The mechanisms regulating exocytosis of the salivary glands of the soft tick,Ornithodoros savignyi

Numerous bioactive compounds are secreted from large dense core granules in tick salivary glands during feeding. Investigations into the signalling pathways regulating secretion indicated that they are similar for Argasidae (fast feeding ticks) and Ixodidae (slow-feeding ticks). In both cases, dopamine is the external signal that activates adenylyl cyclase, subsequently cyclic AMP levels are increased and Protein Kinase A (PKA) is activated, resulting in the phosphorylation of proteins. Secretion was also found to be highly calcium dependant. Firstly, it requires extracellular calcium (via a L-type voltage-gated calcium channel located on the plasma membrane) and secondly, intracellular calcium which is released presumably in response to IP3. In contrast to numerous exocrine cells, membrane depolarisation and elevation of the cAMP levels are not sufficient for inducing exocytosis from O. savignyi salivary glands. Pathways such as the activation of Phospholipase C, inositol-phosphate kinases, Na+K+-ATPases, as well as the disassembly of the actin barrier, have been shown to be essential. Finally, our research also indicated a need for the ATPase NSF, an intact microtubule network and an active cytosolic Phospholipase A2 for exocytosis. A model has been suggested, but a great deal of research is needed to elucidate all the mechanisms of regulated exocytosis. All secretory eukaryotic cells to date require SNARE proteins for fusion of granules with the plasma membrane, leading to the release of granular content. By means of Western blotting we identified the tick homologues of the SNAREs syntaxin, SNAP25 and VAMP, as well as the small GTPase Rab3a, all enriched within the membrane fraction. We also identified the SDS-resistant 20S complex, which forms during the docking of granules and is composed of the three SNARE proteins. Confocal microscopy of the SNARE proteins indicates SNAP25 and VAMP localize to the granule membranes, while syntaxin localises strictly to the plasma membrane. In order to isolate the tick SNARE homologues we exploited protein-protein interactions by means of the yeast two-hybrid system. Screening of an O. savignyi cDNA salivary gland library using rat brain a-SNAP as bait, we identified a transcript encoding a tick syntaxin homologue. It encoded a 126 residue protein which shares 14% identity and 40% similarity with human syntaxin 1. Furthermore, we were able to successfully model the identified protein onto the known crystal structure of human syntaxin 1 and indicate that it shares structural homology with helices 1, 2, 3 and the connecting two loop regions. Following screening of the library with a truncated syntaxin bait construct, two novel domains were identified in all the interacting clones. To date their identity remains unknown. Functional complementation in the syntaxin knockout yeast strain H603 with an O. savignyi cDNA library resulted in the identification of four novel transcripts, which suppressed the temperature sensitive phenotype. Two of these share homology with the N- and C-terminals of syntaxins respectively and were successfully modelled onto the human syntaxin 1 crystal structure. Finally, by exploiting the extensive SNARE binding properties of recombinant rat brain a-SNAP, we were able to isolate the O. savignyi SNAREs, i.e. syntaxin, SNAP25 and VAMP, using pull-down assays. These purified proteins will soon be subjected to amino acid sequencing, and their sequences used to confirm the identified transcripts as true syntaxins. By enhancing our understanding of the molecular basis underlying tick feeding, as well as the proteins involved in the processes, we hope to identify possible targets for the rational design of a viable tick vaccine.Thesis (PhD (Biochemistry))--University of Pretoria, 2007.Biochemistryunrestricte

The evolution of land use and development control and their relevance in a future South Africa

Study project (M. in Town and Regional Planning) -- University of Stellenbosch, 1992.ENGLISH SUMMARY: Modern land use planning and development control originated late in the previous century as a reaction to the poor living conditions created by the industrial revolution. Many types of controlling measures were formed and evolved through the years into the present systems of control. The question is whether these controls still have relevance today. In this study project the evolution of control in Britain, the USA and South Africa is traced, and the present systems in these countries evaluated. The British land use planning system of compulsory development plans and the obtaining of planning permission in any development has experienced some degree of deregulation of planning controls in the 1980s. This led to greater freedom for prospective developers, although there still is a strong feeling that development control must have a stronger, more clear relationship with definite policy. In the absence of policy, control can become arbitrary and ineffective and, instead of reducing uncertainty, enhances and intensifies it. The principle central to US land use planning is the preparation of comprehensive plans, which addresses total socio-economic-physical development, thereby leading development and not restricting it. The most important method of implementing planning is zoning, which can be said to be the 'end' of the planning process and aimed mainly at control. Zoning is therefore the principle legislative land use control instrument in the US. South African land use planning and development control evolved within a unique political framework, whereby different systems were applied for different races. Traditionally, land use planning has been done mainly through town planning, which has zoning as its central principle. Town planning (zoning) schemes create a mechanism whereby the use of land can be controlled. In contrast to American and British practice, South African town planning is often restricted to land use planning and therefore tends to be restrictive rather than offering positive methods or solutions for economic development and the alleviation of poor living conditions. The present situation in South Arica, where mass urbanization, unemployment and poverty affect the lives of millions of people, leads to the belief that less rigid and inflexible control measures can create an environment where informal activities will help in the social upliftment of these deprived people. The existing planning system was therefore found not to be very effective in poor areas. There is a real need for a system to provide for control in highly formal "first world" areas and informal "third world" settlements. The conflict between 'more control' and 'less control' is central in this need. In the study it was, however, concluded that restrictive controls should be kept to a minimum and that land use control should be separated from the current over-abundant control measures in order to help create self-sufficient and sustainable communities. A more innovative approach is need, where town planning is more pro-active and not just an effort to cope with day to day problems.AFRIKAANSE OPSOMMING: Moderne grondgebruikbeplanning en ontwikkelingsbeheer het laat in die vorige eeu ontstaan as In reaksie op die uiters swak lewensomstandighede wat deur die industriele revolusie veroorsaak is. Verskeie tipes beheermaatreels is geskep en het deur die jare verander om die huidige beheersisteme te vorm. Die vraag is of die beheer vandag nog enige relevansie het. In hierdie werkstuk word die evolusie van beheer in Brittanje, die VSA en Suid-Afrika nagespeur en die huidige sisteme in die lande geevalueer. Die Britse grondgebruikbeplanningsisteem van verpligte ontwikkelingsplanne en die verkryging van beplanningstoestemming vir enige ontwikkeling, het gedurende die 1980s In groot mate van deregulering ondergaan. Dit het gelei tot groter vryheid vir voornemende ontwikkelaars. Daar bestaan tog In sterk gevoel dat ontwikkelingsbeheer In sterker, meer duidelike verband met definitiewe beleid moet toon. In die afwesigheid van beleid kan beheer arbitrer en oneffektief word en, in plaas van om onsekerheid te verminder, dit aanmoedig of vererger. Die sentrale beginsel van Amerikaanse grondgebruikbeplanning is die voorbereiding van komprehensiewe of omvattende planne wat totale sosioekonomiese- fisiese ontwikkeling aanspreek en sodoende ontwikkeling lei en nie beperk nie. Die belangrikste metode vir beplanningsimplementering is sonering, wat beskou kan word as die leindpuntl van die beplanningsproses en wat hoofsaaklik op beheer gemik is. Sonering is dus die belangrikste wetlike grondgebruikbeheerinstrument in die VSA. Suid-Afrikaanse grondgebruikbeplanning en ontwikkelingsbeheer het binne 'n unieke politi eke raamwerk, waar verskillende sisteme op verskiUende rasse toegepas is, ontwikkel. Tradisioneel is grondgebruikbeplanning hoofsaaklik deur stadsbeplanning, met sonering as kernbegrip, gedoen. Soneringskemas skep dan In meganisme waarmee grondgebruik beheer kan word. In teenstelling met die VSA en Brittanje, word Suid-Afrikaanse stadsbeplanning gereeld beperk tot grondgebruikbeplanning. Gevolglik neig dit om beperkend te wees, eerder dat dit positiewe metodes en oplossings bied vir ekonomiese ontwikkeling en die verbetering van swak lewensomstandighede. Die huidige situasie in Suid-Afrika, waar massa verstedeliking, werkloosheid en armoede miljoene minderbevoorregte mense daagliks raak, lei tot die gevoel dat minder streng en onbuigsame beheermaatreels 'n omgewing kan skep waar informele aktiwiteite kan bydra tot die sosiale opheffing van die minderbevoorregtes. Dfe huidige beplanningsisteem is egter nie baie effektief ' in arm gebiede nie. Daar is dus 'n groot behoefte aan 'n sisteem wat voorsiening maak vir beheer in formele "eerste wereld" gebiede en vir informele "derde wereld" vestigings. Die konflik tussen 'meer beheer' en 'minder beheer' is sentraal in hierdie behoefte. In die werkstuk is dit bevind dat beperkende beheermeganismes tot 'n minimum beperk moet word en dat grondgebruikbeheer geskei moet word van die huidige oorvloedige beheermeganismes in 'n poging om selfonderhoudende en lewensvatbare gemeenskappe te stig. 'n Meer innovatiewe en verbeeldingryke benadering is nodig, waar stadsbeplanning pro-aktief is en nie net 'n poging om daaglikse probleme te hanteer nie

Insight into the Machinery and Applications for Understanding the Pathogen- Vector Interface

The availability of genome sequencing data in combination with knowledge of expressed genes via transcriptome and proteome data has greatly advanced our understanding of arthropod vectors of disease. Not only have we gained insight into vector biology, but also into their respective vector-pathogen interactions. By combining the strengths of postgenomic databases and reverse genetic approaches such as RNAi, the numbers of available drug and vaccine targets, as well as number of transgenes for subsequent transgenic or paratransgenic approaches, have expanded. These are now paving the way for in- field control strategies of vectors and their pathogens. Basic scientific questions, such as understanding the basic components of the vector RNAi machinery, is vital, as this allows for the transfer of basic RNAi machinery components into RNAi-deficient vectors, thereby expanding the genetic toolbox of these RNAi-deficient vectors and pathogens. In this review, we focus on the current knowledge of arthropod vector RNAi machinery and the impact of RNAi on understanding vector biology and vector-pathogen interactions for which vector genomic data is available on VectorBase

Deep convolutional neural network for mill feed size characterization

Knowing the characteristics of the feed ore size is an important consideration for operations and control of a run-of-mine ore milling circuit. Large feed ore variations are important to detect as they require intervention, whether it be manual by the operator or by an automatic controller. A deep convolutional neural network is used in this work to classify the feed ore images into one of four classes. A VGG16 architecture is used and the classifier is trained making use of transfer learning.The National Research Foundation of South Africahttps://www.journals.elsevier.com/ifac-papersonlineam2020Electrical, Electronic and Computer Engineerin

Comparative microarray analyses of adult female midgut tissues from feeding Rhipicephalus species

The cattle tick, Rhipicephalus microplus, has a debilitating effect on the livestock industry worldwide, owing to its being a vector of the causative agents of bovine babesiosis and anaplasmosis. In South Africa, co-infestation with R. microplus and R. decoloratus, a common vector species on local livestock, occurs widely in the northern and eastern parts of the country. An alternative to chemical control methods is sought in the form of a tick vaccine to control these tick species. However, sequence information and transcriptional data for R. decoloratus is currently lacking. Therefore, this study aimed at identifying genes that are shared between midgut tissues of feeding adult female R. microplus and R. decoloratus ticks. In this regard, a custom oligonucleotide microarray comprising of 13,477 R. microplus sequences was used for transcriptional profiling and 2476 genes were found to be shared between these Rhipicephalus species. In addition, 136 transcripts were found to be more abundantly expressed in R. decoloratus and 1084 in R. microplus. Chi-square analysis revealed that genes involved in lipid transport and metabolism are significantly overrepresented in R. microplus and R. decoloratus. This study is the first transcriptional profiling of R. decoloratus and is an additional resource that can be evaluated further in future studies for possible tick control.The Red Meat Research Development Trust Gauteng Department of Agriculture and Rural Development,University of Pretoria Research Development Program, and the National Research Foundation’s Technology and Human Resourcesfor Industry Program (Grant No. TP2010072300035).http://www.elsevier.com/locate/ttbdishb201

Estimating ore particle size distribution using a deep convolutional neural network

In this work the ore particle size distribution is estimated from an input image of the ore. The normalized weight of ore in each of 10 size classes is reported with good accuracy. A deep convolutional neural network, making use of the VGG16 architecture, is deployed for this task. The goal of using this method is to achieve accurate results without the need for rigorous parameter selection, as is needed with traditional computer vision approaches to this problem. The feed ore particle size distribution has an impact on the performance and control of minerals processing operations. When the ore size distribution undergoes significant changes, operational intervention is usually required (either by the operator or by an automatic controller).The National Research Foundation of South Africahttps://www.journals.elsevier.com/ifac-papersonlinepm2021Electrical, Electronic and Computer Engineerin

A systematic, functional genomics, and reverse vaccinology approach to the identification of vaccine candidates in the cattle tick, Rhipicephalus

In the post-genomic era, reverse vaccinology is proving promising in the development of vaccines against bacterial and viral diseases, with limited application in ectoparasite vaccine design. In this study, we present a systematic approach using a combination of functional genomics (DNA microarrays) techniques and a pipeline incorporating in silico prediction of subcellular localization and protective antigenicity using VaxiJen for the identification of novel anti-tick vaccine candidates. A total of 791 candidates were identified using this approach, of which 176 are membrane-associated and 86 secreted soluble proteins. A preliminary analysis on the antigenicity of selected membrane proteins using anti-gut antisera yielded candidates with an IgG binding capacity greater than previously identified epitopes of Bm86. Subsequent vaccination trials using recombinant proteins will not only validate this approach, but will also improve subsequent reverse vaccinology approaches for the identification of novel anti-tick vaccine candidates.The Red Meat Research Development Trust, University of Pretoria Research Development Programme, and the Technology and Human Resources for Industry Programme.http://www.elsevier.de/ttbdishb201

Transmembrane proteins - mining the cattle tick transcriptome

Managing the spread and load of pathogen-transmitting ticks is an important task worldwide. The cattletick, Rhipicephalus microplus, not only impacts the economy through losses in dairy and meat pro-duction, but also raises concerns for human health in regards to the potential of certain transmittedpathogens becoming zoonotic. However, novel strategies to control R. microplus are hindered by lack ofunderstanding tick biology and the discovery of suitable vaccine or acaricide targets. The importance oftransmembrane proteins as vaccine targets are well known, as is the case in tick vaccines with Bm86 asantigen. In this study, we describe the localization and functional annotation of 878 putative transmem-brane proteins. Thirty proteins could be confirmed in the R. microplus gut using LC-MS/MS analysis andtheir roles in tick biology are discussed. To the best of our knowledge, 19 targets have not been reportedbefore in any proteomics study in various tick species and the possibility of using the identified proteins astargets for tick control are discussed. Although tissue expression of identified putative proteins throughexpansive proteomics is necessary, this study demonstrates the possibility of using bioinformatics forthe identification of targets for further evaluation in tick control strategies.Red Meat Research Development Trust, the University of Pretoria Research Development Programme and the Technology and Human Resources for Industry Programme.http://www.elsevier.com/locate/ttbdis2016-09-30hb201

Gene expression profiling of adult female tissues in feeding Rhipicephalus microplus cattle ticks

The southern cattle tick, Rhipicephalus microplus, is an economically important pest, especially for resource-poor countries, both as a highly adaptive invasive species and prominent vector of disease. The increasing prevalence of resistance to chemical acaricides and variable efficacy of current tick vaccine candidates highlight the need for more effective control methods. In the absence of a fully annotated genome, the wealth of available expressed sequence tag sequence data for this species presents a unique opportunity to study the genes that are expressed in tissues involved in blood meal acquisition, digestion and reproduction during feeding. Utilising a custom oligonucleotide microarray designed from available singletons (BmiGI Version 2.1) and expressed sequence tag sequences of R. microplus, the expression profiles in feeding adult female midgut, salivary glands and ovarian tissues were compared. From 13,456 assembled transcripts, 588 genes expressed in all three tissues were identified from fed adult females 20 days post infestation. The greatest complement of genes relate to translation and protein turnover. Additionally, a number of unique transcripts were identified for each tissue that relate well to their respective physiological/biological function/role(s). These transcripts include secreted anti-hemostatics and defense proteins from the salivary glands for acquisition of a blood meal, proteases as well as enzymes and transporters for digestion and nutrient acquisition from ingested blood in the midgut, and finally proteins and associated factors involved in DNA replication and cell-cycle control for oogenesis in the ovaries. Comparative analyses of adult female tissues during feeding enabled the identification of a catalogue of transcripts that may be essential for successful feeding and reproduction in the cattle tick, R. microplus. Future studies will increase our understanding of basic tick biology, allowing the identification of shared proteins/pathways among different tissues that may offer novel targets for the development of new tick control strategies.The Red Meat Research Development Trust, University of Pretoria Research Development Programme and the Technology and Human Resources for Industry Programme, South Africa. Additional funding and student support was obtained from the National Research Foundation of South Africa.http://www.elsevier.com/locate/ijpar

Probing the Rhipicephalus bursa sialomes in potential anti-tick vaccine candidates : a reverse vaccinology approach

In the wake of the ‘omics’ explosion of data, reverse vaccinology approaches are being applied more readily as an alternative for the discovery of candidates for next generation diagnostics and vaccines. Promising protective antigens for the control of ticks and tick-borne diseases can be discovered by mining available omics data for immunogenic epitopes. The present study aims to explore the previously obtained Rhipicephalus bursa sialotranscriptome during both feeding and Babesia infection, to select antigenic targets that are either membrane-associated or a secreted protein, as well as unique to the ectoparasite and not present in the mammalian host. Further, they should be capable of stimulating T and B cells for a potential robust immune response, and be non-allergenic or toxic to the host. From the R. bursa transcriptome, 5706 and 3025 proteins were identified as belonging to the surfaceome and secretome, respectively. Following a reverse genetics immunoinformatics pipeline, nine preferred candidates, consisting of one transmembrane-related and eight secreted proteins, were identified. These candidates showed a higher predicted antigenicity than the Bm86 antigen, with no homology to mammalian hosts and exposed regions. Only four were functionally annotated and selected for further in silico analysis, which examined their protein structure, surface accessibility, flexibility, hydrophobicity, and putative linear B and T-cell epitopes. Regions with overlapping coincident epitopes groups (CEGs) were evaluated to select peptides that were further analyzed for their physicochemical characteristics, potential allergenicity, toxicity, solubility, and potential propensity for crystallization. Following these procedures, a set of three peptides from the three R. bursa proteins were selected. In silico results indicate that the designed epitopes could stimulate a protective and long-lasting immune response against those tick proteins, reflecting its potential as anti-tick vaccines The immunogenicity of these peptides was evaluated in a pilot immunization study followed by tick feeding to evaluate its impact on tick behavior and pathogen transmission. Combining in silico methods with in vivo immunogenicity evaluation enabled the screening of vaccine candidates prior to expensive infestation studies on the definitive ovine host animals.Spreadsheet S1 – SurfaceomeSpreadsheet S2 – SecretomeSpreadsheet S3 – MARVELSpreadsheet S4 – EVASINSpreadsheet S5 - RICINFundação para a Ciência e Tecnologia (FCT)http://www.mdpi.com/journal/biomedicinespm2021BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant PathologyPlant Production and Soil Scienc