Novel Anti-Campylobacter Compounds Identified Using High Throughput Screening of a Pre-selected Enriched Small Molecules Library

Campylobacter is a leading cause of foodborne bacterial gastroenteritis worldwide and infections can be fatal. The emergence of antibiotic-resistant Campylobacter spp. necessitates the development of new antimicrobials. We identified novel anti-Campylobacter small molecule inhibitors using a high throughput growth inhibition assay. To expedite screening, we made use of a “bioactive” library of 4,182 compounds that we have previously shown to be active against diverse microbes. Screening for growth inhibition of Campylobacter jejuni, identified 781 compounds that were either bactericidal or bacteriostatic at a concentration of 200 µM. Seventy nine of the bactericidal compounds were prioritized for secondary screening based on their physico-chemical properties. Based on the minimum inhibitory concentration against a diverse range of C. jejuni and a lack of effect on gut microbes, we selected 12 compounds. No resistance was observed to any of these 12 lead compounds when C. jejuni was cultured with lethal or sub-lethal concentrations suggesting that C. jejuni is less likely to develop resistance to these compounds. Top 12 compounds also possessed low cytotoxicity to human intestinal epithelial cells (Caco-2 cells) and no hemolytic activity against sheep red blood cells. Next, these 12 compounds were evaluated for ability to clear C. jejuni in vitro. A total of 10 compounds had an anti-C. jejuni effect in Caco-2 cells with some effective even at 25 µM concentrations. These novel 12 compounds belong to five established antimicrobial chemical classes; piperazines, aryl amines, piperidines, sulfonamide and pyridazinone. Exploitation of analogues of these chemical classes may provide Campylobacter specific drugs that can be applied in both human and animal medicine.

Processing of oil enriched fish (Hypophthalmichthys molitrix) sausage using emulsion technology

In most countries along with various food products, fish sausage is supplied in different formulas. Unfortunately, in our country because of different reasons, production and supply of fish sausage in industrial level has not yet been successful and some efforts taken, has also been doomed to failure or not welcomed. Fat fish is a rich source of poly unsaturated fatty acids (PUFA) and co-3. In this research, efforts have been made to produce and enrich sausage with fish oil and maintenance of fatty acids has also been experimented using gas chromatography along with heating process. The stages of producing ground fish and fish sausage are as the following: Transferring and preparing fish, washing the cleared fish, filleting, separating fillet steak, washing and drying them, Refining meat, Producing and homogenizing mixture from basic ingredients in a cutter, filling, knotting and heat processing. The fish sausage produced by this method tried and welcomed by the subjects. In the product in which fish meat was used, the subjects was not recognized fish flavor and taste and when in addition to fish meat, fish oil was used during enrichment, the flavor and taste of fish was considered as highly acceptable. TVN measurement of the produced fish sausage was kept in the refrigerator in two month was at a maximum of 16.5, the amount of peroxide was at a maximum 1.5% after the period of two months. During this period the Colony count was at maximum of 19.5 x 104, the high maximum of the number of coliforms was 10/gr, and for mold and yeast 83/gr , but Escherichia coli, Staphylococcus aureus, Salmonella and Clostridium perfringens were not found. The protein of the resulting product was 15-18%, lipid at about 11-15% and moisture 60-65%. Comparing fatty acids, including unsaturated fatty acids in ground and oil fish used in producing fish sausage with those of fish sausage showed that the heat used in processing had the least effect on fatty acids of the meat and oil used here and the resulting fish sausage is considered as food for good health.

APP RERMS enriched matrix as an adhesion substrate for neuritic outgrowth

Specific domains can determine protein structural functional relationships. For the Alzheimer’s Amyloid Precursor Protein (APP) several domains have been described, both in its intracellular and extracellular fragments. Many functions have been attributed to APP including an important role in cell adhesion and cell to cell recognition. This places APP at key biological responses, including synaptic transmission. To fulfil these functions, extracellular domains take on added significance. The APP extracellular domain RERMS is in fact a likely candidate to be involved in the aforementioned physiological processes. A multidisciplinary approach was employed to address the role of RERMS. The peptide RERMS was crosslinked to PEG (Polyethylene glycol) and the reaction validated by FTIR (Fourier transform infrared spectrometry). FTIR proved to be the most efficient at validating this reaction because it requires only a drop of sample, and it gives information about the reactions occurred in a mixture. The data obtained consist in an infrared spectra of the sample, where peaks positions give information about the structure of the molecules, and the intensity of peaks is related to the concentration of the molecules. Subsequently substrates of PEG impregnated with RERMS were prepared and SH-SY5Y (human neuroblastoma cell line) cells were plated and differentiated on the latter. Several morphological alterations were clearly evident. The RERMS peptide provoked cells to take on a flatter appearance and the cytoskeletal architecture changed, with the appearance of stress fibres, a clear indicator of actin reorganization. Given that focal adhesions play a key role in determining cellular structure the latter were directly investigated. Focal adhesion kinase (FAK) is one of the most highly expressed proteins in the CNS (central nervous system) during development. It has been described to be crucial for radial migration of neurons. FAK can be localized in growth cones and mediated the response to attractive and repulsive cues during migration. One of the mechanisms by which FAK becomes active is by auto phosphorylation at tyrosine 397. It became clearly evident that in the presence of the RERMS peptide pFAK staining at focal adhesions intensified and more focal adhesions became apparent. Furthermore speckled structures in the nucleus, putatively corresponding to increased expression activity, also increased with RERMS. Taken together these results indicate that the RERMS domain in APP plays a critical role in determining cellular physiological responses. Here is suggested a model by which RERMS domain is recognized by integrins and mediate intracellular responses involving FAK, talin, actin filaments and vinculin. This mechanism probably is responsible for mediating cell adhesion and neurite outgrowth on neurons.

Use of inert gas-enriched atmospheres and post-packaging irradiation in shelf-life extension of watercress

Watercress (Nasturtium officinale R. Br.) is a semi-aquatic plant of the Brassicaceae family highly appreciated in the Mediterranean cuisine. It features sharp, peppery and slightly tangy taste and contains health-promoting phytochemicals. Its consumption as a fresh-cut product has increased in recent years, as well as the global market of minimally processed vegetables. This demand is driven by the growing interest in the role of food in promoting the human health and wellbeing and to meet consumer needs for fresh-like and more convenient foods. Due to the reduced shelf-life of this plant, the suitability of inert gas-enriched atmospheres and ionizing irradiation for preserving visual, nutritional and functional quality attributes during cold storage was studied. Watercress samples were gathered in the Northeast region of Portugal, rinsed in tap water and a portion was immediately analyzed (non-stored control). The remaining fresh material was packaged in polyethylene bags under N2- and Ar-enriched atmospheres, conventional atmosphere (air) and vacuum (no atmosphere). Samples under conventional atmosphere were irradiated at 1, 2 and 5 kGy of gamma-rays (predicted doses) in a 60Co experimental chamber. A non-irradiated control followed all the experiment. Then, all packaged samples were stored at 4 ºC for 7 days. The studied quality parameters included the colour that was measured with a Konica Minolta colorimeter, and total soluble solids and pH determined in squeezed juice. The proximate composition (moisture, proteins, fat, ash, carbohydrates and energy) was evaluated using the AOA C procedures. Organic acids, free sugars, fatty acids and tocopherols were analyzed by chromatographic techniques. Samples were also evaluated for its DPPH• scavenging activity, reducing power, and lipid peroxidation inhibition capacity trough the inhibition of the β-carotene bleaching and thiobarbituric acid reactive substances (TBAR S) formation. Differences among treatments were analyzed using the one-way analysis of variance (ANO VA) and a linear discriminant analysis (LDA ) was used to evaluate the effects on the overall postharvest quality. After evaluating the effect on the individual quality parameters, the LDA revealed that the Ar-enriched atmosphere and the irradiation at 2 kGy were suitable processing choices for preserving the integrity of the non-stored control samples during cold storage. Thus, these non-thermal treatments were highlighted for shelf-life extension of fresh-cut watercress.

Peat humic substances enriched with nutrients for agricultural applications: Competition between nutrients and non-essential metals present in tropical soils

Improved agricultural productivity, and reduction of environmental impacts, require studies of the interactions between different soil components. Fertilizers marketed as "organic" or "natural", such as peats or humic substances (HS) extracted from peats, are enriched with macro and micronutrients that, according to the manufacturers, are released to the plant in accordance with its needs. This work investigates the complexation capacity of HS for macro and micronutrient metal species, considering the competition, for HS complexation sites, between non-essential metals (aluminium and lead), present in the soil, and the nutrients. Humic substances were found to possess strong affinities for Pb(II) and Al(III), forming stable complexes, with concomitant release of complexed nutrients. Although HS are already used commercially as organic fertilizers, further studies of methods of HS enrichment, aimed at avoiding losses, are highly desirable from environmental and economic perspectives. (C) 2009 Elsevier B.V. All rights reserved.

Genome-wide copy number variation (CNV) detection in Nelore cattle reveals highly frequent variants in genome regions harboring QTLs affecting production traits.

Background: Copy number variations (CNVs) have been shown to account for substantial portions of observed genomic variation and have been associated with qualitative and quantitative traits and the onset of disease in a number of species. Information from high-resolution studies to detect, characterize and estimate population-specific variant frequencies will facilitate the incorporation of CNVs in genomic studies to identify genes affecting traits of importance. Results: Genome-wide CNVs were detected in high-density single nucleotide polymorphism (SNP) genotyping data from 1,717 Nelore (Bos indicus) cattle, and in NGS data from eight key ancestral bulls. A total of 68,007 and 12,786 distinct CNVs were observed, respectively. Cross-comparisons of results obtained for the eight resequenced animals revealed that 92 % of the CNVs were observed in both datasets, while 62 % of all detected CNVs were observed to overlap with previously validated cattle copy number variant regions (CNVRs). Observed CNVs were used for obtaining breed-specific CNV frequencies and identification of CNVRs, which were subsequently used for gene annotation. A total of 688 of the detected CNVRs were observed to overlap with 286 non-redundant QTLs associated with important production traits in cattle. All of 34 CNVs previously reported to be associated with milk production traits in Holsteins were also observed in Nelore cattle. Comparisons of estimated frequencies of these CNVs in the two breeds revealed 14, 13, 6 and 14 regions in high (>20 %), low (<20 %) and divergent (NEL > HOL, NEL < HOL) frequencies, respectively. Conclusions: Obtained results significantly enriched the bovine CNV map and enabled the identification of variants that are potentially associated with traits under selection in Nelore cattle, particularly in genome regions harboring QTLs affecting production traits.

The proximal first exon architecture of the murine ghrelin gene is highly similar to its human orthologue

BACKGROUND: The murine ghrelin gene (Ghrl), originally sequenced from stomach tissue, contains five exons and a single transcription start site in a short, 19 bp first exon (exon 0). We recently isolated several novel first exons of the human ghrelin gene and found evidence of a complex transcriptional repertoire. In this report, we examined the 5' exons of the murine ghrelin orthologue in a range of tissues using 5' RACE. -----FINDINGS: 5' RACE revealed two transcription start sites (TSSs) in exon 0 and four TSSs in intron 0, which correspond to 5' extensions of exon 1. Using quantitative, real-time RT-PCR (qRT-PCR), we demonstrated that extended exon 1 containing Ghrl transcripts are largely confined to the spleen, adrenal gland, stomach, and skin. -----CONCLUSION: We demonstrate that multiple transcription start sites are present in exon 0 and an extended exon 1 of the murine ghrelin gene, similar to the proximal first exon organisation of its human orthologue. The identification of several transcription start sites in intron 0 of mouse ghrelin (resulting in an extension of exon 1) raises the possibility that developmental-, cell- and tissue-specific Ghrl mRNA species are created by employing alternative promoters and further studies of the murine ghrelin gene are warranted.

Investigation of highly regulated promoters for the production of recombinant proteins in plants

Plants have been identified as promising expression systems for the commercial production of recombinant proteins. Plant-based protein production or “biofarming” offers a number of advantages over traditional expression systems in terms of scale of production, the capacity for post-translation processing, providing a product free of contaminants and cost effectiveness. A number of pharmaceutically important and commercially valuable proteins, such as antibodies, biopharmaceuticals and industrial enzymes are currently being produced in plant expression systems. However, several challenges still remain to improve recombinant protein yield with no ill effect on the host plant. The ability for transgenic plants to produce foreign proteins at commercially viable levels can be directly related to the level and cell specificity of the selected promoter driving the transgene. The accumulation of recombinant proteins may be controlled by a tissue-specific, developmentally-regulated or chemically-inducible promoter such that expression of recombinant proteins can be spatially- or temporally- controlled. The strict control of gene expression is particularly useful for proteins that are considered toxic and whose expression is likely to have a detrimental effect on plant growth. To date, the most commonly used promoter in plant biotechnology is the cauliflower mosaic virus (CaMV) 35S promoter which is used to drive strong, constitutive transgene expression in most organs of transgenic plants. Of particular interest to researchers in the Centre for Tropical Crops and Biocommodities at QUT are tissue-specific promoters for the accumulation of foreign proteins in the roots, seeds and fruit of various plant species, including tobacco, banana and sugarcane. Therefore this Masters project aimed to isolate and characterise root- and seed-specific promoters for the control of genes encoding recombinant proteins in plant-based expression systems. Additionally, the effects of matching cognate terminators with their respective gene promoters were assessed. The Arabidopsis root promoters ARSK1 and EIR1 were selected from the literature based on their reported limited root expression profiles. Both promoters were analysed using the PlantCARE database to identify putative motifs or cis-acting elements that may be associated with this activity. A number of motifs were identified in the ARSK1 promoter region including, WUN (wound-inducible), MBS (MYB binding site), Skn-1, and a RY core element (seed-specific) and in the EIR1 promoter region including, Skn-1 (seed-specific), Box-W1 (fungal elicitor), Aux-RR core (auxin response) and ABRE (ABA response). However, no previously reported root-specific cis-acting elements were observed in either promoter region. To confirm root specificity, both promoters, and truncated versions, were fused to the GUS reporter gene and the expression cassette introduced into Arabidopsis via Agrobacterium-mediated transformation. Despite the reported tissue-specific nature of these promoters, both upstream regulatory regions directed constitutive GUS expression in all transgenic plants. Further, similar levels of GUS expression from the ARSK1 promoter were directed by the control CaMV 35S promoter. The truncated version of the EIR1 promoter (1.2 Kb) showed some differences in the level of GUS expression compared to the 2.2 Kb promoter. Therefore, this suggests an enhancer element is contained in the 2.2 Kb upstream region that increases transgene expression. The Arabidopsis seed-specific genes ATS1 and ATS3 were selected from the literature based on their seed-specific expression profiles and gene expression confirmed in this study as seed-specific by RT-PCR analysis. The selected promoter regions were analysed using the PlantCARE database in order to identify any putative cis elements. The seed-specific motifs GCN4 and Skn-1 were identified in both promoter regions that are associated with elevated expression levels in the endosperm. Additionaly, the seed-specific RY element and the ABRE were located in the ATS1 promoter. Both promoters were fused to the GUS reporter gene and used to transform Arabidopsis plants. GUS expression from the putative promoters was consitutive in all transgenic Arabidopsis tissue tested. Importantly, the positive control FAE1 seed-specific promoter also directed constitutive GUS expression throughout transgenic Arabidopsis plants. The constitutive nature seen in all of the promoters used in this study was not anticipated. While variations in promoter activity can be caused by a number of influencing factors, the variation in promoter activity observed here would imply a major contributing factor common to all plant expression cassettes tested. All promoter constructs generated in this study were based on the binary vector pCAMBIA2300. This vector contains the plant selection gene (NPTII) under the transcriptional control of the duplicated CaMV 35S promoter. This CaMV 35S promoter contains two enhancer domains that confer strong, constitutive expression of the selection gene and is located immediately upstream of the promoter-GUS fusion. During the course of this project, Yoo et al. (2005) reported that transgene expression is significantly affected when the expression cassette is located on the same T-DNA as the 35S enhancer. It was concluded, the trans-acting effects of the enhancer activate and control transgene expression causing irregular expression patterns. This phenomenon seems the most plausible reason for the constitutive expression profiles observed with the root- and seed-specific promoters assessed in this study. The expression from some promoters can be influenced by their cognate terminator sequences. Therefore, the Arabidopsis ARSK1, EIR1, ATS1 and ATS3 terminator sequences were isolated and incorporated into expression cassettes containing the GUS reporter gene under the control of their cognate promoters. Again, unrestricted GUS activity was displayed throughout transgenic plants transformed with these reporter gene fusions. As previously discussed constitutive GUS expression was most likely due to the trans-acting effect of the upstream CaMV 35S promoter in the selection cassette located on the same T-DNA. The results obtained in this study make it impossible to assess the influence matching terminators with their cognate promoters have on transgene expression profiles. The obvious future direction of research continuing from this study would be to transform pBIN-based promoter-GUS fusions (ie. constructs containing no CaMV 35S promoter driving the plant selection gene) into Arabidopsis in order to determine the true tissue specificity of these promoters and evaluate the effects of their cognate 3’ terminator sequences. Further, promoter truncations based around the cis-elements identified here may assist in determining whether these motifs are in fact involved in the overall activity of the promoter.

Channel tracking algorithms for highly efficient wireless broadband communications in rural areas

High-speed broadband internet access is widely recognised as a catalyst to social and economic development, having a significant impact on global economy. Rural Australia’s inherent dispersed population over a large geographical area make the delivery of efficient, well-maintained and cost-effective internet a challenging task. The novel and highly-efficient Multi-User-Single-Antenna for MIMO (MUSA-MIMO) broadband wireless communication technology can effectively be used to deliver wireless broadband access to rural areas. This research aims to develop for the first time, an efficient and accurate algorithm for the tracking and prediction of Channel State Information (CSI) at the transmitter, by characterising time variation effects of the wireless communication channel on the performance of a highly-efficient MUSA-MIMO technology particularly suited for rural communities, improving their quality of life and economic prosperity.

Development of rapid and highly resolving combinatorial genotyping schemes for Campylobacter jejuni and Campylobacter coli

Campylobacter jejuni followed by Campylobacter coli contribute substantially to the economic and public health burden attributed to food-borne infections in Australia. Genotypic characterisation of isolates has provided new insights into the epidemiology and pathogenesis of C. jejuni and C. coli. However, currently available methods are not conducive to large scale epidemiological investigations that are necessary to elucidate the global epidemiology of these common food-borne pathogens. This research aims to develop high resolution C. jejuni and C. coli genotyping schemes that are convenient for high throughput applications. Real-time PCR and High Resolution Melt (HRM) analysis are fundamental to the genotyping schemes developed in this study and enable rapid, cost effective, interrogation of a range of different polymorphic sites within the Campylobacter genome. While the sources and routes of transmission of campylobacters are unclear, handling and consumption of poultry meat is frequently associated with human campylobacteriosis in Australia. Therefore, chicken derived C. jejuni and C. coli isolates were used to develop and verify the methods described in this study. The first aim of this study describes the application of MLST-SNP (Multi Locus Sequence Typing Single Nucleotide Polymorphisms) + binary typing to 87 chicken C. jejuni isolates using real-time PCR analysis. These typing schemes were developed previously by our research group using isolates from campylobacteriosis patients. This present study showed that SNP + binary typing alone or in combination are effective at detecting epidemiological linkage between chicken derived Campylobacter isolates and enable data comparisons with other MLST based investigations. SNP + binary types obtained from chicken isolates in this study were compared with a previously SNP + binary and MLST typed set of human isolates. Common genotypes between the two collections of isolates were identified and ST-524 represented a clone that could be worth monitoring in the chicken meat industry. In contrast, ST-48, mainly associated with bovine hosts, was abundant in the human isolates. This genotype was, however, absent in the chicken isolates, indicating the role of non-poultry sources in causing human Campylobacter infections. This demonstrates the potential application of SNP + binary typing for epidemiological investigations and source tracing. While MLST SNPs and binary genes comprise the more stable backbone of the Campylobacter genome and are indicative of long term epidemiological linkage of the isolates, the development of a High Resolution Melt (HRM) based curve analysis method to interrogate the hypervariable Campylobacter flagellin encoding gene (flaA) is described in Aim 2 of this study. The flaA gene product appears to be an important pathogenicity determinant of campylobacters and is therefore a popular target for genotyping, especially for short term epidemiological studies such as outbreak investigations. HRM curve analysis based flaA interrogation is a single-step closed-tube method that provides portable data that can be easily shared and accessed. Critical to the development of flaA HRM was the use of flaA specific primers that did not amplify the flaB gene. HRM curve analysis flaA interrogation was successful at discriminating the 47 sequence variants identified within the 87 C. jejuni and 15 C. coli isolates and correlated to the epidemiological background of the isolates. In the combinatorial format, the resolving power of flaA was additive to that of SNP + binary typing and CRISPR (Clustered regularly spaced short Palindromic repeats) HRM and fits the PHRANA (Progressive hierarchical resolving assays using nucleic acids) approach for genotyping. The use of statistical methods to analyse the HRM data enhanced sophistication of the method. Therefore, flaA HRM is a rapid and cost effective alternative to gel- or sequence-based flaA typing schemes. Aim 3 of this study describes the development of a novel bioinformatics driven method to interrogate Campylobacter MLST gene fragments using HRM, and is called ‘SNP Nucleated Minim MLST’ or ‘Minim typing’. The method involves HRM interrogation of MLST fragments that encompass highly informative “Nucleating SNPS” to ensure high resolution. Selection of fragments potentially suited to HRM analysis was conducted in silico using i) “Minimum SNPs” and ii) the new ’HRMtype’ software packages. Species specific sets of six “Nucleating SNPs” and six HRM fragments were identified for both C. jejuni and C. coli to ensure high typeability and resolution relevant to the MLST database. ‘Minim typing’ was tested empirically by typing 15 C. jejuni and five C. coli isolates. The association of clonal complexes (CC) to each isolate by ‘Minim typing’ and SNP + binary typing were used to compare the two MLST interrogation schemes. The CCs linked with each C. jejuni isolate were consistent for both methods. Thus, ‘Minim typing’ is an efficient and cost effective method to interrogate MLST genes. However, it is not expected to be independent, or meet the resolution of, sequence based MLST gene interrogation. ‘Minim typing’ in combination with flaA HRM is envisaged to comprise a highly resolving combinatorial typing scheme developed around the HRM platform and is amenable to automation and multiplexing. The genotyping techniques described in this thesis involve the combinatorial interrogation of differentially evolving genetic markers on the unified real-time PCR and HRM platform. They provide high resolution and are simple, cost effective and ideally suited to rapid and high throughput genotyping for these common food-borne pathogens.