Reference Protocol to Assess Analytical Performance of Higher Order Structural Analysis Measurements: Results from an Interlaboratory Comparison.

Measurements of protein higher order structure (HOS) provide important information on stability, potency, efficacy, immunogenicity, and biosimilarity of biopharmaceuticals, with a significant number of techniques and methods available to perform these measurements. The comparison of the analytical performance of HOS methods and the standardization of the results is, however, not a trivial task, due to the lack of reference protocols and reference measurement procedures. Here, we developed a protocol to structurally alter and compare samples of somatropin, a recombinant biotherapeutic, and describe the results obtained by using a number of techniques, methods and in different laboratories. This, with the final aim to provide tools and generate a pool of data to compare and benchmark analytical platforms and define method sensitivity to structural changes. Changes in somatropin HOS, induced by the presence of zinc at increasing concentrations, were observed, both globally and at more localized resolution, across many of the methods utilized in this study and with different sensitivities, suggesting the suitability of the protocol to improve understanding of inter- and cross-platform measurement comparability and assess analytical performance as appropriate.

Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase.

Improvement of thermostability in engineered enzymes can allow biocatalysis on substrates with poor aqueous solubility. Denaturation of the cofactor-binding loops of Escherichia coli transketolase (TK) was previously linked to the loss of enzyme activity under conditions of high pH or urea. Incubation at temperatures just below the thermal melting transition, above which the protein aggregates, was also found to anneal the enzyme to give an increased specific activity. The potential role of cofactor-binding loop instability in this process remained unclear. In this work, the two cofactor-binding loops (residues 185-192 and 382-392) were progressively mutated towards the equivalent sequence from the thermostable Thermus thermophilus TK and variants assessed for their impact on both thermostability and activity. Cofactor-binding loop 2 variants had detrimental effects on specific activity at elevated temperatures, whereas the H192P mutation in cofactor-binding loop 1 resulted in a two-fold improved stability to inactivation at elevated temperatures, and increased the critical onset temperature for aggregation. The specific activity of H192P was 3-fold and 19-fold higher than that for wild-type at 60°C and 65°C respectively, and also remained 2.7-4 fold higher after re-cooling from pre-incubations at either 55°C or 60°C for 1h. Interestingly, H192P was also 2-times more active than wild-type TK at 25°C. Optimal activity was achieved at 60°C for H192P compared to 55°C for wild type. These results show that cofactor-binding loop 1, plays a pivotal role in partial denaturation and aggregation at elevated temperatures. Furthermore, a single rigidifying mutation within this loop can significantly improve the enzyme specific activity, as well as the stability to thermal denaturation and aggregation, to give an increased temperature optimum for activity.

Comparison of vestipitant with ondansetron for the treatment of breakthrough postoperative nausea and vomiting after failed prophylaxis with ondansetron.

BACKGROUND: Postoperative nausea and vomiting (PONV) is common; ondansetron is often used as prophylaxis or for breakthrough episodes. Vestipitant is a neurokinin 1 (NK-1) receptor antagonist that is effective for prophylaxis, but its efficacy for treating established PONV is unknown. This study was performed to evaluate the efficacy and safety of vestipitant, compared with ondansetron for the treatment of breakthrough PONV in patients who had already received prophylactic ondansetron before surgery. METHODS: A multicentre, randomized, single-blind (sponsor-open), parallel group study. Of 527 surgical patients, 130 (25%) had breakthrough PONV and were equally randomized to one of six i.v. doses of vestipitant (4-36 mg) or ondansetron 4 mg. The primary endpoint was the rate of patients exhibiting complete response, defined as no emesis and no further rescue medication from 10 min after infusion up to 24 h after surgery or hospital discharge. RESULTS: All doses of vestipitant were non-inferior to ondansetron in treating PONV after failed prophylaxis with ondansetron. However, vestipitant was superior to ondansetron in decreasing episodes of postoperative emesis and retching. The complete response rate analysis using Bayesian model averaging indicated that no vestipitant dose was superior to ondansetron. Nausea numerical rating scale scores and the times-to-PONV or discharge were similar between the vestipitant and ondansetron treatment groups. CONCLUSIONS: Although overall efficacy was non-inferior between vestipitant and ondansetron, the rate of emesis was lower with vestipitant. These data suggest that vestipitant may be a useful agent for the management of PONV, similar to other NK-1 antagonists. CLINICAL TRIAL REGISTRATION: NCT01507194.

Structural stability of an enzyme biocatalyst.

TK (transketolase) undergoes inactivation during biocatalytic processes due to oxidation, substrate and product inhibition, reactivity of aldehyde substrates, irreversible inactivation at low pH, and dissociation of cofactors. However, the contribution of protein denaturation to each of these mechanisms is not fully understood. The urea-induced reversible denaturations of the apo- and holo-enzyme forms of the homodimeric Escherichia coli TK have been characterized, along with the reconstitution of holo-TK from the apoenzyme and cofactors. An unusual cofactor-bound yet inactive intermediate occurs on both the reconstitution and holo-TK denaturation pathways. The denaturation pathways of the holo- and apoenzymes converge at a second intermediate consisting of a partially denatured apo-homodimer. Preliminary investigation of the denaturation under oxidizing conditions reveals further complexity in the mechanisms of enzyme deactivation that occur under biocatalytic conditions.

One-pot synthesis of amino-alcohols using a de-novo transketolase and beta-alanine: pyruvate transaminase pathway in Escherichia coli.

Biocatalysis continues to emerge as a powerful technique for the efficient synthesis of optically pure pharmaceuticals that are difficult to access via conventional chemistry. The power of biocatalysis can be enhanced if two or more reactions can be achieved by a single whole cell biocatalyst containing a pathway designed de-novo to facilitate a required synthetic sequence. The enzymes transketolase (TK) and transaminase (TAm) respectively catalyze asymmetric carbon--carbon bond formation and amine group addition to suitable substrate molecules. The ability of a transaminase to accept the product of the transketolase reaction can allow the two catalysts to be employed in series to create chiral amino-alcohols from achiral substrates. As proof of principle, the beta-alanine: pyruvate aminotransferase (beta-A:P TAm) from Pseudomonas aeruginosa has been cloned, to create plasmid pQR428, for overexpression in E.coli strain BL21gold(DE3). Production of the beta-A:P TAm alongside the native transketolase (overexpressed from plasmid pQR411), in a single E.coli host, has created a novel biocatalyst capable of the synthesis of chiral amino alcohols via a synthetic two-step pathway. The feasibility of using the biocatalyst has been demonstrated by the formation of a single diastereoisomer of 2-amino-1,3,4-butanetriol (ABT) product, in up to 21% mol/mol yield, by the beta-A:P TAm, via transamination of L-erythrulose synthesized by TK, from achiral substrates glycolaldehyde (GA) and beta-hydroxypyruvate (beta-HPA). ABT synthesis was achieved in a one-pot process, using either whole cells of the dual plasmid strain or cell lysate, while the dual alcohol-amine functionality of ABT makes it an excellent synthon for many pharmaceutical syntheses.

Is there a process of spiritual change or development associated with ageing? A critical review of research.

This review considers whether research shows a process of spiritual change or development associated with ageing. Spirituality was understood as that which is central to a sense of meaning and purpose in an individual's life and pertains to the sacred or transcendent. Electronic literature searches were conducted to find research published 1985-2003 aimed at understanding spiritual change, themes and tasks in later life. A total of 13 studies were reviewed that looked at changes in spirituality over time, spiritual themes and tasks in a lifespan development context and Tornstam's (Torstam, L. (1996). Gerotranscendence--a theory about maturing into old age. Journal of Aging & Identity, 1, 37-50) theory of gerotranscendence. The research reviewed suggested that some aspects of spirituality remain stable into old age but that there are identifiable spiritual tasks, needs and changes associated with ageing. Some common spiritual themes identified across the research were integrity, humanistic concern, changing relationships with others and concern for younger generations, relationship with a transcendent being or power, self transcendence, and coming to terms with death. These were not related to age per se, but to some of the challenges that age presents, and were mediated by cultural factors and individual differences. The findings and their limitations were discussed.

A randomized, double-blind, close-ranging, pilot study of intravenous granisetron in the prevention of postoperative nausea and vomiting in patients abdominal hysterectomy.

BACKGROUND AND OBJECTIVE: Postoperative nausea and vomiting (PONV) is a frequent and unpleasant experience that may increase postoperative complications and costs. For surgical procedures with a high risk of PONV, prevention is preferable to treatment. In this study, the authors explore the dose-response relationship between granisetron administered just prior to the end of surgery and post-operative nausea and vomiting in patients undergoing abdominal hysterectomy. METHODS: This was a randomized, double-blind, placebo-controlled, pilot study of post-operative nausea and vomiting prevention. Patients undergoing elective open abdominal hysterectomy requiring general anaesthesia received a single dose of granisetron 0.1, 0.2 or 0.3 mg or placebo administered approximately 15 min prior to the end of surgery. The primary efficacy end-point was the proportion of patients with no vomiting in the 0--6 h interval following medication administration. No inferential statistics were planned. RESULTS: The proportion of patients with no vomiting episode in the 0--6 h interval after administration of study medication was higher in each granisetron treatment group (>90%) than in the placebo group (77%). Proportions of patients with no vomiting episodes in the 0--24 h interval were similar across treatment groups. Results of analyses of proportions of patients with no moderate or severe nausea episodes, proportions of those requiring rescue medication and times to first use of rescue medication suggested a treatment effect of granisetron relative to placebo in both the 0--6 and 0--24 h intervals. Similar proportions of patients in each treatment group reported at least one adverse event. CONCLUSIONS: Granisetron at doses of 0.1, 0.2 and 0.3 mg administered just prior to the end of surgery suggested a trend of improved efficacy compared to placebo in preventing postoperative nausea and vomiting in the first 6 h after abdominal hysterectomy. This pilot study did not identify a dose-response relationship.

Substance P (Neurokinin-1) antagonist prevents postoperative vomiting after abdominal hysterectomy procedures.

BACKGROUND: The safety and antiemetic efficacy of CP-122,721, a novel neurokinin-1 antagonist, was evaluated when administered alone or in combination with ondansetron. METHODS: Using a randomized, double-blind, placebo-controlled study design, CP-122,721 was initially compared with placebo and subsequently to ondansetron alone and in combination for prophylaxis against postoperative nausea and vomiting in 243 women undergoing abdominal hysterectomy. In the dose-ranging studies (n = 86), patients received either CP-122,721 100 mg (vs. placebo) or 200 mg (vs. placebo) orally 60-90 min before induction of anesthesia. In the interaction study (n = 157), patients received CP-122,721 200 mg or placebo 60-90 min before induction of anesthesia, and ondansetron 4 mg or saline 2 ml intravenously 15-30 min before the end of surgery. Patients assessed their level of nausea and pain on arrival in the postanesthesia care unit and at 0.5-, 1-, 1.5-, 2-, 4-, 8-, 12-, and 24-h intervals postoperatively. Emetic episodes, need for rescue antiemetic-antinausea medication, postoperative complications, and patient satisfaction were recorded. RESULTS: In the initial dose-ranging study, only 10% of the patients experienced emesis within the first 8 h after surgery with CP-122,721 200 mg compared with 50% in the placebo group. CP-122,721 200 mg also decreased the need for rescue medication (25% vs. 48%). CP-122,721 100 mg was less effective than 200 mg in decreasing the incidence of repeated episodes of emesis. In the interaction study, 6% of the patients receiving CP-122,721 200 mg orally experienced emesis less than 2 h after surgery compared with 17% with ondansetron alone. With combined therapy, only 2% experienced emesis. In addition, the median times for 75% of patients to remain free from postoperative nausea and vomiting were 82, 75, and 362 min in the ondansetron, CP-122,721, and combination groups, respectively. CONCLUSIONS: Oral CP-122,721 200 mg decreased emetic episodes compared with ondansetron (4 mg intravenously) during the first 24 h after gynecologic surgery; however, there was no difference in patient satisfaction.

Evolution of binding affinity in a WW domain probed by phage display.

The WW domain is an approximately 38 residue peptide-binding motif that binds a variety of sequences, including the consensus sequence xPPxY. We have displayed hYAP65 WW on the surface of M13 phage and randomized one-third of its three-stranded antiparallel beta-sheet. Improved binding to the hydrophobic peptide, GTPPPPYTVG (WW1), was selected in the presence of three different concentrations of proteinase K to simultaneously drive selection for improved stability as well as high-affinity binding. While some of the selected binders show cooperative unfolding transitions, others show noncooperative thermal unfolding curves. Two novel WW consensus sequences have been identified, which bind to the xPPxY motif with higher affinity than the wild-type hYAP65 WW domain. These WW domain sequences are not precedented in any natural WW domain sequence. Thus, there appear to be a large number of motifs capable of recognizing the target peptide sequence, only a subset of which appear to be used in natural proteins.

Folding intermediates of wild-type and mutants of barnase. I. Use of phi-value analysis and m-values to probe the cooperative nature of the folding pre-equilibrium.

It is difficult to determine whether transient folding intermediates have a cooperative (or first-order) folding transition without measuring their rates of formation directly. An intermediate I could be formed by a second-order transition from a denatured state D that is progressively changed into I as conditions are changed. We have not been able to monitor the rate of formation of the folding intermediate of barnase directly, but have analysed its reactivity and the equilibrium constant for its formation over a combination of wide ranges of temperature, concentration of denaturant and structural variation. Phase diagrams have been constructed for wild-type and 16 mutant proteins to map out the nature of the energy landscape of the denatured state. The free energy of unfolding of I, delta GD-I, changes with [urea] according to a highly cooperative transition. Further, mD-I (= delta delta GD-I/delta [urea]) for wild-type and several mutants is relatively insensitive to temperature, as would be expected for an intermediate that is formed cooperatively, rather than one that melts out according to a second-order transition. The phi-values for the formation of I change abruptly through the folding transitions rather than have the smooth changes expected for a second-order transition. There is a subset of mutants for which both mD-I and phi-value analysis indicate that a second intermediate becomes populated close to the melting temperatures of the native proteins. The folding intermediate of barnase is, thus, a relatively discrete and compact entity which is formed cooperatively.

Folding intermediates of wild-type and mutants of barnase. II. Correlation of changes in equilibrium amide exchange kinetics with the population of the folding intermediate.

There is an unanswered question from previous studies of 1H/2H-exchange of amide protons of barnase. Under certain conditions, there is a relatively abrupt change from EX2 towards EX1 kinetics as the temperature is slightly increased. The change in kinetics for different mutants is not directly related to their changes in stability. We have measured the stability of the folding intermediate of barnase (I) in 2H2O under a variety of conditions and calculated its population at different temperatures. The change in kinetics correlates with the change in the population of the folding intermediate. At higher temperatures and pH, the free energy of I becomes higher than that of the denatured state, D, and the kinetics becomes EX1. The data fit a simple kinetic scheme. Such changes in kinetics may be used to detect the presence of intermediates in the folding reaction at equilibrium in native conditions, but cannot distinguish whether they are on or off-pathway.

Movement of the intermediate and rate determining transition state of barnase on the energy landscape with changing temperature.

Barnase folds cooperatively via an intermediate, followed by a rate-limiting transition state. We have probed possible movements of the intermediate and transition state on the energy landscape with changing temperature, from the temperature dependence of phi-values. These measure interaction energies at the level of individual residues. The results suggest that single destabilizing mutations can redistribute the structures in each ensemble on the energy landscape as the temperature is varied. The results were also analyzed in terms of the bulk properties of each ensemble and their movements on the energy landscape. These movements can be described in terms of the "new view" or equivalently in terms of the classical "Hammond" or "anti-Hammond" effects, observed previously for the transition states of barnase at 7.25 M urea and chymotrypsin inhibitor 2 (CI2) at 0.3 and 6 M GdmCl. The results presented here are under more relevant physiological conditions, free of chemical denaturants. The "average" structures of the intermediate and the transition state do not appear to move on the energy landscape as the temperature is varied. However, there are small rearrangements in the major alpha-helix of the transition state, its average structure moving closer to the native state as the temperature is increased, in agreement with the Hammond effect observed previously.

Factorial structure of the Waterloo Handedness Questionnaire for control and learning-disabled adults.

This study examined the factorial structure of the 32-item version of the Waterloo Handedness Questionnaire (Steenhuis & Bryden, 1989) with a sample of control (n = 325) and learning-disabled (LD) (n = 147) subjects. On the basis of Principal Components Analyses, we have replicated the work of Steenhuis and Bryden (1989) who suggested that hand preference factors for control subjects are multifactorial in nature and are related more to "skilled" and "less skilled" activities rather than on distal/proximal musculature. Further, we have found that the factor structure for hand preference in LD adults is somewhat different than of normally achieving adults. The primary difference occurred within Factor 2. Unlike control subjects, whose Factor 2 items were classified as "less skilled" and preference was less lateralized, items loading on this factor for LD subjects were classified as "skilled" and showed strongly lateralized responses for both left and right handers. Presumably, LD adults show a less marked distinction between "skilled" and "less skilled" unimanual motor activities than control subjects suggesting different degrees of manual laterality between the groups.

Reproductive biology and postnatal development of the neotropical climbing rat, Tylomys.

Neotropical climbing rats (Tylomys), offspring of animals from Mexico and Panama, were maintained in the Michigan State University Museum-Live Animal Colony for the purpose of collecting data on reproduction. Vaginal smears indicated an estrous cycle 6.8 +/- 0.4 da under natural winter lighting, 12.0 +/- 2.5 da when exposed to a 12:12 light:dark cycle, and 4.3 +/- 0.3 da when utilizing an activity wheel and under a 14:10 lighting scheme. The earliest recorded mating for a female Tylomys was 90 da of age. The gestation period was 40.6 +/- 0.5 da, and there was no statistical difference between the gestation lengths of lactating and non-lactating females. The average litter size was 2.3 with a range of 1-4. Weight at birth averaged 20.2 +/- 0.3 g, or about 8% (litter about 16%) the weight of the adult. The daily instantaneous rate of growth (weight) for the first wk was about 5.6%. Graphs were presented indicating changes with time of weight, total body length, and tail, hind foot, and ear lengths. Young respond to loud noises at 6-14 da of age, and eye opening occurs slightly later, at 7-15 da of age. The young cling tenaciously to the female's nipples nearly continuously for the first 3 wk. Females did not perform any nest-building activities. Weaning occurred at approximately 3-4 wk of age.