Engineering RNA phage MS2 virus-like particles for peptide display

Phage display is a powerful and versatile technology that enables the selection of novel binding functions from large populations of randomly generated peptide sequences. Random sequences are genetically fused to a viral structural protein to produce complex peptide libraries. From a sufficiently complex library, phage bearing peptides with practically any desired binding activity can be physically isolated by affinity selection, and, since each particle carries in its genome the genetic information for its own replication, the selectants can be amplified by infection of bacteria. For certain applications however, existing phage display platforms have limitations. One such area is in the field of vaccine development, where the goal is to identify relevant epitopes by affinity-selection against an antibody target, and then to utilize them as immunogens to elicit a desired antibody response. Today, affinity selection is usually conducted using display on filamentous phages like M13. This technology provides an efficient means for epitope identification, but, because filamentous phages do not display peptides in the high-density, multivalent arrays the immune system prefers to recognize, they generally make poor immunogens and are typically useless as vaccines. This makes it necessary to confer immunogenicity by conjugating synthetic versions of the peptides to more immunogenic carriers. Unfortunately, when introduced into these new structural environments, the epitopes often fail to elicit relevant antibody responses. Thus, it would be advantageous to combine the epitope selection and immunogen functions into a single platform where the structural constraints present during affinity selection can be preserved during immunization. This dissertation describes efforts to develop a peptide display system based on the virus-like particles (VLPs) of bacteriophage MS2. Phage display technologies rely on (1) the identification of a site in a viral structural protein that is present on the surface of the virus particle and can accept foreign sequence insertions without disruption of protein folding and viral particle assembly, and (2) on the encapsidation of nucleic acid sequences encoding both the VLP and the peptide it displays. The experiments described here are aimed at satisfying the first of these two requirements by engineering efficient peptide display at two different sites in MS2 coat protein. First, we evaluated the suitability of the N-terminus of MS2 coat for peptide insertions. It was observed that random N-terminal 10-mer fusions generally disrupted protein folding and VLP assembly, but by bracketing the foreign sequences with certain specific dipeptides, these defects could be suppressed. Next, the suitability of a coat protein surface loop for foreign sequence insertion was tested. Specifically, random sequence peptides were inserted into the N-terminal-most AB-loop of a coat protein single-chain dimer. Again we found that efficient display required the presence of appropriate dipeptides bracketing the peptide insertion. Finally, it was shown that an N-terminal fusion that tended to interfere specifically with capsid assembly could be efficiently incorporated into mosaic particles when co-expressed with wild-type coat protein

Research in the Restricted Problems of Three and Four Bodies Final Scientific Report

Seven studies have been conducted on research in the existence and nature of solutions of the restricted problems of three and four bodies. The details and results of five of these research investigations have already been published, and the latest two studies will be published shortly. A complete bibliography of publications is included in this report. This research has been primarily qualitative and has yielded new information on the behavior of trajectories near the libration points in the Earth-Moon-Sun and Sun-Jupiter-Saturn systems, and on the existence of periodic trajectories about the libration points of the circular and elliptical restricted four-body models. We have also implemented Birkhoff's normalization process for conservative and nonconservative Hamiltonian systems with equilibrium points. This makes available a technique for analyzing stability properties of certain nonlinear dynamical systems, and we have applied this technique to the circular and elliptical restricted three-body models. A related study was also conducted to determine the feasibility of using cislunar periodic trajectories for various space missions. Preliminary results suggest that this concept is attractive for space flight safety operations in cislunar space. Results of this research will be of interest to mathematicians, particularly those working in ordinary differential equations, dynamical systems and celestial mechanics; to astronomers; and to space guidance and mission analysts

Plasmalogen Deficiency: A Risk Factor for Dementias and Potential Treatment Target

Altered lipid metabolism is implicated in the risk of sporadic Alzheimer’s disease (AD) and related dementias (ADRD); however, the precise mechanisms accounting for findings from observational studies remains to be fully elucidated. Plasmalogens are a subclass of integral membrane phospholipids with unique properties that appear to play important roles relevant to the pathophysiology of AD and ADRD, including vesicle fusion necessary for synaptic neurotransmitter release, modulation of membrane fluidity and microdomain dynamics, membrane antioxidant functions, and neuroprotection. Like the more familiar phosphatides, plasmalogens are synthesized on a 3-carbon glycerol backbone; however, they differ from phosphatides by the presence of a vinyl ether linkage at the 1st (sn1) glycerol carbon atom in place of the acyl ester linkage present at sn1 in phosphatides, and at sn2 in both lipid subclasses. Plasmalogens bearing the omega-3 fatty acid docosahexaenoic acid (DHA), a key component of fish oils, are the most abundant plasmalogen species in cerebral cortex membranes. Circulating plasmalogen levels are decreased in older individuals, and are further decreased in AD and Mild Cognitive Impairment (MCI). In addition, reduced indices of plasmalogen biosynthesis and/or remodeling are significantly correlated with elevated cerebrospinal fluid (CSF) concentrations of total tau, which is a biomarker of AD and certain other neurodegenerative diseases. This correlation suggests a functional relationship between reduced plasmalogen availability and neurodegeneration. Endogenous plasmalogen synthesis requires the integrity of peroxisomes for the attachment of an alkyl side chain to the sn1 glycerol carbon. Decreased peroxisome function may be a key factor underlying the decrease in circulating plasmalogens with aging and with neurodegenerative diseases such as AD and ADRD. Preclinical data indicate that oral administration of a precursor phospholipid compound, DHA-containing alkyl-diacylglycerol, or DHA-AAG, can increase circulating DHA-containing plasmalogens in a peroxisome-independent manner, as conversion to plasmalogens from this precursor requires only the endoplasmic reticulum. We present here data showing that: 1) oral administration of a single dose of DHA-AAG at 100mg/kg to 6 (4M/2F) healthy subjects aged 23-56 increased circulating plasmalogen levels by 80% within 24 hours; and 2) daily oral administration of DHA-AAG to 22 persons (11M/11F), aged 37-84 (mean= 69) yr, with mild to moderate cognitive impairment [CDR: 0.5 (N=14); 1 (N=4); 2 (N = 4)] on an ascending-dose schedule of 1.0 ml/day for 30 days, followed by 2.0 ml/day for 60 days, followed by 4.0 ml for 30 days, increased serum DHA plasmalogens by \u3e 2-fold by the end of the treatment period. DHA-AAG was well-tolerated by both groups of individuals in these 2 studies. These findings suggest that DHA-AAG may be a useful agent for correcting plasmalogen deficiency associated with aging and aging-associated cognitive disorders. Future studies will examine the effect of plasmalogen repletion with DHA-AAG on cerebrospinal fluid plasmalogen concentrations, and effects on cognitive function and other clinical outcomes

The Draw-A-Clock Contest: A Strategy for Improving Cognitive Status Assessment by Trainees

Background: Historically, psychiatrists have been less inclined than neurologists to utilize pencil and paper tasks during bedside cognitive assessments. Objective: The Draw-AClock Contest was established in 1986 at the University of Massachusetts to promote use of cognitive assessment tasks by psychiatry residents. Methods: Used in neuropsychological assessments since the 1930’s, clock tasks have been popular screening tools for executive function, praxis, visuospatial and constructive ability, often as part of dementia screening. Given its broad utility as a screening tool and the ease and speed of its administration, the Draw-A-Clock task (with hands set to 11:10 and no circle provided) was selected for use by UMass psychiatry residents, with further bedside assessment encouraged to explore any detected deficits. To encourage participation and foster clinical inquiry, residents are asked to submit clinically interesting de-identified patient clocks. For 21 years, clock contest entries have been collected each spring, with basic demographic, diagnostic, and process notes. Resident names are encoded, and entries are judged by a neuropsychiatrist (SB) and a neuropsychologist (EK). A “clock trophy” and detailed analysis of the submission is presented to the winner at the annual graduation banquet. Results: As a result of this contest, mental status examinations by trainees have become more comprehensive and an atmosphere of neuropsychiatric inquiry has been maintained. Faculty members have also incorporated this task into their mental status assessments, thus establishing a culture of cognitive inquiry and an academic tradition. Examples of winning clocks and common findings will be presented. Published abstract: Sullivan J, Benjamin S, Case Report: CADASIL with Cysteine-Sparing Notch-3 Mutation, American Neuropsychiatric Association, abstract, Journal of Neuropsychiatry and Clinical Neuroscience 21(2):221, 2009. DOI 10.1176/appi.neuropsych.21.2.221

Mental disorder and the outcome of HIV/AIDS in low-income and middle-income countries: a systematic review.

OBJECTIVES: To conduct a systematic review of the literature to examine the interrelationship between mental health and treatment outcomes in low-income and middle-income settings; to update the work of Collins et al. (2006). DESIGN: Systematic review of peer-reviewed articles that examined one of the following: the effects of mental disorders (including cognitive impairment) upon engagement with treatment and/or adherence; their influence upon HIV-related clinical outcomes; and the impact of interventions for mental disorder. METHODS: Articles about mental health and HIV/AIDS were included if they were published after 2005 and addressed one of the areas of interest described above. Systematic methods were used for searching, screening, and data extraction. Studies employing quantitative measures of exposures and outcomes wherein all participants had a diagnosis of HIV/AIDS were included. RESULTS: This review found ample and moderately consistent evidence that adverse mental health and alcohol consumption are associated with reduced adherence. Variation in measurement and the relative paucity of work meant that interpretation of studies examining engagement with care and other clinical outcomes was difficult. Evidence on the efficacy and effectiveness of mental health interventions in low-income and middle-income settings was very limited. CONCLUSION: This review suggests that psychosocial factors, namely, depression and alcohol may have adverse effects upon HIV-related outcomes. However, further large, high-quality studies examining outcomes other than adherence are needed. There is also an urgent need for randomized controlled trials of interventions for mental disorder and a need to investigate their impact upon HIV-related outcomes

CD4 inhibits helper T cell activation at lower affinity threshold for full-length T cell receptors than single chain signaling constructs

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Collateral Consequences: Living in the Shadow of a Conviction

Over the course of fifteen weeks, fourteen students and two professors explored the Criminal Justice System and the immense challenges faced by citizens as they attempt to reintegrate into their communities after being incarcerated. Through the lens of Systems Thinking, a system pioneered and described by Peter Senge, we dissected seemingly unrelated experiences to identify behaviors and patterns that have been intrinsic in the Criminal Justice System since “tough on crime” policies emerged into the early 1970’s. During this time, criminal justice and public opinion moved from a more rehabilitative stance to one of punishment and punitive vengeance on those that have wronged society, particularly those who committed drug offenses.. Along with extended sentences came a slew of collateral consequences that kept people impoverished and oppressed even after leaving prison. This specific aspect was the focus of our class’ research and discussion. By breaking this down into subsystems, the class characterized the challenges faced by citizens trying to return to their communities and normal life, and provided areas and suggestions for improving the system and in turn, the lives of these people. This book is the culmination of that research

Communication between secondary and primary care following self-harm: are National Institute of Clinical Excellence (NICE) guidelines being met?

<p>Abstract</p> <p>Background</p> <p>Most patients contact their general practitioner (GP) following presentation to an Emergency Department (ED) after a self-harm incident, and strategies to help GPs manage these patients include efficient communication between services. The aim of this study was to assess the standard of documentation and communication to primary care from secondary care as recommended by the National Institute of Clinical Excellence (NICE) guidelines on the short-term management of people who self-harm.</p> <p>Methods</p> <p>An audit of medical records (ED and Psychiatric) on people aged 16 years and over who had presented to the ED following self-harm, benchmarked according to government guidelines, was performed. Data were collected over a 4-week period at a general teaching hospital.</p> <p>Results</p> <p>We collected data on 93 consecutive episodes of self-harm; 62% of episodes were communicated to primary care, 58% of these communications were within 24 h and most within 3 days. Patient identifying details and follow-up arrangements were specified in most cases. Communication via psychiatric staff was most detailed. ED clinicians provided few communications and were of limited content. Communication with the patient's GP was not made in half of those cases seen by a mental health specialist.</p> <p>Conclusion</p> <p>Government guidelines are only partially being met. Reliance on communication by ED staff would leave a substantial proportion of patients discharged from the ED with no or minimal communication to primary care. Psychiatric services need to improve the rate of communication to the patient's GP following assessment A national sample of National Health Service (NHS) trusts would establish if this is a problem elsewhere.</p

Mu and Delta opioid receptors activate the same G proteins in human neuroblastoma SH-SY5Y cells

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72251/1/sj.bjp.0704430.pd