institutes of Health Consensus Development Program against the eras which shaped it

This research study offers a critical assessment of NIH's Consensus Development Program (CDP), focusing upon its historical and valuative bases and its institutionalization in response to social and political forces. The analysis encompasses systems-level, as well as interpersonal factors in the adoption of consensus as the mechanism for resolving scientific controversies in clinical practice application. Further, the evolution of the CDP is also considered from an ecological perspective as a reasoned adaptation by NIH to pressures from its supporters and clients for translating biomedical research into medical practice. The assessment examines federal science policy and institutional designs for the inclusion of the public interest and democratic deliberation.^ The study relies on three distinct approaches to social research. Conventional historical methods were utilized in the interpretation of social and political influences across eras on the evolution of the National Institutes of Health and its response to demands for accountability and relevance through its Consensus Development Program. An embedded single-case study was utilized for an empirical examination of the CDP mechanism through five exemplar conferences. Lastly, a sociohistorical approach was taken to the CDP in order to consider its responsiveness to the values of the eras which created and shaped it. An exploration of organizational behavior with considerations for institutional reform as a response to continuing political and social pressure, it is a study of organizational birth, growth, and response to demands from its environment. The study has explanatory import in its attempt to account for the creation, timing, and form of the CDP, relative to political, institutional, and cultural pressures, and predictive import thorough its historical view which provides a basis for informed speculation on the playing out of tensions between extramural and intermural scientists and the current demands for health care reform. ^

Regulation of Protein Degradation in the Heart by AMP-Activated Protein Kinase

The degradation of proteins by the ubiquitin proteasome system is essential for cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). During nutrient deprivation, AMPK is activated and intracellular proteolysis is enhanced through the ubiquitin proteasome system (UPS). Whether AMPK plays a role in protein degradation through the UPS in the heart is not known. Here I present data in support of the hypothesis that AMPK transcriptionally regulates key players in the UPS, which, under extreme conditions can be detrimental to the heart. The ubiquitin ligases MAFbx /Atrogin-1 and MuRF1, key regulators of protein degradation, and AMPK activity are increased during nutrient deprivation. Pharmacologic and genetic activation of AMPK is sufficient for the induction of MAFbx/Atrogin-1 and MuRF1 in cardiomyocytes and in the heart in vivo. Comprehensive experiments demonstrate that the molecular mechanism by which AMPK regulates MuRF1 expression is through the transcription factor myocyte enhancer factor 2 (MEF2), which is involved in stress response and cardiomyocyte remodeling. MuRF1 is required for AMPK-mediated protein degradation through the UPS in cardiomyocytes. Consequently, the absence of MuRF1 during chronic fasting preserves cardiac function, possibly by limiting degradation of critical metabolic enzymes. Furthermore, during cardiac hypertrophy, chronic activation of AMPK also leads to cardiac dysfunction, possibly through enhanced protein degradation and metabolic dysregulation. Collectively, my findings demonstrate that AMPK regulates expression of ubiquitin ligases which are required for UPS-mediated protein degradation in the heart. Based on these results, I propose that specific metabolic signals may serve as modulators of intracellular protein degradation in the heart.

Regulation of Protein Degradation in the Heart by AMP-Activated Protein Kinase

The degradation of proteins by the ubiquitin proteasome system is essential for cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). During nutrient deprivation, AMPK is activated and intracellular proteolysis is enhanced through the ubiquitin proteasome system (UPS). Whether AMPK plays a role in protein degradation through the UPS in the heart is not known. Here I present data in support of the hypothesis that AMPK transcriptionally regulates key players in the UPS, which, under extreme conditions can be detrimental to the heart. The ubiquitin ligases MAFbx /Atrogin-1 and MuRF1, key regulators of protein degradation, and AMPK activity are increased during nutrient deprivation. Pharmacologic and genetic activation of AMPK is sufficient for the induction of MAFbx/Atrogin-1 and MuRF1 in cardiomyocytes and in the heart in vivo. Comprehensive experiments demonstrate that the molecular mechanism by which AMPK regulates MuRF1 expression is through the transcription factor myocyte enhancer factor 2 (MEF2), which is involved in stress response and cardiomyocyte remodeling. MuRF1 is required for AMPK-mediated protein degradation through the UPS in cardiomyocytes. Consequently, the absence of MuRF1 during chronic fasting preserves cardiac function, possibly by limiting degradation of critical metabolic enzymes. Furthermore, during cardiac hypertrophy, chronic activation of AMPK also leads to cardiac dysfunction, possibly through enhanced protein degradation and metabolic dysregulation. Collectively, my findings demonstrate that AMPK regulates expression of ubiquitin ligases which are required for UPS-mediated protein degradation in the heart. Based on these results, I propose that specific metabolic signals may serve as modulators of intracellular protein degradation in the heart.