ProxiMAX randomisation:a new technology for non-degenerate saturation mutagenesis of contiguous codons

Back in 2003, we published ‘MAX’ randomisation, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. ‘MAX’ randomisation saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an alpha-helix, as in zinc finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple, contiguous codons in a non-degenerate manner. We have now developed ‘ProxiMAX’ randomisation, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomisation uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialised chemistry, reagents nor equipment and simply relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle. The process can encode both unbiased representation of selected amino acids or else encode them in pre-defined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomisation is particularly relevant to antibody engineering.

ProxiMAX Randomisation:a new technology for non-degenerate saturation mutagenesis of contiguous codons

ProxiMAX randomisation achieves saturation mutagenesis of contiguous codons without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, it uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents and as such, requires no specialised chemistry, reagents nor equipment. When particular residues are known to affect protein activity/specificity, their combinatorial replacement with all 20 amino acids, or a subset thereof, can provide a rapid route to generating proteins with desirable characteristics. Conventionally, saturation mutagenesis replaced key codons with degenerate ones. Although simple to perform, that procedure resulted in unnecessarily large libraries, termination codons and inherent uneven amino acid representation. ProxiMAX randomisation is an enzyme-based technique that can encode unbiased representation of all or selected amino acids or else can provide required codons in pre-defined ratios. Each saturated position can be defined independently of the others. ProxiMAX randomisation is achieved via saturation cycling: an iterative process comprising blunt end ligation, amplification and digestion with a Type IIS restriction enzyme. We demonstrate both unbiased saturation of a short 6-mer peptide and saturation of a hypervariable region of a scfv antibody fragment, where 11 contiguous codons are saturated with selected codons, in pre-defined ratios. As such, ProxiMAX randomisation is particularly relevant to antibody engineering. The development of ProxiMAX randomisation from concept to reality is described.

ProxiMAX randomisation:a new technology for non-degenerate saturation mutagenesis of contiguous codons

Back in 2003, we published ‘MAX’ randomisation, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. ‘MAX’ randomisation saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an alpha-helix, as in zinc finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple, contiguous codons in a non-degenerate manner. We have now developed ‘ProxiMAX’ randomisation, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomisation uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialised chemistry, reagents nor equipment and simply relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle. The process can encode both unbiased representation of selected amino acids or else encode them in pre-defined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomisation is particularly relevant to antibody engineering.

The impact of flash intensity on retinal vessel oxygen saturation measurements using dual wavelength oximetry

PURPOSE. To establish the optimal flash settings for retinal vessel oxygen saturation parameters using dual-wavelength imaging in a multiethnic group. METHODS. Twelve healthy young subjects (mean age 32 years [SD 7]; three Mediterranean, two South Asian, and seven Caucasian individuals) underwent retinal vessel oxygen saturation measurements using dual-wavelength oximetry, noncontact tonometry, and manual sphygmomanometry. In order to evaluate the impact of flash intensity, we obtained three images (fundus camera angle 30°, ONH centered) per flash setting. Flash settings of the fundus camera were increased in steps of 2 (initial setting of 6 and the final of 22), which reflect logarithmic increasing intensities from 13.5 to 214 Watt seconds (Ws). RESULTS. Flash settings below 27 Ws were too low to obtain saturation measurements, whereas flash settings of more than 214 Ws resulted in overexposed images. Retinal arteriolar and venular oxygen saturation was comparable at flash settings of 27 to 76 Ws (arterioles' range: 85%-92%; venules' range: 45%-53%). Higher flash settings lead to increased saturation measurements in both retinal arterioles (up to 110%) and venules (up to 92%), with a more pronounced increase in venules. CONCLUSIONS. Flash intensity has a significant impact on retinal vessel oxygen saturation measurements using dual-wavelength retinal oximetry. High flash intensities lead to supranormal oxygen saturation measurements with a magnified effect in retinal venules compared with arteries. In addition to even retinal illumination, the correct flash setting is of paramount importance for clinical acquisition of images in retinal oximetry. We recommend flash settings between 27 to 76 Ws. © 2013 The Association for Research in Vision and Ophthalmology, Inc.

Experimental data of CO2, CO3, Omega calcite saturation, pH values, alpha and epsilon fractionation factors, and Dd18O calctite-water

The oxygen isotopic composition (d18O) of calcium carbonate of planktonic calcifying organisms is a key tool for reconstructing both past seawater temperature and salinity. The calibration of paloeceanographic proxies relies in general on empirical relationships derived from field experiments on extant species. Laboratory experiments have more often than not revealed that variables other than the target parameter influence the proxy signal, which makes proxy calibration a challenging task. Understanding these secondary or "vital" effects is crucial for increasing proxy accuracy. We present data from laboratory experiments showing that oxygen isotope fractionation during calcification in the coccolithophore Calcidiscus leptoporus and the calcareous dinoflagellate Thoracosphaera heimii is dependent on carbonate chemistry of seawater in addition to its dependence on temperature. A similar result has previously been reported for planktonic foraminifera, supporting the idea that the [CO3]2- effect on d18O is universal for unicellular calcifying planktonic organisms. The slopes of the d18O/[CO3]2- relationships range between -0.0243 per mil/(µmol/kg) (calcareous dinoflagellate T. heimii) and the previously published -0.0022 per mil/(µmol/kg) (non-symbiotic planktonic foramifera Orbulina universa), while C. leptoporus has a slope of -0.0048 per mil/(µmol/kg). We present a simple conceptual model, based on the contribution of d18O-enriched [HCO3]- to the [CO3]2- pool in the calcifying vesicle, which can explain the [CO3]2- effect on d18O for the different unicellular calcifiers. This approach provides a new insight into biological fractionation in calcifying organisms. The large range in d18O/[CO3]2- slopes should possibly be explored as a means for paleoreconstruction of surface [CO3]2-, particularly through comparison of the response in ecologically similar planktonic organisms.

Le ralentissement de l'activité électrique cérébrale dans le trouble comportemental en sommeil paradoxal

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Expérience de pères d'un enfant avec une défience motrice cérébrale

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Troubles spécifiques de la reconnaissance musicale chez des personnes avec ou sans lésion cérébrale

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Activation cérébrale en fonction de l'âge et de la complexité du traitement sémantique des mots par imagerie par résonance magnétique fonctionnelle

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Asymétrie cérébrale et traitement de l'ironie

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Hypoxémie et inflammation systémique après ischémie cérébrale aiguë chez le rat Wistar

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Le ralentissement de l'activité électrique cérébrale dans le trouble comportemental en sommeil paradoxal

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Expérience de pères d'un enfant avec une défience motrice cérébrale

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.