Targeted Secretion Inhibitors—Innovative Protein Therapeutics

Botulinum neurotoxins are highly effective therapeutic products. Their therapeutic success results from highly specific and potent inhibition of neurotransmitter release with a duration of action measured in months. These same properties, however, make the botulinum neurotoxins the most potent acute lethal toxins known. Their toxicity and restricted target cell activity severely limits their clinical utility. Understanding the structure-function relationship of the neurotoxins has enabled the development of recombinant proteins selectively incorporating specific aspects of their pharmacology. The resulting proteins are not neurotoxins, but a new class of biopharmaceuticals, Targeted Secretion Inhibitors (TSI), suitable for the treatment of a wide range of diseases where secretion plays a major role. TSI proteins inhibit secretion for a prolonged period following a single application, making them particularly suited to the treatment of chronic diseases. A TSI for the treatment of chronic pain is in clinical development

Values Affecting Collaboration Among Psychologists and Evangelical Clergy

Previous research has shown that shared values are important to both clergy and psychologists when considering the possibility of collaborating with one another, but it is not clear which values must be shared. Eighty-one psychologists and 56 evangelical Protestant clergy were surveyed using a values questionnaire developed by Jensen and Bergin (1988) with some additional items specifically pertaining to evangelical beliefs, revealing differences within value themes between clergy and psychologists. The epistemological foundations of the two professions create obstacles to collaboration, suggesting a need for psychologists to develop trusting relationships with clergy, engage in specialized training, and reevaluate the post-modern distinction between facts in the public domain and privately held values

Relationship between mode of sport training and general cognitive performance

Purpose - To investigate whether athletes who engage in different modes of sports training correspondingly exhibit different patterns of performance on general cognition tasks. Methods - Sixty participants were recruited into an endurance, motorically complex, or control group, and were administered a series of physical tests and neuropsychological assessments. Results - Athletes in the endurance group demonstrated the highest levels of cardiovascular fitness and those in the motorically complex group exhibited the highest levels of motor fitness. Nonetheless, no differences in cognitive performance were observed between the three groups. Conclusion - These findings indicate that the mode of sport training, which results in either high cardiovascular or high motor fitness, bears no relationship to measures of general cognition in elite athletes. The present findings suggest that coaches and athletic trainers should be encouraged to monitor athletes' stress levels during training in order to maximize the beneficial effects of such training on general cognitive performance.This study was supported, in part, by a grant from the Ministry of Science and Technology, Taiwan, China, for Yu-Kai Chang (NSC102-2420-H-179-001-MY3)

Cemeteries as archives: who says dead men tell no tales?

Cemeteries are more than just the final resting place of our ancestors; many scholarly fields have found the cemetery to be a valuable historical resource. The cemetery contains a wealth of information, including the personal stories of those buried there, the actions of the organization that created it, and the beliefs of the people in the community to which it belongs. In many cases, the cemetery is the only remaining documentary evidence about a person or a group of people. The archival profession has tasked itself with preserving the documentary heritage of the full spectrum of society, but it has yet to recognize the archival value of the physical cemetery, due in part to its non-traditional format as immovable, threedimensional objects contextually bound to a physical landscape. This thesis outlines the ways in which the cemetery fits the definition of archives and how the characteristics of the cemetery can align with various aspects of archival theory. This thesis argues for the archival profession to recognize that cemeteries are archives and to use their unique perspective to help preserve the evidential and informational value at the cemetery for future generations

Neural and behavioral correlates of aberrant salience in individuals at risk for psychosis

Correction to the original article published in Schizophrenia Bulletin, Volume 39, Issue 6, 1 November 2013, Pages 1328–1336; https://doi.org/10.1093/schbul/sbs147

Structure-function relationships of ricin A-chain

This thesis represents e study of the reletionships between the structure and function of the A-chain of ricin. Ricin is one member of a family of ribosome- inactivating proteins (RIPs) that inactivate protein synthesis by modification of the ribosomal RNA. The mechanism of this catalytic effect is unknown. This project was designed to investigate which amino acid residues were important in the mechanism of catalysis. Using site-directed mutagenesis of specific ricin A- chain residues, eight mutants were created for biochemical analysis. Residues were targeted for autogenesis by analysis of the position of conserved amino acids in the ricin 3-D structure. Glul77 was mutated to Lys, Ala and Asp. and Argl8O was mutated to Gin, Ala and Met. The arginine residue at position 29 was altered to an alanine, and a deletion of residues Serl76 to Argl80 was performed. Mutant recombinant ricin A-chain (rRTA) constructs were prepared for in vitro and in vivo expression in the vectors pGEMl and pDS5/3 respectively. Initially, mutants were translated in a cell-free wheatgerm translation system to assess the size of the mutant polypeptides. All the constructs produced polypeptides of the correct else. The N-glycosidase activity of the mutants was then assessed with two methods of analysis using rabbit reticulocyte ribosomes. It was shown that some of the mutants were devoid of detectable activity and some had reduced activity. Mutant constructs were expressed in Escherichia coli in order to isolate protein for quantitative activity measurements. Crude E. coli extracts containing rRTA and rRTA mutants were tested for activity. It was found that the relative activities of mutant proteins produced in E. coli was similar to that seen previously with the in vitro measurements. Soluble, active protein was recovered for a few of the mutant proteins, although no expression was observed from some constructs. Various purification procedures were assessed end ion-exchange chromatography was determined to be most suitable. Mutant D177 was tested for its N-glycosidase activity towards salt-washed yeast ribosomes and related to the activity of wild-type rRTA. It was shown that the activity of D177 was approximately 60 fold lower than that of wild- type rRTA with the majority of the difference being observed with the kcat of the reaction. It was also shown that the data produced in this study correlated with a recently published putative mechanism of action of ricin A-chain

White matter integrity as a predictor of response to treatment in first episode psychosis

The integrity of brain white matter connections is central to a patient's ability to respond to pharmacological interventions. This study tested this hypothesis using a specific measure of white matter integrity, and examining its relationship to treatment response using a prospective design in patients within their first episode of psychosis. Diffusion tensor imaging data were acquired in 63 patients with first episode psychosis and 52 healthy control subjects (baseline). Response was assessed after 12 weeks and patients were classified as responders or non-responders according to treatment outcome. At this second time-point, they also underwent a second diffusion tensor imaging scan. Tract-based spatial statistics were used to assess fractional anisotropy as a marker of white matter integrity. At baseline, non-responders showed lower fractional anisotropy than both responders and healthy control subjects (P < 0.05; family-wise error-corrected), mainly in the uncinate, cingulum and corpus callosum, whereas responders were indistinguishable from healthy control subjects. After 12 weeks, there was an increase in fractional anisotropy in both responders and non-responders, positively correlated with antipsychotic exposure. This represents one of the largest, controlled investigations of white matter integrity and response to antipsychotic treatment early in psychosis. These data, together with earlier findings on cortical grey matter, suggest that grey and white matter integrity at the start of treatment is an important moderator of response to antipsychotics. These findings can inform patient stratification to anticipate care needs, and raise the possibility that antipsychotics may restore white matter integrity as part of the therapeutic response

Aerobic fitness is associated with greater white matter integrity in children

Aerobic fitness has been found to play a positive role in brain and cognitive health of children. Yet, many of the neural biomarkers related to aerobic fitness remain unknown. Here, using diffusion tensor imaging, we demonstrated that higher aerobic fitness was related to greater estimates of white matter microstructure in children. Higher fit 9- and 10-year-old children showed greater fractional anisotropy (FA) in sections of the corpus callosum, corona radiata, and superior longitudinal fasciculus, compared to lower fit children. The FA effects were primarily characterized by aerobic fitness differences in radial diffusivity, thereby raising the possibility that estimates of myelination may vary as a function of individual differences in fitness during childhood. White matter structure may be another potential neural mechanism of aerobic fitness that assists in efficient communication between gray matter regions as well as the integration of regions into networks. © 2014 Chaddock-Heyman, Erickson, Holtrop, Voss, Pontifex, Raine, Hillman and Kramer

The isolation and characterisation of temperature-dependent Ricin A chain molecules in Saccharomyces cerevisiae.

Ricin is a heterodimeric plant protein that is potently toxic to mammalian cells. Toxicity results from the catalytic depurination of eukaryotic ribosomes by ricin A chain (RTA) that follows toxin endocytosis to, and translocation across, the endoplasmic reticulum (ER) membrane. To ultimately identify proteins required for these later steps in the entry process, it will be useful to express the catalytic subunit within the ER of yeast cells in a manner that initially permits cell growth. A subsequent switch in conditions to provoke innate toxin action would permit only those strains containing defects in genes normally essential for toxin retro-translocation, refolding or degradation to survive. As a route to such a screen, several RTA mutants with reduced catalytic activity have previously been isolated. Here we report the use of Saccharomyces cerevisiae to isolate temperaturedependent mutants of endoplasmic reticulum-targeted RTA. Two such toxin mutants with opposing phenotypes were isolated. One mutant RTA (RTAF108L/L151P) allowed the yeast cells that express it to grow at 37°C while the same cells did not grow at 23ºC. Both mutations were required for temperature-dependent growth. The second toxin mutant (RTAE177D) allowed cells to grow at 23°C but not at 37°C. Interestingly, RTAE177D has been previously reported to have reduced catalytic activity, but this is the first demonstration of a temperature-sensitive phenotype. To provide a more detailed characterisation of these mutants we have investigated their N-glycosylation, stability, catalytic activity and, where appropriate, a three dimensional structure. The potential utility of these mutants is discussed

Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum

The plant cytotoxin ricin enters target mammalian cells by receptor-mediated endocytosis and undergoes retrograde transport to the endoplasmic reticulum (ER). Here, its catalytic A chain (RTA) is reductively separated from the cell-binding B chain, and free RTA enters the cytosol where it inactivates ribosomes. Cytosolic entry requires unfolding of RTA and dislocation across the ER membrane such that it arrives in the cytosol in a vulnerable, nonnative conformation. Clearly, for such a dislocated toxin to become active, it must avoid degradation and fold to a catalytic conformation. Here, we show that, in vitro, Hsc70 prevents aggregation of heat-treated RTA, and that RTA catalytic activity is recovered after chaperone treatment. A combination of pharmacological inhibition and cochaperone expression reveals that, in vivo, cytosolic RTA is scrutinized sequentially by the Hsc70 and Hsp90 cytosolic chaperone machineries, and that its eventual fate is determined by the balance of activities of cochaperones that regulate Hsc70 and Hsp90 functions. Cytotoxic activity follows Hsc70-mediated escape of RTA from an otherwise destructive pathway facilitated by Hsp90. We demonstrate a role for cytosolic chaperones, proteins typically associated with folding nascent proteins, assembling multimolecular protein complexes and degrading cytosolic and stalled, cotranslocational clients, in a toxin triage, in which both toxin folding and degradation are initiated from chaperone-bound states