Design and evaluation of synthetic silica-based monolithic materials in shrinkable tube for efficient protein extraction

Sample pretreatment is a required step in proteomics in order to remove interferences and preconcentrate the samples. Much research in recent years has focused on porous monolithic materials since they are highly permeable to liquid flow and show high mass transport compared with more common packed beds. These features are due to the micro-structure within the monolithic silica column which contains both macropores that reduce the back pressure, and mesopores that give good interaction with analytes. The aim of this work was to fabricate a continuous porous silica monolithic rod inside a heat shrinkable tube and to compare this with the same material whose surface has been modified with a C(18) phase, in order to use them for preconcentration/extraction of proteins. The performance of the silica-based monolithic rod was evaluated using eight proteins; insulin, cytochrome C, lysozyme, myoglobin, β-lactoglobulin, ovalbumin, hemoglobin, and bovine serum albumin at a concentration of 60 μM. The results show that recovery of the proteins was achieved by both columns with variable yields; however, the C(18) modified silica monolith gave higher recoveries (92.7 to 109.7%) than the non-modified silica monolith (25.5 to 97.9%). Both silica monoliths can be used with very low back pressure indicating a promising approach for future fabrication of the silica monolith inside a microfluidic device for the extraction of proteins from biological media

Mathematical model of the Lux luminescence system in the terrestrial bacterium Photorhabdus luminescens.

A mathematical model of the Lux luminescence system, governed by the operon luxCDABE in the terrestrial bacterium Photorhabdus luminescens, was constructed using a set of coupled ordinary differential equations. This model will have value in the interpretation of Lux data when used as a reporter in time-course gene expression experiments. The system was tested on time series and stationary data from published papers and the model is in good agreement with the published data. Metabolic control analysis demonstrates that control of the system lies mainly with the aldehyde recycling pathway (LuxE and LuxC). The rate at which light is produced in the steady state model shows a low sensitivity to changes in kinetic parameter values to those measured in other species of luminescent bacteria, demonstrating the robustness of the Lux system

X-ray crystallographic analysis of the pyrophosphate-dependent phosphofructokinase of Spirochaeta thermophilum : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, New Zealand

The structure of a homodimeric, non-allosteric, PPᵢ.-dependent phosphofructokinase from the thermophilic bacterium Spirochaeta thermophilum has been resolved by X-ray crystallography in two distinct conformations at 2.2 (R = 0.1991 [R free = 0.2288]) and 1.85 Å (R = 0.1923 [R free = 0.2035]) resolution. The 554 residue (Mᵣ 61080 g.mol-¹) subunit, a homologue of the plant PPᵢ,-PFK β-subunit exhibits an asymmetrical quaternary structure and shares both sequence and tertiary structure with the N- and C- terminal Rossmann-like domains of prokaryotic ATP-PFKs. Spirochaeta thermophilum PPᵢ-PFK exhibits three major inserts relative to the prokaryotic ATP-PFK of E. coli, an N-terminal insert, a C-terminal insert, and an insert within the PFK C-terminal domain which forms an autonomous α-helical domain. The active site is formed at the interface of the N and C domains. The 'open' and 'closed' subunit asymmetry of the S. thermophilum PPᵢ-PFK 1.85 Å atomic model mirrors that of the B. burgdorferi PPᵢ-PFK (1KZH [Moore et al.2002]) with the exception that the two unique β-hairpins (380-390 [α16-α17] and 485-495 [β14-β15]) of subunit A are not displaced into the active site. Both subunits of the S. thermophilum PPᵢ-PFK 2.2 Å atomic model adopt an 'open', apparently inactive conformation. The conformational change involves concomitant closure of the active site of both subunits via a rigid-body displacement of the C and α- helical domains, relative to the N domain. The N domain of one subunit and the C domain of the opposing subunit can be thought of as a rigid body, therefore closure of one active site dictates closure of the other. Rotation of the small domain forces Met251 of the MGR motif to adopt an active conformation and displacement of the α-helical domain, specifically the 380-390 β-hairpin into the active site 'folds' Arg253 (MGR) into an active conformation. Closure of the active site, which prevents wasteful hydrolysis, involves movement of the β14-β15 β-hairpin into the active site and simultaneous rearrangement of the PPᵢ-binding GGDD motif. The conformational change of the S. thermophilum PPᵢ-PFK is surprisingly complex and unique relative to prokaryotic ATP-PFKs and involves displacement of novel structural elements. These movements change the conformation of conserved motifs at the active site and therefore function to modulate PPᵢ-dependent activity

The role of charge localisation in mass spectrometry.

Ab-Initio molecular orbital calculations have been used to investigate the degree of charge localisation associated with the formation of ground state molecular radical cations upon electron impact for some simple amides, thio-amides, urea, thiourea, and their N-methyl substituted analogues, and guanidine. Some of the thio-amides which were not commercially available, were synthesised for the mass spectrometric studies. Ionisation energies have been calculated from the molecular orbital energies, using Koopmans' Theorem, and related to the predicted site of charge location in the molecular radical cations. The molecular orbital data was also used to study the effect of N-methylation on ionisation energy. The results obtained were found to be in close agreement with results obtained from photoelectron spectroscopy and electron impact mass spectrometry. Mulliken population analysis was used to obtain total atomic charges from the molecular orbital calculations so that the charge distributions in the neutral molecules and the radical cations could be compared. The molecular orbital calculations predict the major change in charge distribution to be equally shared between each nitrogen atom of urea, upon ionisation, with very little charge located on the oxygen; however for thiourea the change in charge distribution is largely located on the sulphur atom with very little change in the charge on either of the nitrogen atoms. These results are in agreement with previous predictions based on observed experimental data. The amides show more delocalisation with the charge more evenly distributed between nitrogen and oxygen, although N-methylation causes the charge to be preferentially located on nitrogen. The thio amides show the charge preferentially located on sulphur throughout. Molecular orbital calculations have also been used to investigate the energetics of the major fragmentation reaction in some of these compounds in relation to the predicted site of charge location in both the ground state molecular radical cations, and the fragment ions. This study has enabled an attempt to be made at rationalising the observed electron impact mass spectra on the basis of the calculated change in charge distribution upon ionisation of the compounds studied

Cloning, expression, and purification of the Drosophila melanogaster dosage compensation complex chromodomains and their Homo sapiens orthologues

Sexual differentiation is a fundamental characteristic of all eukaryotes, dictating sex-specific morphology, physiology and behavior. Diploid organisms with heteromorphic sex chromosomes (XX or XY) require regulatory compensation of the X chromosome to maintain correct levels of genetic expression between the sexes, a process termed sex-specific dosage compensation (SSDC). The fruit fly, Drosophila melanogaster dosage compensates by upregulating transcription of most X-linked genes two-fold. Associated with this two-fold up regulation is the male-specific lethal (MSL) complex, a RNA-protein complex comprised of at least five known proteins; MSL1, MSL2, MSL3, males absent on the first (MOF), and maleless (MLE) and two non-translated RNA molecules; roX1 (RNA on the X chromosome) and roX2. The complex modulates the chromatin structure of the male X chromosome via acetylation of H4K16. MOF and MSL3 both exhibit an N-terminal chromodomain, whose function is unclear. The MSL3 chromodomain has been suggested to bind H3K36Me3. Chromodomains are a paradigm of how a single structural fold has evolved in diverse proteins to bind distinct targets. Chromodomains are common to nuclear regulators, and bind diverse targets including histones, DNA, and RNA. They function as recognition motifs of histone post-translational modifications and facilitate the translation of the histone code into a distinct local chromatin structure via recruiting the appropriate chromatin modulating machinery. The goal of this research is to determine the structure of the D. melanogaster MOF and MSL3 chromodomains by X-ray crystallographic and/or nuclear magnetic resonance techniques, to advance our understanding of the structural characteristics of these diverse domains. Here we report the cloning and reproducible expression and purification of the D. melanogaster MOF and MSL3 chromodomains and their Homo sapiens orthologues. The D. melanogaster MOF chromodomain, whose NMR structure was published during this research, has been crystallized. Attempts to solve the crystal structure by molecular replacement, multiple-wavelength anomalous dispersion, and single-wavelength isomorphous replacement are reported

Barriers to Implementing Research and Technology into the Rail Industry: A Case Study of TPWS and GSM-R in the United Kingdom

There is considerable pressure for the rail industry to deliver and implement technologies which increase the efficiencies of the railway. The Rail Technical Strategy Europe set out by the International Union of Railways (UIC) outlines what technologies are required to meet future demands. Research is key for such projects to develop, but there is an inherent issue of progressing the research through to the rail industry, with many barriers preventing their implementation. This paper has conducted two retrospective case studies on implementation in the UK; Train Protection Warning System (TPWS) and Global Systems for Mobiles - Railways (GSM-R). Performing a literature analysis has allowed this study to view how the research in the two cases developed and by collecting qualitative data, analysis of the case studies around the topics of technology management, stakeholder management, and human factors from industry and technical experts outline the implementation approach taken. This paper uses the two cases to identify ways to overcome the barriers to implementation and suggests bridging the research - industry gap requires a more collaborative approach earlier in the research cycle to reduce the perceived risk of research and technology implementation, which in-turn increases its economic viability

Optimization preparation of the biosynthesis of silver nanoparticles using watermelon and study of its antibacterial activity

The field of nanotechnology is the most active area of research in modern materials science. There is increased demand on nanoparticles because of their wide applicability in various areas; for example, electronics, catalysis, chemistry, energy and medicine. Commonly, silver nanoparticles are fabricated using toxic and flammable chemicals. Therefore, the aim of this work is to find a cost effective and environment-friendly technique for green synthesis of silver nanoparticles from silver nitrate (AgNO3) solution using watermelon extract. The different parameters were optimised for the fabrication of silver nanoparticles including the contact time, concentration of watermelon extract, concentration of silver nitrate solution, reactant ratio, and reaction temperature. The fabricated nanoparticles were characterised using different instruments such as UV-Vis spectrophotometry and their size determined by transmission electron microscopy (TEM), while the element composition of the sample was obtained from the energy dispersive X-ray (EDX) spectrum. The antibacterial effect of the fabricated silver nanoparticles against two human pathogens Escherichia coli (E. coli) and Staphylococcus aureus (S.aureus) was studied and the obtained results confirm the fact that SNPs have the antibacterial property of nanoparticles

Increasing gas sorption onto carbon by milling with alumina

Graphite milled alone for 50 h shows an increase in gas sorption of a factor of three over unmilled powder. When graphite is milled in the presence of a much harder phase, aluminium oxide, the increase in sorption is much greater with a seven fold increase achieved after 5 h milling. Extending the milling time resulted in increased capacity; an estimated capacity of 2.5 g/ g carbon was achieved after 50 h milling