Site-directed mutagenesis and kinetic studies of the West Nile virus NS3 protease identify key enzyme-substrate interactions

The flavivirus West Nile virus (WNV) has spread rapidly throughout the world in recent years causing fever, meningitis, encephalitis, and fatalities. Because the viral protease NS2B/NS3 is essential for replication, it is attracting attention as a potential therapeutic target, although there are currently no antiviral inhibitors for any flavivirus. This paper focuses on elucidating interactions between a hexapeptide substrate (Ae-KPGLKR-p-nitroanilide) and residues at S1 and S2 in the active site of WNV protease by comparing the catalytic activities of selected mutant recombinant proteases in vitro. Homology modeling enabled the predictions of key mutations in VWNV NS3 protease at S1 (V115A/F, D129A/ E/N, S135A, Y150A/F, S160A, and S163A) and S2 (N152A) that might influence substrate recognition and catalytic efficiency. Key conclusions are that the substrate P1 Arg strongly interacts with S1 residues Asp-129, Tyr-150, and Ser-163 and, to a lesser extent, Ser-160, and P2 Lys makes an essential interaction with Asn-152 at S2. The inferred substrate-enzyme interactions provide a basis for rational protease inhibitor design and optimization. High sequence conservation within flavivirus proteases means that this study may also be relevant to design of protease inhibitors for other flavivirus proteases.

Global processing speed as a mediator of developmental changes in children's auditory memory span

This study examined the role of global processing speed in mediating age increases in auditory memory span in 5- to 13-year-olds. Children were tested on measures of memory span, processing speed, single-word speech rate, phonological sensitivity, and vocabulary. Structural equation modeling supported a model in which age-associated increases in processing speed predicted the availability of long-term memory phonological representations for redintegration processes. The availability of long-term phonological representations, in turn, explained variance in memory span. Maximum speech rate did not predict independent variance in memory span. (c) 2005 Elsevier Inc. All rights reserved.

Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins

Failure to express soluble proteins in bacteria is mainly attributed to the properties of the target protein itself, as well as the choice of the vector, the purification tag and the linker between the tag and protein, and codon usage. The expression of proteins with fusion tags to facilitate subsequent purification steps is a widely used procedure in the production of recombinant proteins. However, the additional residues can affect the properties of the protein; therefore, it is often desirable to remove the tag after purification. This is usually done by engineering a cleavage site between the tag and the encoded protein that is recognised by a site-specific protease, such as the one from tobacco etch virus (TEV). In this study, we investigated the effect of four different tags on the bacterial expression and solubility of nine mouse proteins. Two of the four engineered constructs contained hexahistidine tags with either a long or short linker. The other two constructs contained a TEV cleavage site engineered into the linker region. Our data show that inclusion of the TEV recognition site directly downstream of the recombination site of the Invitrogen Gateway vector resulted, in a loss of solubility of the nine mouse proteins. Our work suggests that one needs to be very careful when making modifications to expression vectors and combining different affinity and fusion tags and cleavage sites: (c) 2006 Elsevier Inc. All rights reserved.

Deduced amino acid sequences surrounding the fusion glycoprotein cleavage site and of the carboxyl-terminus of haemagglutinin-neuraminidase protein of the avirulent thermostable vaccine strain I-2 of Newcastle disease virus

A single-tube RT-PCR technique generated a 387 bp or 300 bp cDNA amplicon covering the F-0 cleavage site or the carboxyl (C)-terminus of the HN gene, respectively, of Newcastle disease virus (NDV) strain 1-2. Sequence analysis was used to deduce the amino acid sequences of the cleavage site of F protein and the C-terminus of HN protein, which were then compared with sequences for other NDV strains. The cleavage site of NDV strain 1-2 had a sequence Motif of (112)RKQGRLIG(119), consistent with an avirulent phenotype. Nucleotide sequencing and deduction of amino acids at the C-terminus of HN revealed that strain 1-2 had a 7-amino-acid extension (VEILKDGVREARSSR). This differs from the virulent viruses that caused outbreaks of Newcastle disease in Australia in the 1930s and 1990s, which have HN extensions of 0 and 9 amino acids, respectively. Amino acid sequence analyses of the F and HN genes of strain 1-2 confirmed its avirulent nature and its Australian origin.

Liposomal cationic charge and antigen adsorption are important properties for the efficient deposition of antigen at the injection site and ability of the vaccine to induce a CMI response

With respect to liposomes as delivery vehicles and adjuvants for vaccine antigens, the role of vesicle surface charge remains disputed. In the present study we investigate the influence of liposome surface charge and antigen-liposome interaction on the antigen depot effect at the site of injection (SOI). The presence of liposome and antigen in tissue at the SOI as well as the draining lymphatic tissue was quantified to analyse the lymphatic draining of the vaccine components. Furthermore investigations detailing cytokine production and T-cell antigen specificity were undertaken to investigate the relationship between depot effect and the ability of the vaccine to induce an immune response. Our results suggest that cationic charge is an important factor for the retention of the liposomal component at the SOI, and a moderate to high (>50%) level of antigen adsorption to the cationic vesicle surface was required for efficient antigen retention in the same tissue. Furthermore, neutral liposomes expressing poor levels of antigen retention were limited in their ability to mediate long term (14 days) antigen presentation to circulating antigen specific T-cells and to induce the Th1 and Th17 arms of the immune system, as compared to antigen adsorbing cationic liposomes. The neutral liposomes did however induce the production of IL-5 at levels comparable to those induced by cationic liposomes, indicating that neutral liposomes can induce a weak Th2 response.

Investigations of the site and mechanisms of drug-induced reductions in the reabsorption of divalent cations by the kidney

Disturbances in electrolyte homeostasis are a frequent adverse side-effect of the administration of aminoglycoside antibiotics such as gentamicin, and the antineoplastic agent cis-platinum. The aims of this work were to further elucidate the site(s) and mechanism(s) by which these drugs may produce disturbances in the renal reabsorption of calcium and magnesium. These investigations were undertaken using a range of in vivo and in vitro techniques and models. Initially, a series of in vivo studies was conducted to delineate aspects of the acute and chronic effects of both drugs on renal electrolyte handling and to select and evaluate an appropriate animal model: subsequent investigations were focused on gentamicin. In a study of the acute and chronic effects of cis-platinum administration, there were pronounced acute changes in a variety of indices of nephrotoxic injury, including electrolyte excretion. Most effects resolved but there were chronic increases in the urinary excretion of calcium and magnesium. The renal response of three strains of rat (Fischer 344, Sprague-Dawley (SD), and Wistar) to a ranges of doses of gentamicin was also investigated. Drug administration produced substantially different responses between strains, in particular marked differences in calcium and magnesium excretion. The results suggested that the SD rat was an appropriately sensitive strain for use in further investigations. Acute infusion of gentamicin in the anaesthetised SD rat produced rapid, substantial increases in the fractional excretion of calcium and magnesium, while sodium and potassium output were unaffected, confirming previous results of similar experiments using F344 rats. Studies using lithium clearance measurements in the anaesthetised SD rat were undertaken to investigate the effects of gentamicin on proximal tubular calcium reabsorption. Lithium clearance was unaffected by acute gentamicin infusion, suggesting that the site of acute gentamicin-induced hypercalciuria may not be located in the proximal tubule. Inhibition of Ca2+ ATPase activity was investigated as a potential mechanism by which calcium reabsorption could be affected after aminoglycoside administration. In vitro, both Ca2+ ATPase and Na+/K+ ATPase activity could be similarly inhibited by the presence of aminoglycosides, in a dose-related manner. Whilst inhibition of Na+/K+ ATPase could be demonstrated biochemically after in vivo administration of gentamicin, there were no concurrent effects on Ca2+ ATPase activity, suggesting that inhibition of Ca2+ ATPase activity is unlikely to be a primary mechanism of aminoglycoside-induced reductions of calcium reabsorption. Histochemical studies could not discern inhibition of either Na+/K+ ATPase or Ca2+ ATPase activity after in vivo administration of gentamicin. Selection of renal cell lines for further investigative in vitro studies on the mechanisms of altered cation reabsorption was considered using MTT (3-(4,5,-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Neutral Red cytotoxicity assays. The ability of LLC-PK1 and LLC-RK1 cell lines to correctly rank a series of nephrotoxic compounds with their known nephrotoxic potency in vivo was studied. Using these cell lines grown on semi-permeable inserts, alterations in the paracellular transport of 45Ca was investigated as a possible mechanism by which gentamicin could alter calcium reabsorption in vivo. Short term exposure (I h) of LLC-RK1 cells to gentamicin, via both cell surfaces, resulted in a reduction in paracellular permeability to both transepithelial 3H-mannitol and 45Ca fluxes. When LLC-RK1 cells were exposed via the apical surface only, similar dose-related reductions were seen to those observed when cells were exposed to the drug from both sides. Short-term basal exposure to gentamicin appeared to contribute less to the observed reductions in 3H-mannitol and 45Ca fluxes. Experiments investigating transepithelial movement of 45Ca and 3H-mannitol on LLC-PK1 cells after acute gentamicin exposure were inconclusive. Longer exposure (48 h) to gentamicin caused an increase in the permeability of the monolayer and a consequent increase in transepithelial 45Ca flux in the LLC-RK1 cell line; increases in permeability of LLC-PK1 cells to 45Ca and 3H-mannitol were not apparent under the same conditions. The site and mechanism at which gentamicin, in particular, alters calcium reabsorption cannot be definitively described from these studies. However, indirect evidence from lithium clearance studies suggests that the site of the lesion is unlikely to be located in the proximal tubule. The mechanism by which gentamicin exposure alters calcium reabsorption may be by reducing paracellular permeability to calcium rather than by altering active calcium transport processes.

Manipulation of the surface pegylation in combination with reduced vesicle size of cationic liposomal adjuvants modifies their clearance kinetics from the injection site, and the rate and type of T cell response

The mechanism behind the immunostimulatory effect of the cationic liposomal vaccine adjuvant dimethyldioctadecylammonium and trehalose 6,6′- dibehenate (DDA:TDB) has been linked to the ability of these cationic vesicles to promote a depot after administration, with the liposomal adjuvant and the antigen both being retained at the injection site. This can be attributed to their cationic nature, since reduction in vesicle size does not influence their distribution profile yet neutral or anionic liposomes have more rapid clearance rates. Therefore the aim of this study was to investigate the impact of a combination of reduced vesicle size and surface pegylation on the biodistribution and adjuvanticity of the formulations, in a bid to further manipulate the pharmacokinetic profiles of these adjuvants. From the biodistribution studies, it was found that with small unilamellar vesicles (SUVs), 10% PEGylation of the formulation could influence liposome retention at the injection site after 4 days, whilst higher levels (25 mol%) of PEG blocked the formation of a depot and promote clearance to the draining lymph nodes. Interestingly, whilst the use of 10% PEG in the small unilamellar vesicles did not block the formation of a depot at the site of injection, it did result in earlier antibody response rates and switch the type of T cell responses from a Th1 to a Th2 bias suggesting that the presence of PEG in the formulation not only control the biodistribution of the vaccine, but also results in different types of interactions with innate immune cells. © 2012 Elsevier B.V.

Pleasure, pain and procrastination:reflections on the experience of doing memory-work research

The focus of this article is the process of doing memory-work research. We tell the story of our experience of what it was like to use this approach. We were enthused to work collectively on a "discovery" project to explore a method with which we were unfamiliar. We hoped to build working relationships based on mutual respect and the desire to focus on methodology and its place in our psychological understanding. The empirical activities highlighted methodological and experiential challenges, which tested our adherence to the social constructionist premise of Haug's original description of memory work. Combined with practical difficulties of living across Europe, writing and analyzing the memories became contentious. We found ourselves having to address a number of tensions emanating from the work and our approach to it. We discuss some of these tensions alongside examples that illustrate the research process and the ways we negotiated the collective nature of the memory-work approach. © 2012 Copyright Taylor and Francis Group, LLC.

What checkers actually check:an eye tracking study of inhibitory control and working memory

Background - Not only is compulsive checking the most common symptom in Obsessive Compulsive Disorder (OCD) with an estimated prevalence of 50–80% in patients, but approximately ~15% of the general population reveal subclinical checking tendencies that impact negatively on their performance in daily activities. Therefore, it is critical to understand how checking affects attention and memory in clinical as well as subclinical checkers. Eye fixations are commonly used as indicators for the distribution of attention but research in OCD has revealed mixed results at best. Methodology/Principal Finding - Here we report atypical eye movement patterns in subclinical checkers during an ecologically valid working memory (WM) manipulation. Our key manipulation was to present an intermediate probe during the delay period of the memory task, explicitly asking for the location of a letter, which, however, had not been part of the encoding set (i.e., misleading participants). Using eye movement measures we now provide evidence that high checkers’ inhibitory impairments for misleading information results in them checking the contents of WM in an atypical manner. Checkers fixate more often and for longer when misleading information is presented than non-checkers. Specifically, checkers spend more time checking stimulus locations as well as locations that had actually been empty during encoding. Conclusions/Significance - We conclude that these atypical eye movement patterns directly reflect internal checking of memory contents and we discuss the implications of our findings for the interpretation of behavioural and neuropsychological data. In addition our results highlight the importance of ecologically valid methodology for revealing the impact of detrimental attention and memory checking on eye movement patterns.

The role of working memory in compulsive checking and OCD:a systematic classification of 58 experimental findings

Despite the large body of research regarding the role of memory in OCD, the results are described as mixed at best (Hermans et al., 2008). For example, inconsistent findings have been reported with respect to basic capacity, intact verbal, and generally affected visuospatial memory. We suggest that this is due to the traditional pursuit of OCD memory impairment as one of the general capacity and/or domain specificity (visuospatial vs. verbal). In contrast, we conclude from our experiments (i.e., Harkin & Kessler, 2009, 2011; Harkin, Rutherford, & Kessler, 2011) and recent literature (e.g., Greisberg & McKay, 2003) that OCD memory impairment is secondary to executive dysfunction, and more specifically we identify three common factors (EBL: Executive-functioning efficiency, Binding complexity, and memory Load) that we generalize to 58 experimental findings from 46 OCD memory studies. As a result we explain otherwise inconsistent research – e.g., intact vs. deficient verbal memory – that are difficult to reconcile within a capacity or domain specific perspective. We conclude by discussing the relationship between our account and others', which in most cases is complementary rather than contradictory.

Theorizing accounting and other [calculative] practices in private equity:Experimenting with Schatzki’s ‘site’ ontology

This thesis contributes to social studies of finance and accounting (Vollmer, Mennicken, & Preda, 2009) and the practice theory literatures (Feldman & Orlikowski, 2011) by experimenting (Baxter & Chua, 2008) with concepts developed by Theodore Schatzki and demonstrating their relevance and usefulness in theorizing and explaining accounting and other organizational phenomena. Influenced by Schatzki, I have undertaken a sociological investigation of the practices, arrangements, and nexuses forming (part of) the social ‘site’ of private equity (PE). I have examined and explained the organization of practices within the PE industry. More specifically, I have sought to throw light on the practice organizations animating various PE practices. I have problematized a particular aspect of Schatzki’s practice organization framework: ‘general understanding’, which has so far been poorly understood and taken for granted in the accounting literature. I have tried to further explore the concept to clarify important definitional issues surrounding its empirical application. In investigating the forms of accounting and control practices in PE firms and how they link with other practices forming part of the ‘site’, I have sought to explain how the ‘situated functionality’ of accounting is ‘prefigured’ by its ‘dispersed’ nature. In doing so, this thesis addresses the recent calls for research on accounting and control practices within financial services firms. This thesis contributes to the social studies of finance and accounting literature also by opening the blackbox of investment [e]valuation practices prevalent in the PE industry. I theorize the due diligence of PE funds as a complex of linked calculative practices and bring to fore the important aspects of ‘practical intelligibility’ of the investment professionals undertaking investment evaluation. I also identify and differentiate the ‘causal’ and ‘prefigurational’ relations between investment evaluation practices and the material entities ‘constituting’ those practices. Moreover, I demonstrate the role of practice memory in those practices. Finally, the thesis also contributes to the practice theory literature by identifying and attempting to clarify and/or improve the poorly defined and/or underdeveloped concepts of Schatzki’s ‘site’ ontology framework.

Consistency and accuracy in memory: Effects of schema-typicality, retention interval and question-format

This study examined the effect of schemas on consistency and accuracy of memory across interviews, providing theoretical hypotheses explaining why inconsistencies may occur. The design manipulated schema-typicality of items (schema-typical and atypical), question format (free-recall, cued-recall and recognition) and retention interval (immediate/2 week and 2 week/4 week). Consistency, accuracy and experiential quality of memory were measured. ^ All independent variables affected accuracy and experiential quality of memory while question format was the only variable affecting consistency. These results challenge the commonly held notion in the legal arena that consistency is a proxy for accuracy. The study also demonstrates that other variables, such as item-typicality and retention interval have different effects on consistency and accuracy in memory. ^

Eyewitness memory: Balancing the accuracy, precision, and quantity of information through metacognitive monitoring and control

Historically, memory has been evaluated by examining how much is remembered, however a more recent conception of memory focuses on the accuracy of memories. When using this accuracy-oriented conception of memory, unlike with the quantity-oriented approach, memory does not always deteriorate over time. A possible explanation for this seemingly surprising finding lies in the metacognitive processes of monitoring and control. Use of these processes allows people to withhold responses of which they are unsure, or to adjust the precision of responses to a level that is broad enough to be correct. The ability to accurately report memories has implications for investigators who interview witnesses to crimes, and those who evaluate witness testimony. ^ This research examined the amount of information provided, accuracy, and precision of responses provided during immediate and delayed interviews about a videotaped mock crime. The interview format was manipulated such that a single free narrative response was elicited, or a series of either yes/no or cued questions were asked. Instructions provided by the interviewer indicated to the participants that they should either stress being informative, or being accurate. The interviews were then transcribed and scored. ^ Results indicate that accuracy rates remained stable and high after a one week delay. Compared to those interviewed immediately, after a delay participants provided less information and responses that were less precise. Participants in the free narrative condition were the most accurate. Participants in the cued questions condition provided the most precise responses. Participants in the yes/no questions condition were most likely to say “I don’t know”. The results indicate that people are able to monitor their memories and modify their reports to maintain high accuracy. When control over precision was not possible, such as in the yes/no condition, people said “I don’t know” to maintain accuracy. However when withholding responses and adjusting precision were both possible, people utilized both methods. It seems that concerns that memories reported after a long retention interval might be inaccurate are unfounded. ^

Effect of delay of first interview and repetition of interview on accuracy and confidence of recall of a flashbulb -type memory

Hearing of the news of the death of Diana, Princess of Wales, in a traffic accident, is taken as an analogue for being a percipient but uninvolved witness to a crime, or a witness to another person's sudden confession to some illegal act. This event (known in the literature as a “reception event”) has previously been hypothesized to cause one to form a special type of memory commonly known as a “flashbulb memory” (FB) (Brown and Kulik, 1977). FB's are hypothesized to be especially resilient against forgetting, highly detailed including peripheral details, clear, and inspiring great confidence in the individual for their accuracy. FB's are dependent for their formation upon surprise, emotional valence, and impact, or consequentiality to the witness of the initiating event. FB's are thought to be enhanced by frequent rehearsal. FB's are very important in the context of criminal investigation and litigation in that investigators and jurors usually place great store in witnesses, regardless of their actual accuracy, who claim to have a clear and complete recollection of an event, and who express this confidently. Therefore, the lives, or at least the freedom, of criminal defendants, and the fortunes of civil litigants hang on the testimony of witnesses professing to have FB's. ^ In this study, which includes a large and diverse sample (N = 305), participants were surveyed within 2–4 days after hearing of the fatal accident, and again at intervals of 2 and 4 weeks, 6, 12, and 18 months. Contrary to the FB hypothesis, I found that participants' FB's degraded over time beginning at least as early as two weeks post event. At about 12 months the memory trace stabilized, resisting further degradation. Repeated interviewing did not have any negative affect upon accuracy, contrary to concerns in the literature. Analysis by correlation and regression indicated no effect or predictive power for participant age, emotionality, confidence, or student status, as related to accuracy of recall; nor was participant confidence in accuracy predicted by emotional impact as hypothesized. Results also indicate that, contrary to the notions of investigators and jurors, witnesses become more inaccurate over time regardless of their confidence in their memories, even for highly emotional events. ^

The presence of a non-B-Z-structure enhances an enzyme's reactivity at its recognition site

Alternating (CG) sequences form an unusual conformation in the presence of cobalt hexamine. The oligomer, BZ-IV, containing a (CG)4 run (BZ-IV sequence: 5'TCGACGCGCGCGATCAGTCA- 3') was inserted at the Sal I site of the Escherichia coli pGEM-5zf(+) plasmid producing the plasmid pCW001. Hinf I digestion of pCW001 produced a 367 base pair (bp) fragment containing the BZ-IV insert. For controls, the 452 bp Hinf I fragment from the pCW001 plasmid and the 347 bp Hinf I fragment from the pGEM plasmid were used. Digestion studies were performed using the restriction enzymes Bgl I, EcoRV, Hha I, Mbo I, Not I, Pst I, and Taq I and methylation studies were performed using dam methylase. Data were obtained by beta scanning or ethidium bromide staining the polyacrylamide gels of the digestion or methylation products. The results show that in the presence of 100 uM cobalt hexamine, in which BZ-IV takes on a non-B-Z-structure, the enzyme's ability to react and cleave its recognition site is enhanced.