The integration of ProxiMAX randomisation with CIS display for the production of novel peptides

Saturation mutagenesis is a powerful tool in modern protein engineering, which permits key residues within a protein to be targeted in order to potentially enhance specific functionalities. However, the creation of large libraries using conventional saturation mutagenesis with degenerate codons (NNN or NNK/S) has inherent redundancy and consequent disparities in codon representation. Therefore, both chemical (trinucleotide phosphoramidites) and biological methods (sequential, enzymatic single codon additions) of non-degenerate saturation mutagenesis have been developed in order to combat these issues and so improve library quality. Large libraries with multiple saturated positions can be limited by the method used to screen them. Although the traditional screening method of choice, cell-dependent methods, such as phage display, are limited by the need for transformation. A number of cell-free screening methods, such as CIS display, which link the screened phenotype with the encoded genotype, have the capability of screening libraries with up to 1014 members. This thesis describes the further development of ProxiMAX technology to reduce library codon bias and its integration with CIS display to screen the resulting library. Synthetic MAX oligonucleotides are ligated to an acceptor base sequence, amplified, and digested, subsequently adding a randomised codon to the acceptor, which forms an iterative cycle using the digested product of the previous cycle as the base sequence for the next. Initial use of ProxiMAX highlighted areas of the process where changes could be implemented in order to improve the codon representation in the final library. The refined process was used to construct a monomeric anti-NGF peptide library, based on two proprietary dimeric peptides (Isogenica) that bind NGF. The resulting library showed greatly improved codon representation that equated to a theoretical diversity of ~69%. The library was subsequently screened using CIS display and the discovered peptides assessed for NGF-TrkA inhibition by ELISA. Despite binding to TrkA, these peptides showed lower levels of inhibition of the NGF-TrkA interaction than the parental dimeric peptides, highlighting the importance of dimerization for inhibition of NGF-TrkA binding.

Cascaded Bayesian processes: an account of bias in orientation perception

Following adaptation to an oriented (1-d) signal in central vision, the orientation of subsequently viewed test signals may appear repelled away from or attracted towards the adapting orientation. Small angular differences between the adaptor and test yield 'repulsive' shifts, while large angular differences yield 'attractive' shifts. In peripheral vision, however, both small and large angular differences yield repulsive shifts. To account for these tilt after-effects (TAEs), a cascaded model of orientation estimation that is optimized using hierarchical Bayesian methods is proposed. The model accounts for orientation bias through adaptation-induced losses in information that arise because of signal uncertainties and neural constraints placed upon the propagation of visual information. Repulsive (direct) TAEs arise at early stages of visual processing from adaptation of orientation-selective units with peak sensitivity at the orientation of the adaptor (theta). Attractive (indirect) TAEs result from adaptation of second-stage units with peak sensitivity at theta and theta+90 degrees , which arise from an efficient stage of linear compression that pools across the responses of the first-stage orientation-selective units. A spatial orientation vector is estimated from the transformed oriented unit responses. The change from attractive to repulsive TAEs in peripheral vision can be explained by the differing harmonic biases resulting from constraints on signal power (in central vision) versus signal uncertainties in orientation (in peripheral vision). The proposed model is consistent with recent work by computational neuroscientists in supposing that visual bias reflects the adjustment of a rational system in the light of uncertain signals and system constraints.

Removing the redundancy from randomised gene libraries

Amino acid substitution plays a vital role in both the molecular engineering of proteins and analysis of structure-activity relationships. High-throughput substitution is achieved by codon randomisation, which generates a library of mutants (a randomised gene library) in a single experiment. For full randomisation, key codons are typically replaced with NNN (64 sequences) or NNG CorT (32 sequences). This obligates cloning of redundant codons alongside those required to encode the 20 amino acids. As the number of randomised codons increases, there is therefore a progressive loss of randomisation efficiency; the number of genes required per protein rises exponentially. The redundant codons cause amino acids to be represented unevenly; for example, methionine is encoded just once within NNN, whilst arginine is encoded six times. Finally, the organisation of the genetic code makes it impossible to encode functional subsets of amino acids (e.g. polar residues only) in a single experiment. Here, we present a novel solution to randomisation where genetic redundancy is eliminated; the number of different genes equals the number of encoded proteins, regardless of codon number. There is no inherent amino acid bias and any required subset of amino acids may be encoded in one experiment. This generic approach should be widely applicable in studies involving randomisation of proteins. © 2003 Elsevier Ltd. All rights reserved.

A new randomization assay reveals Unexpected elements of sequence bias in model 'randomized' gene libraries:Implications for biopanning

Although techniques such as biopanning rely heavily upon the screening of randomized gene libraries, there is surprisingly little information available on the construction of those libraries. In general, it is based on the cloning of 'randomized' synthetic oligonucleotides, in which given position(s) contain an equal mixture of all four bases. Yet, many supposedly 'randomized' libraries contain significant elements of bias and/or omission. Here, we report the development and validation of a new, PCR-based assay that enables rapid examination of library composition both prior to and after cloning. By using our assay to analyse model libraries, we demonstrate that the cloning of a given distribution of sequences does not necessarily result in a similarly composed library of clones. Thus, while bias in randomized synthetic oligonucleotide mixtures can be virtually eliminated by using unequal ratios of the four phosphoramidites, the use of such mixtures does not ensure retrieval of a truly randomized library. We propose that in the absence of a technique to control cloning frequencies, the ability to analyse the composition of libraries after cloning will enhance significantly the quality of information derived from those libraries. (C) 2000 Published by Elsevier Science B.V. All rights reserved.

Optic flow in human vision: MEG reveals a foveo-fugal bias in V1, specialization for spiral space in hMSTs, and global motion sensitivity in the IPS

Abstract We recorded MEG responses from 17 participants viewing random-dot patterns simulating global optic flow components (expansion, contraction, rotation, deformation, and translation) and a random motion control condition. Theta-band (3–7 Hz), MEG signal power was greater for expansion than the other optic flow components in a region concentrated along the calcarine sulcus, indicating an ecologically valid, foveo-fugal bias for unidirectional motion sensors in V1. When the responses to the optic flow components were combined, a decrease in MEG beta-band (17–23 Hz) power was found in regions extending beyond the calcarine sulcus to the posterior parietal lobe (inferior to IPS), indicating the importance of structured motion in this region. However, only one cortical area, within or near the V5/hMT+ complex, responded to all three spiral-space components (expansion, contraction, and rotation) and showed no selectivity for global translation or deformation: we term this area hMSTs. This is the first demonstration of an exclusive region for spiral space in the human brain and suggests a functional role better suited to preliminary analysis of ego-motion than surface pose, which would involve deformation. We also observed that the rotation condition activated the cerebellum, suggesting its involvement in visually mediated control of postural adjustment.

Morphology in autism spectrum disorders:local processing bias and language

We conducted a detailed study of a case of linguistic talent in the context of autism spectrum disorder, specifically Asperger syndrome. I.A. displays language strengths at the level of morphology and syntax. Yet, despite this grammar advantage, processing of figurative language and inferencing based on context presents a problem for him. The morphology advantage for I.A. is consistent with the weak central coherence (WCC) account of autism. From this account, the presence of a local processing bias is evident in the ways in which autistic individuals solve common problems, such as assessing similarities between objects and finding common patterns, and may therefore provide an advantage in some cognitive tasks compared to typical individuals. We extend the WCC account to language and provide evidence for a connection between the local processing bias and the acquisition of morphology and grammar.

Development of the MAX randomisation technique

During the last decade the use of randomised gene libraries has had an enormous impact in the field of protein engineering. Such libraries comprise many variations of a single gene in which codon replacements are used to substitute key residues of the encoded protein. The expression of such libraries generates a library of randomised proteins which can subsequently be screened for desired or novel activities. Randomisation in this fashion has predominantly been achieved by the inclusion of the codons NNN or NNGCor T, in which N represents any of the four bases A,C,G, or T. The use of thesis codons however, necessities the cloning of redundant codons at each position of randomisation, in addition to those required to encode the twenty possible amino acid substitutions. As degenerate codons must be included at each position of randomisation, this results in a progressive loss of randomisation efficiency as the number of randomised positions is increased. The ratio of genes to proteins in these libraries rises exponentially with each position of randomisation, creating large gene libraries, which generate protein libraries of limited diversity upon expression. In addition to these problems of library size, the cloning of redundant codons also results in the generation of protein libraries in which substituted amino acids are unevenly represented. As several of the randomised codons may encode the same amino acid, for example serine which is encoded six time using the codon NNN, an inherent bias may be introduced into the resulting protein library during the randomisation procedure. The work outlined here describes the development of a novel randomisation technique aimed at a eliminating codon redundancy from randomised gene libraries, thus addressing the problems of library size and bias, associated with the cloning of redundant codons.

A demonstration of attentional bias, using a novel dual task paradigm, towards clinically salient material in recovering alcohol abuse patients?

Background. This study examined whether alcohol abuse patients are characterized either by enhanced schematic processing of alcohol related cues or by an attentional bias towards the processing of alcohol cues. Method. Abstinent alcohol abusers (N = 25) and non-clinical control participants (N = 24) performed a dual task paradigm in which they had to make an odd/even decision to a centrally presented number while performing a peripherally presented lexical decision task. Stimuli on the lexical decision task comprised alcohol words, neutral words and non-words. In addition, participants completed an incidental recall task for the words presented in the lexical decision task. Results. It was found that, in the presence of alcohol related words, the performance of patients on the odd/even decision task was poorer than in the presence of other stimului. In addition, patients displayed slower lexical decision times for alcohol related words. Both groups displayed better recall for alcohol words than for other stimuli. Conclusions. These results are interpreted as supporting neither model of drug cravings. Rather, it is proposed that, in the presence of alcohol stimuli, alcohol abuse patients display a breakdown in the ability to focus attention.

Techniques for determining bias and stability in satellite altimeters

The case for monitoring large-scale sea level variability is established in the context of the estimation of the extent of anthropogenic climate change. Satellite altimeters are identified as having the potential to monitor this change with high resolution and accuracy. Possible sources of systematic errors and instabilities in these instruments which would be hurdles to the most accurate monitoring of such ocean signals are examined. Techniques for employing tide gauges to combat such inaccuracies are proposed and developed. The tide gauge at Newhaven in Sussex is used in conjunction with the nearby satellite laser ranger and high-resolution ocean models to estimate the absolute bias of the TOPEX, Poseidon, ERS 1 and ERS 2 altimeters. The theory which underlies the augmentation of altimeter measurements with tide gauge data is developed. In order to apply this, the tide gauges of the World Ocean Circulation Experiment are assessed and their suitability for altimeter calibration is determined. A reliable subset of these gauges is derived. A method of intra-altimeter calibration is developed using these tide gauges to remove the effect of variability over long time scales. In this way the long-term instability in the TOPEX range measurement is inferred and the drift arising from the on-board ultra stable oscillator is thus detected. An extension to this work develops a method for inter-altimeter calibration, allowing the systematic differences between unconnected altimeters to be measured. This is applied to the TOPEX and ERS 1 altimeters.

Improving recombinant eukaryotic membrane protein yields in Pichia pastoris:the importance of codon optimization and clone selection

In the last 15 years, 80% of all recombinant proteins reported in the literature were produced in the bacterium, Escherichia coli, or the yeast, Pichia pastoris. Nonetheless, developing effective general strategies for producing recombinant eukaryotic membrane proteins in these organisms remains a particular challenge. Using a validated screening procedure together with accurate yield quantitation, we therefore wished to establish the critical steps contributing to high yields of recombinant eukaryotic membrane protein in P. pastoris. Whilst the use of fusion partners to generate chimeric constructs and directed mutagenesis have previously been shown to be effective in bacterial hosts, we conclude that this approach is not transferable to yeast. Rather, codon optimization and the preparation and selection of high-yielding P. pastoris clones are effective strategies for maximizing yields of human aquaporins.

Memory bias for emotional facial expressions in major depression

Sixteen clinically depressed patients and sixteen healthy controls were presented with a set of emotional facial expressions and were asked to identify the emotion portrayed by each face. They, were subsequently given a recognition memory test for these faces. There was no difference between the groups in terms of their ability to identify emotion between from faces. All participants identified emotional expressions more accurately than neutral expressions, with happy expressions being identified most accurately. During the recognition memory phase the depressed patients demonstrated superior memory for sad expressions, and inferior memory for happy expressions, relative to neutral expressions. Conversely, the controls demonstrated superior memory for happy expressions, and inferior memory for sad expressions, relative to neutral expressions. These results are discussed in terms of the cognitive model of depression proposed by Williams, Watts, MacLeod, and Mathews (1997).

A working memory bias for alcohol-related stimuli depends on drinking score

We tested 44 participants with respect to their working memory (WM) performance on alcohol-related versus neutral visual stimuli. Previously an alcohol attentional bias (AAB) had been reported using these stimuli, where the attention of frequent drinkers was automatically drawn toward alcohol-related items (e.g., beer bottle). The present study set out to provide evidence for an alcohol memory bias (AMB) that would persist over longer time-scales than the AAB. The WM task we used required memorizing 4 stimuli in their correct locations and a visual interference task was administered during a 4-sec delay interval. A subsequent probe required participants to indicate whether a stimulus was shown in the correct or incorrect location. For each participant we calculated a drinking score based on 3 items derived from the Alcohol Use Questionnaire, and we observed that higher scorers better remembered alcohol-related images compared with lower scorers, particularly when these were presented in their correct locations upon recall. This provides first evidence for an AMB. It is important to highlight that this effect persisted over a 4-sec delay period including a visual interference task that erased iconic memories and diverted attention away from the encoded items, thus the AMB cannot be reduced to the previously reported AAB. Our finding calls for further investigation of alcohol-related cognitive biases in WM, and we propose a preliminary model that may guide future research. © 2012 American Psychological Association.

Genetic modulation of the response bias towards facial displays of anger and happiness

Background: Investigating genetic modulation of emotion processing may contribute to the understanding of heritable mechanisms of emotional disorders. The aim of the present study was to test the effects of catechol- O-methyltransferase (COMT) val158met and serotonin-transporter-linked promoter region (5-HTTLPR) polymorphisms on facial emotion processing in healthy individuals. Methods: Two hundred and seventy five (167 female) participants were asked to complete a computerized facial affect recognition task, which involved four experimental conditions, each containing one type of emotional face (fearful, angry, sad or happy) intermixed with neutral faces. Participants were asked to indicate whether the face displayed an emotion or was neutral. The COMT-val158met and 5-HTTLPR polymorphisms were genotyped. Results: Met homozygotes (COMT) showed a stronger bias to perceive neutral faces as expressions of anger, compared with val homozygotes. However, the S-homozygotes (5-HTTLPR) showed a reduced bias to perceive neutral faces as expressions of happiness, compared to L-homozygotes. No interaction between 5-HTTLPR and COMT was found. Conclusions: These results add to the knowledge of individual differences in social cognition that are modulated via serotonergic and dopaminergic systems. This potentially could contribute to the understanding of the mechanisms of susceptibility to emotional disorders. © 2013 Elsevier Masson SAS.