Expression, purification and characterisation of recombinant peptide:N-glycosidase F. : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Biochemistry at Massey University

PNGase F (Peptide-N4-(N-acetyl-D-glucosaminyl) asparagine amidase F) is an amidohydrolase isolated from the extracellular medium of the Gram-negative bacterium Flavobacterium meningosepticum. The 34.8-kDa enzyme catalyses the complete and intact cleavage of asparagine-linked oligosaccharide chains from their associated proteins. A T7 promoter-based E. coli expression system was developed in which PNGase F was expressed as a fusion protein with a leader sequence from the ompA gene. The hexa-histidine-tagged PNGase F was correctly processed and exported to the E. coli periplasm and had a calculated molecular weight of 36.2 kDa. A single step purification using immobilised metal affinity chromatography yielded 8 mg of pure protein per litre of culture. The sequence of the PNGase F coding region from the CDC strain 3352 of F. meningosepticum was found to differ from a published sequence from another strain of the bacterium (ATCC 33958) in 57 positions. These differences between the two strains result in eight amino acid substitutions, which are mostly conservative in nature and are on the surface of the protein. Moreover, three potential N-glycosylation sites not present in the ATCC strain 33958 were detected in CDC strain 3352. The recombinant enzyme has similar characteristics of the native enzyme with a pH optimum of 8.5 and is strongly inhibited by Ag+, Cu2+, Fe3+ ions but not by sulfhydryl-targeting agents such as DTT and NEM. This indicates inhibition by these ions is probably through interactions with a histidine residue at position 193 that may be involved in substrate recognition or catalysis. The specific activity of the native PNGase F is about four times that of the recombinant protein which may be contributed to inhibition by components of the CompleteTM protease inhibitor tablets used in the enzyme preparation or due to modifications for cloning and purification. Using a discontinuous assay and a non-labelled 11-mer ovalbumin-derived glycopeptide as substrate, a rough estimate of the Michaelis constant (Km) for the recombinant PNGase F was determined to be 2.1 μM. An intriguing observation with the activity assays was the apparent product inhibition of enzyme activity and the inhibitor may be either peptide and/or glycan components, which require further investigations into the cause of the inhibition

Common security issues and challenges in wireless sensor networks and IEEE 802.11 wireless mesh networks

Both Wireless Mesh Network (WMN) and Wireless Sensor Network (WSN) are multi-hop wireless networks. WMN is an emerging community based integrated broadband wireless network which ensures high bandwidth ubiquitous internet provision to users, while, WSN is application specific and ensures large scale real-time data processing in complex environment. Both these wireless networks have some common vulnerable features which may increase the chances of different sorts of security attacks. Wireless sensor nodes have computation, memory and power limitations, which do not allow for implementation of complex security mechanism. In this paper, we discuss the common limitations and vulnerable features of WMN and WSN, along with the associated security threats and possible countermeasures. We also propose security mechanisms keeping in view the architecture and limitations of both. This article will serve as a baseline guide for the new researchers who are concern with the security aspects of WMN and WSN

Addressing the needs of traumatic brain injury with clinical proteomics.

BackgroundNeurotrauma or injuries to the central nervous system (CNS) are a serious public health problem worldwide. Approximately 75% of all traumatic brain injuries (TBIs) are concussions or other mild TBI (mTBI) forms. Evaluation of concussion injury today is limited to an assessment of behavioral symptoms, often with delay and subject to motivation. Hence, there is an urgent need for an accurate chemical measure in biofluids to serve as a diagnostic tool for invisible brain wounds, to monitor severe patient trajectories, and to predict survival chances. Although a number of neurotrauma marker candidates have been reported, the broad spectrum of TBI limits the significance of small cohort studies. Specificity and sensitivity issues compound the development of a conclusive diagnostic assay, especially for concussion patients. Thus, the neurotrauma field currently has no diagnostic biofluid test in clinical use.ContentWe discuss the challenges of discovering new and validating identified neurotrauma marker candidates using proteomics-based strategies, including targeting, selection strategies and the application of mass spectrometry (MS) technologies and their potential impact to the neurotrauma field.SummaryMany studies use TBI marker candidates based on literature reports, yet progress in genomics and proteomics have started to provide neurotrauma protein profiles. Choosing meaningful marker candidates from such 'long lists' is still pending, as only few can be taken through the process of preclinical verification and large scale translational validation. Quantitative mass spectrometry targeting specific molecules rather than random sampling of the whole proteome, e.g., multiple reaction monitoring (MRM), offers an efficient and effective means to multiplex the measurement of several candidates in patient samples, thereby omitting the need for antibodies prior to clinical assay design. Sample preparation challenges specific to TBI are addressed. A tailored selection strategy combined with a multiplex screening approach is helping to arrive at diagnostically suitable candidates for clinical assay development. A surrogate marker test will be instrumental for critical decisions of TBI patient care and protection of concussion victims from repeated exposures that could result in lasting neurological deficits