Murine coronavirus ubiquitin-like domain is important for papain-like protease stability and viral pathogenesis

Ubiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation. IMPORTANCE Introducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis.

Cesare Lombroso

Sylvester

The Right to Die Debate: The Demonization of Dr. Kevorkian and the Creation of a Moral Panic Surrounding Physician-Assisted Suicide in the United States

The Right to Die Debate is a recent but highly controversial moral matter. In particular, physician-assisted suicide (PAS) is an issue that has been evaded by the medical community for years. As of 1990, most states had never encountered the issue before and therefore did not have any laws in place to prohibit PAS (Strate et. al, 2005). Dr. Jack Kevorkian, a retired pathologist from Royal Oak Michigan was the first to publicly address PAS. He brought the issue into the limelight through a bizarre and crude series of assisted deaths that had a lasting impact on not only the Right to Die Debate as whole, but on public policy and both federal and state governmental agendas. This study focuses on the way in which the media, in particular the New York Times (NYT) has portrayed Dr. Jack Kevorkian as incompetent, morally culpable and in an overall negative light in the past twenty years. Applying Stanley Cohen’s 1972 theory of moral panic, a content analysis of NYT media publications between 1990 and 1999 supports Cohen’s theory and reveals that the media has created a moral panic surrounding Kevorkian. This has in turn led to public policy that prevents both terminally ill individuals and their doctors from having a desirable choice; that of voluntary euthanasia and PAS.

Volume 11, Number 3: September/October 2005

Page 2 – The University and its Libraries celebrate 125 years of growth and service to the citizens of Connecticut. • UConn joins the Center for Research Libraries, the nation’s oldest and most significant library consortium. Page 3 - A profile of Stanley Israelite recounts his civic contributions and his role as a creator of the Dodd Research Center. Page 4 - The Thomas J. Dodd Research Center celebrates its 10th anniversary with a series of special events. • The Pharmacy Library occupies new quarters in UConn’s new Pharmacy/Biology Building. Page 5 - The 2005 Connecticut Children’s Book Fair features a great lineup of authors and illustrators. Page 6 - Staff News • The Wharton Data Research Service is now available for financial research. Page 7 - Donors to the University Libraries, January 1 – June 30, 2005

Soliman der Zweyte, oder die drey Sultaninnen : Ein Singspiel in zwey Aufzügen ; für die k. k. Hoftheater

nach dem Französischen des Herrn Favart bearbeitet von Franz Xaver Huber. Die Musik ist von Hrn. Franz Xav. Süßmayer, Kapellmeister in wirklichen Diensten der k. k. Theatral-Hof-Direktion

Ms.Ff.F.Stoltze 3. 1 - Brief von Alexander Askenasy an Lyda Stoltze

Frankfurter Latern, Sylvester & Cie., Alexander Askenasy: Stoltze -Sammlung, Franz Rittweger

Ms.Ff.F.Stoltze 3. 443 - Brief von Friedrich Stoltze an Hermann Stoltze

"Sylvester & Cie.", "Der Sperrbatzen", Glückwünsche und Feiern zum 70. Geburtstag, Marie Stoltze, Nachrichten aus Frankfurt, Gelegenheitsarbeiten, Artikel für "Über Land und Meer"

Emin Pascha und die Meuterei in Äquatoria : neunmonatlicher Aufenthalt und Gefangenschaft in den letzten der Sudan-Provinzen

von A. J. Mounteney Jephson und Henry M. Stanley

Henry M. Stanleys Reise durch den dunklen Weltteil : nach Stanleys Berichten für weitere Kreise

Henry M. Stanley. Bearb. von Berthold Volz

The illness experience of persons with Parkinson's Disease

Background. Parkinson's disease is a chronic, progressive, age-related, neurodegenerative disorder with no known cause or promising cure. While substantial information is known about the pathophysiology of Parkinson's disease, little is known about the illness experience of persons living with the disease. The purpose of this study was to understand how persons with Parkinson's disease construct their illness experience and manage living with their illness on a daily basis. ^ Method. A qualitative study with an ethnographic approach employed the strategies of participant observations and fieldwork. Field data were generated from a two year exposure to two Parkinson's disease support groups in east Texas. Open-ended semi-structured interviews with seven men and seven women with Parkinson's disease were also conducted. These data were combined and analyzed using thematic analysis. ^ Findings. The illness experience is described through the metaphor "Sailing the Sea in The Eye of the Storm." This metaphor served as the overarching theme that covered the two interacting content themes of the voyage of Daily Negotiations in the Midst of Uncertainty and Reconstruction of the Self with Parkinson's Disease. Daily negotiations incorporated navigating daily activities with the uncertainty of both the progression and daily vicissitudes of the disease. Participants described their symptoms as progressive imprisonment that interfered with daily activities. The progressive nature of the disease required the participants to reconstruct their perceptions of themselves. Reconstructing the self involved the paradoxical balancing of preserving the self while simultaneously releasing aspects of the former self to reconstruct the self with Parkinson's disease. This process was reflected in four exemplars: I Know Me." "It's Still Me," "See Me." and "Remember Me." ^ Conclusions. This qualitative study illuminated the struggle of persons in dealing with the uncertainties and fluctuations of Parkinson's disease and the process of reconstructing their perceptions of themselves. The meaning and reconstruction of the illness experience expressed by participants will inform understanding beyond the disease itself to the illness experience that these participants must deal with on a daily basis. ^

Safety, pharmacokinetics, and antiretroviral activity of multiple doses of ibalizumab (formerly TNX-355), an anti-CD4 monoclonal antibody in HIV-1 infected adults

Context: Despite tremendous strides in HIV treatment over the past decade, resistance remains a major problem. A growing number of patients develop resistance and require new therapies to suppress viral replication. ^ Objective: To assess the safety of multiple administrations of the anti-CD4 receptor (anti-CD4) monoclonal antibody ibalizumab given as intravenous (IV) infusions, in three dosage regimens, in subjects infected with human immunodeficiency virus (HIV-1). ^ Design: Phase 1, multi-center, open-label, randomized clinical trial comparing the safety, pharmacokinetics and antiviral activity of three dosages of ibalizumab. ^ Setting: Six clinical trial sites in the United States. ^ Participants: A total of twenty-two HIV-positive patients on no anti-retroviral therapy or a stable failing regimen. ^ Intervention: Randomized to one of two treatment groups in Arms A and B followed by non-randomized enrollment in Arm C. Patients randomized to Arm A received 10 mg/kg of ibalizumab every 7 days, for a total of 10 doses; patients randomized to Arm B received a total of six doses of ibalizumab; a single loading dose of 10 mg/kg on Day 1 followed by five maintenance doses of 6 mg/kg every 14 days, starting at Week 1. Patients assigned to Arm C received 25 mg/kg of ibalizumab every 14 days for a total of 5 doses. All patients were followed for safety for an additional 7 to 8 weeks. ^ Main Outcome Measures: Clinical and laboratory assessments of safety and tolerability of multiple administrations of ibalizumab in HIV-infected patients. Secondary measures of efficacy include HIV-1 RNA (viral load) measurements. ^ Results: 21 patients were treatment-experienced and 1 was naïve to HIV therapy. Six patients were failing despite therapy and 15 were on no current HIV treatment. Mean baseline viral load (4.78 log 10; range 3.7-5.9) and CD4+ cell counts (332/μL; range 89-494) were similar across cohorts. Mean peak decreases in viral load from baseline of 0.99 log10(1.11 log10, and 0.96 log 10 occurred by Wk 2 in Cohorts A, B and C, respectively. Viral loads decreased by >1.0 log10 in 64%; 4 patients viral loads were suppressed to < 400 copies/mL. Viral loads returned towards baseline by Week 9 with reduced susceptibility to ibalizumab. CD4+ cell counts rose transiently and returned toward baseline. Maximum median elevations above BL in CD4+ cell counts for Cohorts A, B and C were +257, +198 and +103 cells/μL, respectively and occurred within 3 Wks in 16 of 22 subjects. The half-life of ibalizumab was 3-3.5 days and elimination was characteristic of capacity-limited kinetics. Administration of ibalizumab was well tolerated. Four serious adverse events were reported during the study. None of these events were related to study drug. Headache, nausea and cough were the most frequently reported treatment emergent adverse events and there were no laboratory abnormalities related to study drug. ^ Conclusions: Ibalizumab administered either weekly or bi-weekly was safe, well tolerated, and demonstrated antiviral activity. Further studies with ibalizumab in combination with standard antiretroviral treatments are warranted.^

Sexual initiation of U.S. Latino and Colombian high school students: Toward a new theory of adolescent sexuality and implications for sexual health prevention and promotion

The general research question for this dissertation was: do the data on adolescent sexual experiences and sexual initiation support the explicit or implicit adolescent sexuality theories informing the sexual health interventions currently designed for youth? To respond to this inquiry, three different studies were conducted. The first study included a conceptual and historical analysis of the notion of adolescence introduced by Stanley Hall, the development of an alternative model based on a positive view of adolescent sexuality, and the rationale for introducing to adolescent sexual health prevention programs the new definitions of sexual health and the social determinants of health approach. The second one was a quantitative study aimed at surveying not only adolescents' risky sexual behaviors but also sexual experiences associated with desire/pleasure which have been systematically neglected when investigating the sexual and reproductive health of the youth. This study was conducted with a representative sample of the adolescents attending public high schools in the State of Caldas in the Republic of Colombia. The third study was a qualitative analysis of 22 interviews conducted with male and female U.S. Latino adolescents on the reasons for having had or having not had vaginal sex. The more relevant results were: most current adolescent sexual health prevention programs are still framed in a negative approach to adolescent sexuality developed a century ago by Stanley Hall and Sigmund Freud which do not accept the adolescent sexual experience and propose its sublimation. In contrast, the Colombian study indicates that, although there are gender differences, adolescence is for males and females a normal period of sexual initiation not limited to coital activity, in which sexual desire/pleasure is strongly associated with sexual behavior. By the same token, the study about the reasons for having had or not had initiated heterosexual intercourse indicated that curiosity, sexual desire/pleasure, and love are basic motivations for deciding to have vaginal sexual intercourse for the first time and that during adolescence, young women and men reach the cognitive development necessary for taking conscious decisions about their sexual acts. The findings underline the importance of asking pertinent questions about desire/pleasure when studying adolescent sexuality and adopting an evidence-based approach to sexual health interventions.^

STRATEGIES FOR THE TRANSFER OF APPROPRIATE TECHNOLOGY TO DEVELOPING COUNTRIES: THE IMPLEMENTATION AND DEVELOPMENT OF ENVIRONMENTAL HEALTH PROGRAMS IN TUNISIA

This dissertation focuses on Project HOPE, an American medical aid agency, and its work in Tunisia. More specifically this is a study of the implementation strategies of those HOPE sponsored projects and programs designed to solve the problems of high morbidity and infant mortality rates due to environmentally related diarrheal and enteric diseases. Several environmental health programs and projects developed in cooperation with Tunisian counterparts are described and analyzed. These include (1) a paramedical manpower training program; (2) a national hospital sanitation and infection control program; (3) a community sewage disposal project; (4) a well reconstruction project; and (5) a solid-waste disposal project for a hospital.^ After independence, Tunisia, like many developing countries, encountered several difficulties which hindered progress toward solving basic environmental health problems and prompted a request for aid. This study discusses the need for all who work in development programs to recognize and assess those difficulties or constraints which affect the program planning process, including those latent cultural and political constraints which not only exist within the host country but within the aid agency as well. For example, failure to recognize cultural differences may adversely affect the attitudes of the host staff towards their work and towards the aid agency and its task. These factors, therefore, play a significant role in influencing program development decisions and must be taken into account in order to maximize the probability of successful outcomes.^ In 1969 Project HOPE was asked by the Tunisian government to assist the Ministry of Health in solving its health manpower problems. HOPE responded with several programs, one of which concerned the training of public health nurses, sanitary technicians, and aids at Tunisia's school of public health in Nabeul. The outcome of that program as well as the strategies used in its development are analyzed. Also, certain questions are addressed such as, what should the indicators of success be, and when is the time right to phase out?^ Another HOPE program analyzed involved hospital sanitation and infection control. Certain generic aspects of basic hospital sanitation procedures were documented and presented in the form of a process model which was later used as a "microplan" in setting up similar programs in other Tunisian hospitals. In this study the details of the "microplan" are discussed. The development of a nation-wide program without any further need of external assistance illustrated the success of HOPE's implementation strategies.^ Finally, although it is known that the high incidence of enteric disease in developing countries is due to poor environmental sanitation and poor hygiene practices, efforts by aid agencies to correct these conditions have often resulted in failure. Project HOPE's strategy was to maximize limited resources by using a systems approach to program development and by becoming actively involved in the design and implementation of environmental health projects utilizing "appropriate" technology. Three innovative projects and their implementation strategies (including technical specifications) are described.^ It is advocated that if aid agencies are to make any progress in helping developing countries basic sanitation problems, they must take an interdisciplinary approach to progrm development and play an active role in helping counterparts seek and identify appropriate technologies which are socially and economically acceptable. ^

UTILIZATION OF EVALUATION RESULTS

The issue over utilization of evaluation results has been receiving increasing attention lately. However, a continuing problem has been the lack of knowledge of the specific determinants of the utilization of evaluation results, the extent that these results are used, and the absence in the literature of empirical investigations of those determinants. The purposes of the current study were to examine the patterns of utilization of the recommendations made to an organization following an extensive evaluation, and to quantitatively examine some determinants of that utilization. During the course of the ten-month study, a number of important points regarding evaluation studies, and the utilization of their results were identified. Primary among these was the fact that a definition of utilization that requires adoption of recommendations is limited. Tremendous amounts of organizational activity may accrue from the results of an evaluation regardless of whether the results are adopted. In addition, none of the characteristics of the recommendations that were examined proved to be significant predictors of the extent of their utilization other than whether adoption was considered an authority decision or the results of staff concensus. ^