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.

Longitudinal parametric oscillation in photorefractive sillenites: comparison between theory and experiment

By using an alternative setup for photorefractive parametric oscillation in which wave mixing between the recording beams is avoided it has become possible to make more detailed comparisons with the space-charge wave theory. In the present paper we compare the experimental features of longitudinal parametric oscillation observed in a crystal of Bi12SiO20 with the theoretical predictions.

16,000 km, 10 Gbits-1 soliton transmission over standard fibre by reduction of interactions through optimum amplifier positioning

We show that by optimizing the amplifier position in a two-stage dispersion map, the (dispersion-managed) soliton-soliton interaction can be reduced, enabling transmission of 10-Gbits-1 solitons over standard fiber over 16,000 km

Influence of beam-coupling on photorefractive parametric oscillation in a dc-field-biased Bi12SiO20 crystal

The influence of beam coupling on photorefractive parametric oscillation generated in a Bi12SiO20 crystal is investigated experimentally by comparing two configurations with and without the presence of beam coupling. It is shown that beam coupling has a great influence; for example, the transversal split of the K/2 subharmonic grating is seen only in the beam-coupling geometry. A case that resembles K/4 subharmonic generation can, however, still be found in the absence of beam coupling.

43Gbit/s RZ-DQPSK transmission over 1000km of G.652 ultra-low-loss fibre with 250km amplifier spans

We demonstrate 40x43Gbit/s RZ-DQPSK transmission over 1000km of ultra-low-loss G.652 fibre with 250km amplifier spacing. Hybrid Raman-EDFA amplification with co- and contra-directional Raman pumping enables 27dB Raman gain per span and error-free post-FEC performance. ©2010 IEEE.

Noise and gain optimisation in bi-directionally pumped dispersion compensating amplifier modules

We perform optimisation of bi-directionally pumped dispersion compensating Raman amplifier modules. Optimal forward and backward pump powers for basic configurations using different commercially available fibers are presented for both single- and multi-channel systems. Optical signal-to-noise ratio improvement of up to 8 dB is achieved as a result of optimisation. © 2003 Published by Elsevier B.V.

Tunable master-oscillator power-amplifier based on chirped quantum-dot structures

A broadly tunable master-oscillator power-amplifier (MOPA) picosecond optical pulse source is demonstrated, consisting of an external cavity passively mode-locked laser diode with a tapered semiconductor amplifier. By employing chirped quantum-dot structures on both the oscillator's gain chip and amplifier, a wide tunability range between 1187 and 1283 nm is achieved. Under mode-locked operation, the highest output peak power of 4.39 W is achieved from the MOPA, corresponding to a peak power spectral density of 31.4 dBm/nm. © 1989-2012 IEEE.

Tunable master-oscillator power-amplifier using all chirped quantum-dot structures

A tunable master-oscillator power-amplifier (MOPA) picosecond optical pulse source using all chirped quantum dot (QD) structures is demonstrated (60nm tunability). Under fundamental mode-locked operation, the highest peak power of 4.39 W is achieved.

Enhanced Raman amplifier flatness with nonlinear broadening over non-standard transmission fibre

Nonlinear CW pump broadening over non-standard transmission fibre is used for the first time to achieve improved gain flatness in a single-pump broadband Raman amplifier. As an illustration of the benefits that can be obtained from this approach, a threefold increase in the bandwidth for 0.1 dB gain variation is reported when the broadened pump is used to produce 9.2 dB on-off gain over 25 km LEAF fibre. © 2005 Elsevier B.V. All rights reserved.

A non-parametric data envelopment analysis approach for improving energy efficiency of grape production

Grape is one of the world's largest fruit crops with approximately 67.5 million tonnes produced each year and energy is an important element in modern grape productions as it heavily depends on fossil and other energy resources. Efficient use of these energies is a necessary step toward reducing environmental hazards, preventing destruction of natural resources and ensuring agricultural sustainability. Hence, identifying excessive use of energy as well as reducing energy resources is the main focus of this paper to optimize energy consumption in grape production.In this study we use a two-stage methodology to find the association of energy efficiency and performance explained by farmers' specific characteristics. In the first stage a non-parametric Data Envelopment Analysis is used to model efficiencies as an explicit function of human labor, machinery, chemicals, FYM (farmyard manure), diesel fuel, electricity and water for irrigation energies. In the second step, farm specific variables such as farmers' age, gender, level of education and agricultural experience are used in a Tobit regression framework to explain how these factors influence efficiency of grape farming.The result of the first stage shows substantial inefficiency between the grape producers in the studied area while the second stage shows that the main difference between efficient and inefficient farmers was in the use of chemicals, diesel fuel and water for irrigation. The use of chemicals such as insecticides, herbicides and fungicides were considerably less than inefficient ones. The results revealed that the more educated farmers are more energy efficient in comparison with their less educated counterparts. © 2013.

Universality in the merging dynamics of parametric active contours:a study in MRI based lung segmentation

Measurement of lung ventilation is one of the most reliable techniques in diagnosing pulmonary diseases. The time-consuming and bias-prone traditional methods using hyperpolarized H 3He and 1H magnetic resonance imageries have recently been improved by an automated technique based on 'multiple active contour evolution'. This method involves a simultaneous evolution of multiple initial conditions, called 'snakes', eventually leading to their 'merging' and is entirely independent of the shapes and sizes of snakes or other parametric details. The objective of this paper is to show, through a theoretical analysis, that the functional dynamics of merging as depicted in the active contour method has a direct analogue in statistical physics and this explains its 'universality'. We show that the multiple active contour method has an universal scaling behaviour akin to that of classical nucleation in two spatial dimensions. We prove our point by comparing the numerically evaluated exponents with an equivalent thermodynamic model. © IOP Publishing Ltd and Deutsche Physikalische Gesellschaft.