Molecular characterisation of the HMG CoA reductase gene from Neotyphodium Lolii : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Genetics at Massey University, Palmerston North, New Zealand

3-Hydroxy-3-methylglutaryl Coenzyme A reductase (Hmg) catalyses the conversion of HMG CoA to mevalonic acid: the first step of the isoprenoid biosynthetic pathway. This pathway produces a wide variety of primary metabolites which are involved in many different cellular processes. Neotyphodium endophytes in association with the grass host are known to produce a range of secondary metabolites including the indole diterpenoids (eg paxilline and lolitrem) and the ergopeptine alkaloids (eg ergovaline). Given that these pathways are upregulated in planta the availability of mevalonic acid, be it from fungal or plant source, will be important in controlling the levels of the different toxins synthesised. The aim of this work was to clone the fungal endophyte hmg and characterise the promoter to enable study of its regulation in planta via reporter gene studies. Using degenerate primers designed against conserved regions of other hmg genes a 359 bp fragment was amplified from the Neotyphodium lolii isolate Lpl9, which grows in perennial ryegrass (Lolium perenne). DNA sequencing confirmed that the sequence amplified was part of a unique hmg gene. Southern hybridisations suggest that there is a single copy of hmg in strain Lpl9 (a haploid) but two copies in strain Lpl (an interspecific hybrid; Schardl et al. 1994). The fragment of N. lolii hmg was used to screen a λGEM-12 genomic library of Lpl9 and four positive overlapping clones were isolated. Fragments of one clone. λJD12, were subcloned, sequenced and a physical map of this region of the genome was constructed. The entire sequence of hmg was determined using primer walking and was found to encode a 1188 amino acid polypeptide. From comparison to other Hmg proteins the catalytic domain has been shown to be highly conserved while the amino-terminal domain, containing transmembrane regions is divergent with very little sequence similarity near the translation start site and promoter region. Using RT-PCR analysis the hmg gene was shown to consist of two open reading frames separated by a 73 bp intron. RT-PCR was also used to determine the location of the transcriptional start site and this is supported by the presence of putative CAAT and TATA consensus sequences. With the promoter region identified and characterised further analysis of the regulation of hmg in planta can be undertaken

An Examination of Privacy Policies of Global On-line E-pharmacies

This paper investigates the differences in privacy policy functions among 90 online pharmacy websites in nine countries in Europe, Asia and North America. Results from this study show that the majority of websites do have privacy policies, but the level of functional protection of consumers varies widely. Even in those countries where strong privacy laws exist, the level of privacy protection adherence is often very low. Most studies of privacy policy issues have concentrated on websites from developed nations, with few studies of the pharmacy industry. A better understanding of this industry, as well as understanding the differences in privacy policy implementation among developing and developed countries, provides important lessons for both businesses and consumers

Increased survival of experimentally evolved antimicrobial peptide-resistant Staphylococcus aureus in an animal host.

Antimicrobial peptides (AMPs) have been proposed as new class of antimicrobial drugs, following the increasing prevalence of bacteria resistant to antibiotics. Synthetic AMPs are functional analogues of highly evolutionarily conserved immune effectors in animals and plants, produced in response to microbial infection. Therefore, the proposed therapeutic use of AMPs bears the risk of ‘arming the enemy’: bacteria that evolve resistance to AMPs may be cross‐resistant to immune effectors (AMPs) in their hosts. We used a panel of populations of Staphylococcus aureus that were experimentally selected for resistance to a suite of individual AMPs and antibiotics to investigate the ‘arming the enemy’ hypothesis. We tested whether the selected strains showed higher survival in an insect model (Tenebrio molitor) and cross‐resistance against other antimicrobials in vitro. A population selected for resistance to the antimicrobial peptide iseganan showed increased in vivo survival, but was not more virulent. We suggest that increased survival of AMP‐resistant bacteria almost certainly poses problems to immune‐compromised hosts

Practical Pedagogy for Embedding Drone Technology into a Business and Computing Curriculum

This paper outlines the design of an undergraduate module in "Applied Drone Technology‟ to enhance student engagement and learning of a new technology within a business school curriculum. It focuses on the development strategy and issues the team encountered when trying to create something outside the usual core computing and business curriculum. Although there are barriers and issues to integrating drones into a curriculum, it can be accomplished with proper planning and a strategic vision. The result was a module that can be used by students in a business school, but with the capability of being used by students in other academic units

Recon 2.2: from reconstruction to model of human metabolism.

IntroductionThe human genome-scale metabolic reconstruction details all known metabolic reactions occurring in humans, and thereby holds substantial promise for studying complex diseases and phenotypes. Capturing the whole human metabolic reconstruction is an on-going task and since the last community effort generated a consensus reconstruction, several updates have been developed.ObjectivesWe report a new consensus version, Recon 2.2, which integrates various alternative versions with significant additional updates. In addition to re-establishing a consensus reconstruction, further key objectives included providing more comprehensive annotation of metabolites and genes, ensuring full mass and charge balance in all reactions, and developing a model that correctly predicts ATP production on a range of carbon sources.MethodsRecon 2.2 has been developed through a combination of manual curation and automated error checking. Specific and significant manual updates include a respecification of fatty acid metabolism, oxidative phosphorylation and a coupling of the electron transport chain to ATP synthase activity. All metabolites have definitive chemical formulae and charges specified, and these are used to ensure full mass and charge reaction balancing through an automated linear programming approach. Additionally, improved integration with transcriptomics and proteomics data has been facilitated with the updated curation of relationships between genes, proteins and reactions.ResultsRecon 2.2 now represents the most predictive model of human metabolism to date as demonstrated here. Extensive manual curation has increased the reconstruction size to 5324 metabolites, 7785 reactions and 1675 associated genes, which now are mapped to a single standard. The focus upon mass and charge balancing of all reactions, along with better representation of energy generation, has produced a flux model that correctly predicts ATP yield on different carbon sources.ConclusionThrough these updates we have achieved the most complete and best annotated consensus human metabolic reconstruction available, thereby increasing the ability of this resource to provide novel insights into normal and disease states in human. The model is freely available from the Biomodels database (http://identifiers.org/biomodels.db/MODEL1603150001)

A Risk Score for Predicting Multiple Sclerosis

Multiple sclerosis (MS) develops as a result of environmental influences on the genetically susceptible. Siblings of people with MS have an increased risk of both MS and demonstrating asymptomatic changes in keeping with MS. We set out to develop an MS risk score integrating both genetic and environmental risk factors. We used this score to identify siblings at extremes of MS risk and attempted to validate the score using brain MRI.78 probands with MS, 121 of their unaffected siblings and 103 healthy controls were studied. Personal history was taken, and serological and genetic analysis using the illumina immunochip was performed. Odds ratios for MS associated with each risk factor were derived from existing literature, and the log values of the odds ratios from each of the risk factors were combined in an additive model to provide an overall score. Scores were initially calculated using log odds ratio from the HLA-DRB1*1501 allele only, secondly using data from all MS-associated SNPs identified in the 2011 GWAS. Subjects with extreme risk scores underwent validation studies. MRI was performed on selected individuals.There was a significant difference in the both risk scores between people with MS, their unaffected siblings and healthy controls (p<0.0005). Unaffected siblings had a risk score intermediate to people with MS and controls (p<0.0005). The best performing risk score generated an AUC of 0.82 (95%CI 0.75–0.88).The risk score demonstrates an AUC on the threshold for clinical utility. Our score enables the identification of a high-risk sibling group to inform pre-symptomatic longitudinal studies

Epidemiological Interactions between Urogenital and Intestinal Human Schistosomiasis in the Context of Praziquantel Treatment across Three West African Countries

© 2015 Knowles et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The attached file is the published version of the article

Paediatric Investigators Collaborative Network on Infections in Canada (PICNIC) study of aseptic meningitis

BACKGROUND: The seasonality, clinical and radiographic features and outcome of aseptic meningitis have been described for regional outbreaks but data from a wider geographic area is necessary to delineate the epidemiology of this condition. METHODS: A retrospective chart review was completed of children presenting with aseptic meningitis to eight Canadian pediatric hospitals over a two-year period. RESULTS: There were 233 cases of proven enteroviral (EV) meningitis, 495 cases of clinical aseptic meningitis and 74 cases of possible aseptic meningitis with most cases occurring July to October. Headache, vomiting, meningismus and photophobia were more common in children ≥ 5 years of age, while rash, diarrhea and cough were more common in children < 5 years of age. Pleocytosis was absent in 22.3% of children < 30 days of age with proven EV meningitis. Enterovirus was isolated in cerebrospinal fluid (CSF) from 154 of 389 patients (39.6%) who had viral culture performed, and a nucleic acid amplification test for enterovirus was positive in CSF from 81 of 149 patients (54.3%). Imaging of the head by computerized tomography or magnetic resonance imaging was completed in 96 cases (19.7%) and 24 had abnormal findings that were possibly related to meningitis while none had changes that were definitely related to meningitis. There was minimal morbidity and there were no deaths. CONCLUSION: The clinical presentation of aseptic meningitis varies with the age of the child. Absence of CSF pleocytosis is common in infants < 30 days of age. Enterovirus is the predominant isolate, but no etiologic agent is identified in the majority of cases of aseptic meningitis in Canadian children

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy