LacI-mediated sequence-specific affinity purification of plasmid DNA for therapeutic applications

Affinity purification of plasmid DNA is an attractive option for the biomanufacture of therapeutic plasmids, which are strictly controlled for levels of host protein, DNA, RNA, and endotoxin. Plasmid vectors are considered to be a safer alternative than viruses for gene therapy, but milligram quantities of DNA are required per dose. Previous affinity approaches have involved triplex DNA formation and a sequence-specific zinc finger protein. We present a more generically applicable protein-based approach, which exploits the lac operator, present in a wide diversity of plasmids, as a target sequence. We used a GFP/His-tagged Lacl protein, which is precomplexed with the plasmid, and the resulting complex was immobilized on a solid support (TALON resin). Ensuing elution gives plasmid DNA, in good yield (>80% based on recovered starting material, 35-50% overall process), free from detectable RNA and protein and with minimal genomic DNA contamination. Such an affinity-based process should enhance plasmid purity and ultimately, after appropriate development, may simplify the biomanufacturing process of therapeutic plasmids.

Sequential grouping of pure-tone percepts evoked by the segregation of components from a complex tone

A sudden change applied to a single component can cause its segregation from an ongoing complex tone as a pure-tone-like percept. Three experiments examined whether such pure-tone-like percepts are organized into streams by extending the research of Bregman and Rudnicky (1975). Those authors found that listeners struggled to identify the presentation order of 2 pure-tone targets of different frequency when they were flanked by 2 lower frequency “distractors.” Adding a series of matched-frequency “captor” tones, however, improved performance by pulling the distractors into a separate stream from the targets. In the current study, sequences of discrete pure tones were substituted by sequences of brief changes applied to an otherwise constant 1.2-s complex tone. Pure-tone-like percepts were evoked by applying 6-dB increments to individual components of a complex comprising harmonics 1–7 of 300 Hz (Experiment 1) or 0.5-ms changes in interaural time difference to individual components of a log-spaced complex (range 160–905 Hz; Experiment 2). Results were consistent with the earlier study, providing clear evidence that pure-tone-like percepts are organized into streams. Experiment 3 adapted Experiment 1 by presenting a global amplitude increment either synchronous with, or just after, the last captor prior to the 1st distractor. In the former case, for which there was no pure-tone-like percept corresponding to that captor, the captor sequence did not aid performance to the same extent as previously. It is concluded that this change to the captor-tone stream partially resets the stream-formation process, and so the distractors and targets became likely to integrate once more. (PsycINFO Database Record (c) 2011 APA, all rights reserved)

The stratigraphy of the Fishguard volcanic complex and associated sediments, SW Dyfed, Wales

This thesis describes the geology of a Lower Palaeozoic terrain, situated west of the town of Fishguard, SW Dyfed, Wales. The area is dominated by the Fishguard Volcanic Complex (Upper Llanvirn), and sediments that range in age from the Middle Cambrian to the Lower Llandeilo. The successions represent an insight into sedimentation and volcanism for c. 100 Ma. along the south-western margin of the Lower Palaeozoic Welsh Basin. The stratigraphy of the sedimentary sequence has been completely revised and the existing volcanostratigraphy modified. The observed complexity of the stratigraphy is primarily the consequence of Caldedonide deformation which resulted in large scale repetition. Fold-thrust tectonics dominates the structural style of the area. Caledonide trending (NE-SW) cross-faults complicate preexisting structures. Middle Cambrian (?) sedimentation is documented by shallow marine clastics and red shales deposited within tidal - subtidal environments. Upper Cambrian sedimentation was dominated by shallow marine `storm' and `fair weather' sedimentation within a muddy shelf environment. Shallow marine conglomerates and heterolithic intertidal siliciclastics mark the onset of Ordovician sedimentation during the lower Arenig transgression. Mid-Arenig sediments reflect deposits influenced by storm, fair-weather and wave related processes in various shallow marine environments, including; shoreface, inner shelf, shoaling bar, and deltaic. Graptolitic marine shales were deposited from the upper mid-Arenig through to the lower Llandeilo; during which time sediments accumulated by pelagic processes and fine grained turbidites. The varied nature of sedimentation reflects both localised change within the depositional system and the influence of larger regional eustatic events. Ordovician subaqueous volcanic activity produced thick accumulations of lavas, pyroclastics, hydroclastics, and hyaloclastics. The majority of volcanism was effusive in nature, erupted below the Pressure Compensation Level. Basaltic volcanism was characterised by pillowed lavas and tube networks, whilst sheet-flow lavas, pillow breccias and minor hyaloclastites developed locally. Silicic volcanism was dominated by rhyolitic clastics of various affinities, although coherent silicic obsidian lavas, sheet-flow lavas and pyroclastics developed. Hypabyssal intrusives of variable composition and habit occur throughout the volcanic successions. Low-grade regional metamorphism has variably affected the area, conditions of the prehnite-pumpellyite and greenschist facies having been attained. Numerous secondary phases developed in response to the conditions imposed, which collectively indicate that P-T conditions were of low-pressure facies series in the range P= 1.2-2.0 kbars and T= 230-350oC, under an elevated geothermal gradient of 40-45oC km-1. Polymineralic cataclastites associated with Caledonide deformation indicate that tectonism and metamorphism were in part contemporaneous.

Build-up and resetting of auditory stream segregation in quiet and in complex-tone backgrounds

Thirteen experiments investigated the dynamics of stream segregation. Experiments 1-6b used a similar method, where a same-frequency induction sequence (usually 10 repetitions of an identical pure tone) promoted segregation in a subsequent, briefer test sequence (of alternating low- and high-frequency tones). Experiments 1-2 measured streaming using a direct report of perception and a temporal-discrimination task, respectively. Creating a single deviant by altering the final inducer (e.g. in level or replacement with silence) reduced segregation, often substantially. As the prior inducers remained unaltered, it is proposed that the single change actively reset build-up. The extent of resetting varied gradually with the size of a frequency change, once noticeable (experiments 3a-3b). By manipulating the serial position of a change, experiments 4a-4b demonstrated that resetting only occurred when the final inducer was replaced with silence, as build-up is very rapid during a same-frequency induction sequence. Therefore, the observed resetting cannot be explained by fewer inducers being presented. Experiment 5 showed that resetting caused by a single deviant did not increase when prior inducers were made unpredictable in frequency (four-semitone range). Experiments 6a-6b demonstrated that actual and perceived continuity have a similar effect on subsequent streaming judgements promoting either integration or segregation, depending on listening context. Experiment 7 found that same-frequency inducers were considerably more effective at promoting segregation than an alternating-frequency inducer, and that a trend for deviant-tone resetting was only apparent for the same-frequency case. Using temporal-order judgments, experiments 8-9 demonstrated the stream segregation of pure-tone-like percepts, evoked by sudden changes in amplitude or interaural time difference for individual components of a complex tone, Active resetting was observed when a deviant was inserted into a sequence of these percepts (Experiment 10). Overall, these experiments offer new insight into the segregation-promotIng effect of induction sequences, and the factors which can reset this effect.

A novel multilocus sequence typing scheme for the opportunistic pathogen Propionibacterium acnes and characterisation of type 1 cell surface-associated antigens

We have developed a novel multilocus sequence typing (MLST) scheme and database (http://pubmlst.org/pacnes/) for Propionibacterium acnes based on the analysis of seven core housekeeping genes. The scheme, which was validated against previously described antibody, single locus and random amplification of polymorphic DNA typing methods, displayed excellent resolution and differentiated 123 isolates into 37 sequence types (STs). An overall clonal population structure was detected with six eBURST groups representing the major clades I, II and III, along with two singletons. Two highly successful and global clonal lineages, ST6 (type IA) and ST10 (type IB1), representing 64?% of this current MLST isolate collection were identified. The ST6 clone and closely related single locus variants, which comprise a large clonal complex CC6, dominated isolates from patients with acne, and were also significantly associated with ophthalmic infections. Our data therefore support an association between acne and P. acnes strains from the type IA cluster and highlight the role of a widely disseminated clonal genotype in this condition. Characterization of type I cell surface-associated antigens that are not detected in ST10 or strains of type II and III identified two dermatan-sulphate-binding proteins with putative phase/antigenic variation signatures. We propose that the expression of these proteins by type IA organisms contributes to their role in the pathophysiology of acne and helps explain the recurrent nature of the disease. The MLST scheme and database described in this study should provide a valuable platform for future epidemiological and evolutionary studies of P. acnes.

Toward the prediction of class I and II mouse major histocompatibility complex-peptide-binding affinity:in silico bioinformatic step-by-step guide using quantitative structure-activity relationships

Quantitative structure-activity relationship (QSAR) analysis is a cornerstone of modern informatics. Predictive computational models of peptide-major histocompatibility complex (MHC)-binding affinity based on QSAR technology have now become important components of modern computational immunovaccinology. Historically, such approaches have been built around semiqualitative, classification methods, but these are now giving way to quantitative regression methods. We review three methods--a 2D-QSAR additive-partial least squares (PLS) and a 3D-QSAR comparative molecular similarity index analysis (CoMSIA) method--which can identify the sequence dependence of peptide-binding specificity for various class I MHC alleles from the reported binding affinities (IC50) of peptide sets. The third method is an iterative self-consistent (ISC) PLS-based additive method, which is a recently developed extension to the additive method for the affinity prediction of class II peptides. The QSAR methods presented here have established themselves as immunoinformatic techniques complementary to existing methodology, useful in the quantitative prediction of binding affinity: current methods for the in silico identification of T-cell epitopes (which form the basis of many vaccines, diagnostics, and reagents) rely on the accurate computational prediction of peptide-MHC affinity. We have reviewed various human and mouse class I and class II allele models. Studied alleles comprise HLA-A*0101, HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0206, HLA-A*0301, HLA-A*1101, HLA-A*3101, HLA-A*6801, HLA-A*6802, HLA-B*3501, H2-K(k), H2-K(b), H2-D(b) HLA-DRB1*0101, HLA-DRB1*0401, HLA-DRB1*0701, I-A(b), I-A(d), I-A(k), I-A(S), I-E(d), and I-E(k). In this chapter we show a step-by-step guide into predicting the reliability and the resulting models to represent an advance on existing methods. The peptides used in this study are available from the AntiJen database (http://www.jenner.ac.uk/AntiJen). The PLS method is available commercially in the SYBYL molecular modeling software package. The resulting models, which can be used for accurate T-cell epitope prediction, will be made are freely available online at the URL http://www.jenner.ac.uk/MHCPred.

New horizons in mouse immunoinformatics:reliable in silico prediction of mouse class I histocompatibility major complex peptide binding affinity

Quantitative structure–activity relationship (QSAR) analysis is a main cornerstone of modern informatic disciplines. Predictive computational models, based on QSAR technology, of peptide-major histocompatibility complex (MHC) binding affinity have now become a vital component of modern day computational immunovaccinology. Historically, such approaches have been built around semi-qualitative, classification methods, but these are now giving way to quantitative regression methods. The additive method, an established immunoinformatics technique for the quantitative prediction of peptide–protein affinity, was used here to identify the sequence dependence of peptide binding specificity for three mouse class I MHC alleles: H2–Db, H2–Kb and H2–Kk. As we show, in terms of reliability the resulting models represent a significant advance on existing methods. They can be used for the accurate prediction of T-cell epitopes and are freely available online (http://www.jenner.ac.uk/MHCPred).

Deconvolution of magnetic acoustic change complex (mACC)

Objective: The aim of this study was to design a novel experimental approach to investigate the morphological characteristics of auditory cortical responses elicited by rapidly changing synthesized speech sounds. Methods: Six sound-evoked magnetoencephalographic (MEG) responses were measured to a synthesized train of speech sounds using the vowels /e/ and /u/ in 17 normal hearing young adults. Responses were measured to: (i) the onset of the speech train, (ii) an F0 increment; (iii) an F0 decrement; (iv) an F2 decrement; (v) an F2 increment; and (vi) the offset of the speech train using short (jittered around 135. ms) and long (1500. ms) stimulus onset asynchronies (SOAs). The least squares (LS) deconvolution technique was used to disentangle the overlapping MEG responses in the short SOA condition only. Results: Comparison between the morphology of the recovered cortical responses in the short and long SOAs conditions showed high similarity, suggesting that the LS deconvolution technique was successful in disentangling the MEG waveforms. Waveform latencies and amplitudes were different for the two SOAs conditions and were influenced by the spectro-temporal properties of the sound sequence. The magnetic acoustic change complex (mACC) for the short SOA condition showed significantly lower amplitudes and shorter latencies compared to the long SOA condition. The F0 transition showed a larger reduction in amplitude from long to short SOA compared to the F2 transition. Lateralization of the cortical responses were observed under some stimulus conditions and appeared to be associated with the spectro-temporal properties of the acoustic stimulus. Conclusions: The LS deconvolution technique provides a new tool to study the properties of the auditory cortical response to rapidly changing sound stimuli. The presence of the cortical auditory evoked responses for rapid transition of synthesized speech stimuli suggests that the temporal code is preserved at the level of the auditory cortex. Further, the reduced amplitudes and shorter latencies might reflect intrinsic properties of the cortical neurons to rapidly presented sounds. Significance: This is the first demonstration of the separation of overlapping cortical responses to rapidly changing speech sounds and offers a potential new biomarker of discrimination of rapid transition of sound.