Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

In this review, we detail the efforts performed to couple the purification and the immobilization of industrial enzymes in a single step. The use of antibodies, the development of specific domains with affinity for some specific supports will be revised. Moreover, we will discuss the use of domains that increase the affinity for standard matrices (ionic exchangers, silicates). We will show how the control of the immobilization conditions may convert some unspecific supports in largely specific ones. The development of tailor-made heterofunctional supports as a tool to immobilize–stabilize–purify some proteins will be discussed in deep, using low concentration of adsorbent groups and a dense layer of groups able to give an intense multipoint covalent attachment. The final coupling of mutagenesis and tailor made supports will be the last part of the review.

Population Monte Carlo Algorithm in High Dimensions

The population Monte Carlo algorithm is an iterative importance sampling scheme for solving static problems. We examine the population Monte Carlo algorithm in a simplified setting, a single step of the general algorithm, and study a fundamental problem that occurs in applying importance sampling to high-dimensional problem. The precision of the computed estimate from the simplified setting is measured by the asymptotic variance of estimate under conditions on the importance function. We demonstrate the exponential growth of the asymptotic variance with the dimension and show that the optimal covariance matrix for the importance function can be estimated in special cases.

Protein complementation assay as a display system for screening protein libraries in the intracellular environment

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the β-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) *Escherichia coli* proteins were used as model interaction partners for developing the system. These proteins drove effective β-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other β-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent β-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wild-type Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

Bayesian hybrid algorithms and models : implementation and associated issues

This thesis addresses computational challenges arising from Bayesian analysis of complex real-world problems. Many of the models and algorithms designed for such analysis are ‘hybrid’ in nature, in that they are a composition of components for which their individual properties may be easily described but the performance of the model or algorithm as a whole is less well understood. The aim of this research project is to after a better understanding of the performance of hybrid models and algorithms. The goal of this thesis is to analyse the computational aspects of hybrid models and hybrid algorithms in the Bayesian context. The first objective of the research focuses on computational aspects of hybrid models, notably a continuous finite mixture of t-distributions. In the mixture model, an inference of interest is the number of components, as this may relate to both the quality of model fit to data and the computational workload. The analysis of t-mixtures using Markov chain Monte Carlo (MCMC) is described and the model is compared to the Normal case based on the goodness of fit. Through simulation studies, it is demonstrated that the t-mixture model can be more flexible and more parsimonious in terms of number of components, particularly for skewed and heavytailed data. The study also reveals important computational issues associated with the use of t-mixtures, which have not been adequately considered in the literature. The second objective of the research focuses on computational aspects of hybrid algorithms for Bayesian analysis. Two approaches will be considered: a formal comparison of the performance of a range of hybrid algorithms and a theoretical investigation of the performance of one of these algorithms in high dimensions. For the first approach, the delayed rejection algorithm, the pinball sampler, the Metropolis adjusted Langevin algorithm, and the hybrid version of the population Monte Carlo (PMC) algorithm are selected as a set of examples of hybrid algorithms. Statistical literature shows how statistical efficiency is often the only criteria for an efficient algorithm. In this thesis the algorithms are also considered and compared from a more practical perspective. This extends to the study of how individual algorithms contribute to the overall efficiency of hybrid algorithms, and highlights weaknesses that may be introduced by the combination process of these components in a single algorithm. The second approach to considering computational aspects of hybrid algorithms involves an investigation of the performance of the PMC in high dimensions. It is well known that as a model becomes more complex, computation may become increasingly difficult in real time. In particular the importance sampling based algorithms, including the PMC, are known to be unstable in high dimensions. This thesis examines the PMC algorithm in a simplified setting, a single step of the general sampling, and explores a fundamental problem that occurs in applying importance sampling to a high-dimensional problem. The precision of the computed estimate from the simplified setting is measured by the asymptotic variance of the estimate under conditions on the importance function. Additionally, the exponential growth of the asymptotic variance with the dimension is demonstrated and we illustrates that the optimal covariance matrix for the importance function can be estimated in a special case.

Vulnerabilities in the courtroom

Book summary: In a constantly evolving context of performance management, accountability and risk assessment, police organisations and frontline police officers are required to pay careful attention to what has come to be known as ‘at risk people’, ‘vulnerable populations’ or ‘vulnerable people’. Vulnerable people have become a key focus of policy. Concurrently, there have been stronger demands on police, and a steep increase in police powers in relation to their interaction with vulnerable people. The premise of this protectionist and interventionist agenda is threefold: to protect the rights of vulnerable individuals proactively cater for their vulnerability within the justice system; and to secure police operations and protocols within strict guidelines. This collection unpacks ‘vulnerable people policing’ in theory and practice and guides the reader through the policing process as it is experienced by police officers, victims, offenders, witnesses and justice stakeholders. Each chapter features a single step of the policing process: from police recruit education through to custody, and the final transfer of vulnerable people to courts and sentencing. This edited collection provides analytical, theoretical and empirical insights on vulnerable people policing, and reflects on critical issues in a domain that is increasingly subject to speedy conversion from policy to practice, and heightened media and political scrutiny. It breaks down policing practices, operations and procedures that have vulnerable populations as a focus, bringing together original and innovative academic research and literature, practitioner experience and discussion of policy implications (from local and international perspectives). The particular nature of this collection highlights the multi-disciplinary nature of police work, sheds light on how specific, mandatory policies guide police officers steps in their interaction with vulnerable populations, and discusses the practicalities of police decision making at key points in this process.

Combined electrophoretic deposition–anodization method to fabricate reduced graphene oxide–TiO2 nanotube films

We directly constructed reduced graphene oxide–titanium oxide nanotube (RGO–TNT) film using a single-step, combined electrophoretic deposition–anodization (CEPDA) method. This method, based on the simultaneous anodic growth of tubular TiO2 and the electrophoretic-driven motion of RGO, allowed the formation of an effective interface between the two components, thus improving the electron transfer kinetics. Composites of these graphitic carbons with different levels of oxygen-containing groups, electron conductivity and interface reaction time were investigated; a fine balance of these parameters was achieved.

High-throughput vectors for efficient gene silencing in plants

A major challenge in the post-genome era of plant biology is to determine the functions of all genes in the plant genome. A straightforward approach to this problem is to reduce or knockout expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study but is limited by gene redundancy, lethal knockouts, non-tagged mutants, and the inability to target the inserted element to a specific gene. The efficacy of gene silencing in plants using inverted-repeat transgene constructs that encode a hairpin RNA (hpRNA) has been demonstrated by a number of groups, and has several advantages over insertional mutagenesis. In this paper we describe two improved pHellsgate vectors that facilitate rapid generation of hpRNA-encoding constructs, pHellsgate 4 allows the production of an hpRNA construct in a single step from a single polymerase chain reaction product, while pHellsgate 8 requires a two-step process via an intermediate vector. We show that these vectors are effective at silencing three endogenous genes in Arabidopsis, FLOWERING LOCUS C, PHYTOENE DESATURASE and ETHYLENE INSENSITIVE 2. We also show that a construct of sequences from two genes silences both genes.

Reaction of Aromatic Peroxyl Radicals with Alkynes: A Mass Spectrometric and Computational Study Using the Distonic Radical Ion Approach

The reaction of the aromatic distonic peroxyl radical cations N-methyl pyridinium-4-peroxyl (PyrOO center dot+) and 4-(N,N,N-trimethyl ammonium)-phenyl peroxyl (AnOO center dot+), with symmetrical dialkyl alkynes 10?ac was studied in the gas phase by mass spectrometry. PyrOO center dot+ and AnOO center dot+ were produced through reaction of the respective distonic aryl radical cations Pyr center dot+ and An center dot+ with oxygen, O2. For the reaction of Pyr center dot+ with O2 an absolute rate coefficient of k1=7.1X10-12 cm3 molecule-1 s-1 and a collision efficiency of 1.2?% was determined at 298 K. The strongly electrophilic PyrOO center dot+ reacts with 3-hexyne and 4-octyne with absolute rate coefficients of khexyne=1.5X10-10 cm3 molecule-1 s-1 and koctyne=2.8X10-10 cm3 molecule-1 s-1, respectively, at 298 K. The reaction of both PyrOO center dot+ and AnOO center dot+ proceeds by radical addition to the alkyne, whereas propargylic hydrogen abstraction was observed as a very minor pathway only in the reactions involving PyrOO center dot+. A major reaction pathway of the vinyl radicals 11 formed upon PyrOO center dot+ addition to the alkynes involves gamma-fragmentation of the peroxy O?O bond and formation of PyrO center dot+. The PyrO center dot+ is rapidly trapped by intermolecular hydrogen abstraction, presumably from a propargylic methylene group in the alkyne. The reaction of the less electrophilic AnOO center dot+ with alkynes is considerably slower and resulted in formation of AnO center dot+ as the only charged product. These findings suggest that electrophilic aromatic peroxyl radicals act as oxygen atom donors, which can be used to generate alpha-oxo carbenes 13 (or isomeric species) from alkynes in a single step. Besides gamma-fragmentation, a number of competing unimolecular dissociative reactions also occur in vinyl radicals 11. The potential energy diagrams of these reactions were explored with density functional theory and ab initio methods, which enabled identification of the chemical structures of the most important products.

Reactive stepping behaviour in response to forward loss of balance predicts future falls in community-dwelling older adults

Background: a fall occurs when an individual experiences a loss of balance from which they are unable to recover. Assessment of balance recovery ability in older adults may therefore help to identify individuals at risk of falls. The purpose of this 12-month prospective study was to assess whether the ability to recover from a forward loss of balance with a single step across a range of lean magnitudes was predictive of falls. Methods: two hundred and one community-dwelling older adults, aged 65–90 years, underwent baseline testing of sensorimotor function and balance recovery ability followed by 12-month prospective falls evaluation. Balance recovery ability was defined by whether participants required either single or multiple steps to recover from forward loss of balance from three lean magnitudes, as well as the maximum lean magnitude participants could recover from with a single step. Results: forty-four (22%) participants experienced one or more falls during the follow-up period. Maximal recoverable lean magnitude and use of multiple steps to recover at the 15% body weight (BW) and 25%BW lean magnitudes significantly predicted a future fall (odds ratios 1.08–1.26). The Physiological Profile Assessment, an established tool that assesses variety of sensori-motor aspects of falls risk, was also predictive of falls (Odds ratios 1.22 and 1.27, respectively), whereas age, sex, postural sway and timed up and go were not predictive. Conclusion: reactive stepping behaviour in response to forward loss of balance and physiological profile assessment are independent predictors of a future fall in community-dwelling older adults. Exercise interventions designed to improve reactive stepping behaviour may protect against future falls.

Plasma break-down and re-build : same functional vertical graphenes from diverse natural precursors

Plasmas, the 4th state of matter, uniformly transform natural precursors with different chemical composition in solid, liquid, and gas states into the same functional vertical graphenes in a single-step process within a few minutes. Functional vertical graphenes show reliable biosensing properties, strong binding with proteins, and improved adhesion to substrates.

Structure-controlled, vertical graphene-based, binder-free electrodes from plasma-reformed butter enhance supercapacitor performance

Vertical graphene nanosheets (VGNS) hold great promise for high-performance supercapacitors owing to their excellent electrical transport property, large surface area and in particular, an inherent three-dimensional, open network structure. However, it remains challenging to materialise the VGNS-based supercapacitors due to their poor specific capacitance, high temperature processing, poor binding to electrode support materials, uncontrollable microstructure, and non-cost effective way of fabrication. Here we use a single-step, fast, scalable, and environmentally-benign plasma-enabled method to fabricate VGNS using cheap and spreadable natural fatty precursor butter, and demonstrate the controllability over the degree of graphitization and the density of VGNS edge planes. Our VGNS employed as binder-free supercapacitor electrodes exhibit high specific capacitance up to 230 F g−1 at a scan rate of 10 mV s−1 and >99% capacitance retention after 1,500 charge-discharge cycles at a high current density, when the optimum combination of graphitic structure and edge plane effects is utilised. The energy storage performance can be further enhanced by forming stable hybrid MnO2/VGNS nano-architectures which synergistically combine the advantages from both VGNS and MnO2. This deterministic and plasma-unique way of fabricating VGNS may open a new avenue for producing functional nanomaterials for advanced energy storage devices.

Effect of input power and gas pressure on the roughening and selective etching of SiO2/Si surfaces in reactive plasmas

We report on the application low-temperature plasmas for roughening Si surfaces which is becoming increasingly important for a number of applications ranging from Si quantum dots to cell and protein attachment for devices such as "laboratory on a chip" and sensors. It is a requirement that Si surface roughening is scalable and is a single-step process. It is shown that the removal of naturally forming SiO2 can be used to assist in the roughening of the surface using a low-temperature plasma-based etching approach, similar to the commonly used in semiconductor micromanufacturing. It is demonstrated that the selectivity of SiO2 /Si etching can be easily controlled by tuning the plasma power, working gas pressure, and other discharge parameters. The achieved selectivity ranges from 0.4 to 25.2 thus providing an effective means for the control of surface roughness of Si during the oxide layer removal, which is required for many advance applications in bio- and nanotechnology.

Multifunctional three-dimensional T-junction graphene micro-wells: Energy-efficient, plasma-enabled growth and instant water-based transfer for flexible device applications

The “third-generation” 3D graphene structures, T-junction graphene micro-wells (T-GMWs) are produced on cheap polycrystalline Cu foils in a single-step, low-temperature (270 °C), energy-efficient, and environment-friendly dry plasma-enabled process. T-GMWs comprise vertical graphene (VG) petal-like sheets that seemlessly integrate with each other and the underlying horizontal graphene sheets by forming T-junctions. The microwells have the pico-to-femto-liter storage capacity and precipitate compartmentalized PBS crystals. The T-GMW films are transferred from the Cu substrates, without damage to the both, in de-ionized or tap water, at room temperature, and without commonly used sacrificial materials or hazardous chemicals. The Cu substrates are then re-used to produce similar-quality T-GMWs after a simple plasma conditioning. The isolated T-GMW films are transferred to diverse substrates and devices and show remarkable recovery of their electrical, optical, and hazardous NO2 gas sensing properties upon repeated bending (down to 1 mm radius) and release of flexible trasparent display plastic substrates. The plasma-enabled mechanism of T-GMW isolation in water is proposed and supported by the Cu plasma surface modification analysis. Our GMWs are suitable for various optoelectronic, sesning, energy, and biomedical applications while the growth approach is potentially scalable for future pilot-scale industrial production.

Catalyst engineering for lithium ion batteries: The catalytic role of Ge in enhancing the electrochemical performance of SnO2(GeO2)0.13/G anodes

The catalytic role of germanium (Ge) was investigated to improve the electrochemical performance of tin dioxide grown on graphene (SnO(2)/G) nanocomposites as an anode material of lithium ion batteries (LIBs). Germanium dioxide (GeO(20) and SnO(2) nanoparticles (<10 nm) were uniformly anchored on the graphene sheets via a simple single-step hydrothermal method. The synthesized SnO(2)(GeO(2))0.13/G nanocomposites can deliver a capacity of 1200 mA h g(-1) at a current density of 100 mA g(-1), which is much higher than the traditional theoretical specific capacity of such nanocomposites (∼ 702 mA h g(-1)). More importantly, the SnO(2)(GeO(2))0.13/G nanocomposites exhibited an improved rate, large current capability (885 mA h g(-1) at a discharge current of 2000 mA g(-1)) and excellent long cycling stability (almost 100% retention after 600 cycles). The enhanced electrochemical performance was attributed to the catalytic effect of Ge, which enabled the reversible reaction of metals (Sn and Ge) to metals oxide (SnO(2) and GeO(2)) during the charge/discharge processes. Our demonstrated approach towards nanocomposite catalyst engineering opens new avenues for next-generation high-performance rechargeable Li-ion batteries anode materials.

Surface chemical studies of Thiobacillus ferrooxidans with reference to copper tolerance

A strain of Thiobacillus ferrooxidans was adapted to grow at higher concentrations of copper by single step culturing in the presence of 20 g/L (0.314 mol/L) cupric ions added to 9K medium. Exposure to copper results in change in the surface chemistry of the microorganism. The isoelectric point of the adapted strain (pI=4.7) was observed to be at a higher pH than that of the wild unadapted strain(pI=2.0). Compared to the wild strain, the copper adapted strain was found to be more hydrophobic and showed enhanced attachment efficiency to the pyrite mineral. The copper adsorption ability of the adapted strain was also found to be higher than that of the wild strain. Fourier transform infrared spectroscopy of adapted cells suggested that a proteinaceous new cell surface component is synthesized by the adapted strain. Treatment of adapted cells with proteinase-K, resulted in complete loss of tolerance to copper, reduction in copper adsorption and hydrophobicity of the adapted cells. These observations strongly suggest a role played by cell surface modifications of Thiobacillus ferrooxidans in imparting the copper tolerance to the cells and bioleaching of sulphide minerals.