enabling technologies for health - CSIR

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enabling technologies for health - CSIR

Palesa Molukanele andDr Ted Roberts eradiatea virus with a laser beam“Once we are able to deactivate the M13virus, there’s a variety of viruses we would beinterested in experimenting with, opportunisticviruses that attack immuno-compromised individualslike people infected with HIV or pregnantwomen.”Molukanele says the advantage of using lasertherapy as opposed to drug delivery, is thatlaser therapy would not evoke problems ofdrug resistance. “With this technique, wewould be able to be very selective and deactivateonly the affected areas,” she says.Lasersexplored forthe deactivationof virusesCSIR laser scientist Dr Ted Roberts says lasertechnology is one of many possible ways ofdeactivating viruses. “The research we areworking on at the moment will allow the laserbeam to interact with the virus and subsequentlydeactivate it,” he says.The project draws on expertise in laserscience and biosciences. “[The virus] M13 isuseful for tests because it does not infect orpose harm to humans,” says Dr Roberts. “Itthrives or multiplies on E.Coli and what we arehoping to do is to put it in a solution with acertain concentration. We then apply it toE.Coli bacteria and see if it infects the normalcells.”MANY VIRUSES can be harmful and anuisance to mankind, plants and animals. Inhuman beings, they destroy or alter living cellsand cause diseases. In plants, viruses damageplant cells and ruin crops. CSIR scientists arehard at work on a multidisciplinary projecttitled Femtosecond laser deactivation ofviruses. The project is the brainchild of CSIRlaser scientist, Dr Anton du Plessis.The aim of the research is to selectively deactivatecertain viruses, while leaving the sensitivematerials, such as the host cells, unharmed,by manipulating and controlling with a femtosecondlaser system. This group is repeatingexperiments conducted in the USA to see ifthey can deactivate viruses under the sameconditions. Further experiments will then beconducted to see if they can optimise theprocess by using a very large variation oflaser parameters that can be achieved at theCSIR National Laser Centre.PhD student Palesa Molukanele elaborates:“Scientists in the USA managed to deactivatethe M13 virus with lasers. M13 is a viruscomposed of circular, single-stranded DNAthat infects bacteria. It is a non-lytic virus, inother words, it does not kill infected cells bydisrupting their plasma membranes. Infectionis not lethal; however, the infection causes turbidplaques in E. Coli, a gram-negative bacteriumthat is commonly found in the lowerintestine of warm-blooded organisms.“Given the success of the American scientists,we feel that we have a contribution to make inimproving the laser parameters,” she says.The idea, according to him, is to find out howeffective this particular concentration is and touse the same concentration for the laser irradiation.“We vary the laser parameters such aslaser intensity and exposure time in our effortsto find an optimum condition for deactivatingthe virus,” says Dr Roberts.He says limited work is done worldwide in thisarea of research. “It is exciting to be workingon a multidisciplinary project, where biologicaland physics scientists share their knowledgein pursuit of a common objective.”– Mzimasi GcukumanaEnquiries:Palesa Molukanelepmolukanele@csir.co.zaTed Robertstroberts@csir.co.zaS C I E N C E S C O P E N O V E M B E R 2 0 0 965


E N A B L I N G T E C H N O L O G I E S F O R H E A LT HPictured in front are Dr Colin Kenyon, research group leader; AnjoSteyn, PhD student. Pictured back are Caroline Kunene, laboratoryassistant; Marli Botha, PhD student; Michael Mathomu, intern;Clement Nkosi, intern; Robyn Roth, senior researcher and SilindileZunngu, intern.Upsetting thenitrogen metabolismof infection-causing bacteriaS C I E N C E S C O P E N O V E M B E R 2 0 0 966


PhD studentAnjo Steynloads a samplefor analysis byhigh pressureliquid chromatographyRECENT BREAK THROUGHS by theCSIR in the understanding of the biochemistryof infection-causing organisms, such as thetuberculosis (TB)-causing bacteria, will potentiallyenable the development of therapeuticsthat will specifically target the bacteria. Thiscould significantly impact the disease burdenof TB, HIV/Aids and other infectious diseasesin South Africa and around the world.The CSIR’s structural biology group focuses onidentifying, elucidating and clarifying potentialnew drug targets, primarily in infectious diseasessuch as TB, HIV/Aids and malaria.Part of the process of developing new drugsinvolves identifying a 'target' in the organismcausing the disease. This can include specificenzymes or reactions that can be 'upset'. TheCSIR research is focusing on developing novelanti-infectious agents that target these specificenzymes or reactions within the causativeorganism – and specifically inhibit or 'upset'the target organism. Once identified, thesedrug targets form a basis for rational drugdesign and lead drug identification andmodification programmes.One project within the structural biologygroup is focused on clarifying and understandingthe reaction mechanisms and regulationof the enzyme glutamine synthetase (GS).GS is a central enzyme involved in nitrogenmetabolism. It catalyses the reaction of threemolecules, L-glutamate, adenosine triphosphate(ATP) and ammonia, to L-glutamine,adenosine diphosphate (ADP), and inorganicphosphate. This process is important becauseglutamine acts as a precursor for the synthesisof a number of other amino acids as wellas purines and pyrimidines, which are vitalprecursors to the synthesis of nucleic acids.Of the three distinct forms of GS that occur,only GSII occurs in higher organisms (eukaryotes).GSI is found only in bacteria (eubacteria)and archaea (archabacteria). Within GSI,two significant bacterial sub-divisions exist:GSI- and GSI-ß. The TB-causing organism,Mycobacterium tuberculosis, has the GSI-ß enzyme,which is crucial for the fitness of TB andis related to its pathogenecity. The enzyme isregulated or controlled via a complex adenylylation/deadenylylationcascade. The differencebetween the bacterial and human GSenzymes enables scientists to target the bacterialenzyme specifically, without interferingwith the functioning of the human host enzyme.The theory behind the research involves examiningthe reaction mechanisms deployed byGS as well as the adenylylation/deadenylylationcontrol mechanism of GS in biochemicaldetail to identify a way to inhibit specific as-pects of the M. tuberbculosis GS functionality.In practice this has involved comparingadenylyated and deadenylyated GS inEscherichia coli and M. tuberculosis (fourfunctionally distinct enzymes in total). This isof particular value since as humans, we donot have the adenylylation/deadenylylationcascade in our GS, meaning that potentialtherapeutics could target the adenylylatedGS of TB or other causative organism withoutinterfering with normal bodily functions.CSIR researchers have discovered a specificmechanism in which ATP binds to enzymes,which is controlled and regulated in a numberof ways. Dr Colin Kenyon, who leads thisresearch says, “This breakthrough in theunderstanding of the related biochemistry cannow be exploited to produce specific classesof therapeutics that will target this mechanismfor potential control in TB as well as malariaand possibly cancer.”The biochemistry of this research has nowextended into a new area, including theidentification of novel mechanisms targetingspecific kinases to control TB, malaria andcancer.Enquiries:Colin Kenyonckenyon@csir.co.zaS C I E N C E S C O P E N O V E M B E R 2 0 0 967


Traditionally, glycosylated human therapeuticsare produced using mammalian expressionsystems, such as the Chinese hamster ovarycell lines. However, the use of animal-derivedcell lines has proved to be more expensive,less safe, complex and more susceptible toviral contamination. The use of yeast offers significantadvantages over current methodsusing animal-derived cell lines, as well aschemical synthesis. It is cheaper – due to theshorter fermentation time, safer, with a higherquality and quantity and is also locally manufactured.According to CSIR researcherNtsane Moleleki, "it could contribute significantlyto the biotechnology sector by revolutionisingthe way that therapeutic proteins aremade". To date, one group at Dartmouth Collegein the US has successfully humanised theglycosylation pathway in a different yeast.The CSIR started work on Y. lipolytica in 2006with support from BioPAD. The overall aim isto humanise the glycosylation pathway in thisyeast while at the same time improving andcustomising it as a host for expression ofhuman therapeutic proteins and peptides.The yeast Y. lipolytica was selected specificallydue to findings of a comparative studywhich demonstrated the superior nature ofY. lipolytica as a secretion host of activeheterologous proteins (i.e. proteins that arenot normally found in yeast). This yeast hasalso been studied extensively by key CSIRpartners such as the French National Institutefor Agricultural Research (INRA), which is partof the National Centre for Scientific Research(CNRS) in France.The first phase of this project has resulted in anumber of significant outputs. The primary aimhas been fulfilled through the development ofextensive human capacity in yeast geneticengineering, glycobiology techniques, proteinexpression and, in the long term, customisedyeast strains that can express humanisedbiotherapeutics. In addition to the establishmentof a core group of 11 scientists, anumber of additional students (PhDs andMScs) have also benefited. In the next cycleof funding, intensive workshops will be conductedto train research students from previouslydisadvantaged universities around thecountry in techniques such as protein purification,yeast genetic engineering and glycanprofiling.Other outputs of the project include the developmentof standard operating procedures,protocols on how to analyse N-linked glycans,and a 'toolkit' of Y. lipolytica strains thatglycosylate proteins to different extents.Intellectual property is also being generatedfor the methodology used to produce proteinsand peptides.The research team – Pictured (back) areDonald Kahari, Siyavuya Bulani, ZawadiChipeta; (middle) Ntsane Moleleki,Lebogang Nkuna, Ananias Kgopa,Faranani Ramagoma; (front) NokukhanyaMfumo, Wendy Limani, PuseletsoManyapye and Nozipho ZuluThese achievements provide a good foundationfor extending this research into a proteinproductionplatform based on Y. lipolytica,which is being supported by BioPAD for anadditional three years. This future phase ofresearch will focus on glycoengineering anddeveloping the yeast for the production oftherapeutic peptides and proteins.As Moleleki says, “Something great has beenstarted here. More time is needed to refinethe process. This could be a potential way toproduce glycosylated proteins cheaply whilesimultaneously contributing to the scarce skillsset in South Africa and further afield, througha number of PhDs.”Enquiries:Ntsane Molelekinmoleleki@csir.co.zaS C I E N C E S C O P E N O V E M B E R 2 0 0 971


E N A B L I N G T E C H N O L O G I E S F O R H E A LT Hbi·o·ma·te·ri·aln.A biocompatible material thatis used to construct artificialorgans, rehabilitation devices,or prostheses and replacenatural body tissues.THEY ARE HOLLOW, spherical microparticleswith single port-like openings and porescovering their entire surface. They are at theheart of a CSIR innovation in soft tissue fillermaterial, primarily because they are designedto be conducive to the in-growth of humancells and are completely resorbable. They’vebeen named Dermapearl, and CSIRresearchers are now embarking on preclinicaltrials to substantiate their claims conclusively.The researchers believe that their inventioncould quite literally change the face of cosmeticsurgery, and over a longer period, anarray of medical conditions.Lara Kotzé, CSIR researcher and projectleader, says the particles are the perfect dermalfiller, because the hollow, ported particlesserve as both a tissue bulking substance and atissue harbour for cells. “The inside cavity andport of the particle provide space for the cellsto proliferate and for sufficient nutrient andoxygen exchange,” she explains. The materialfrom which the microparticles are made alsodisplays favourable resorption properties.Lara Kotzé“There is documented evidence that long-termimplants have adverse tissue reactions, thereforeyou wouldn’t want a foreign material inyour body permanently. While these particlesare completely resorbable, they take longer tobe absorbed back into the body than someother dermal fillers and their proposed effectof increased cell proliferation therefore giveslonger-lasting and more permanent results,”she says.S C I E N C E S C O P E N O V E M B E R 2 0 0 972


CSIR injectable dermal filler progresses to preclinical trialsMICROPARTICLES WITH UNIQUECELL HARBOUR CONCEPTin the biomaterials domainKotzé says biomaterials are any material, syntheticor natural, that is used as a structuralcomponent that comes into contact with biologicalsystems. It could be artificial skin, contactlenses, joint replacements, tooth implantsor dermal fillers and it could be made of ceramics,polymers, both synthetic and natural,or metals. “The demand for biomaterials israpidly increasing, one reason being the risein human life expectancy,” says Kotzé.Dermal fillers have an obvious use in reducingthe signs of facial aging through non-invasivecosmetic surgery. Such a soft tissue bulkingproduct is also suitable to correct a conditioncalled lipoatrophy, which is the loss of fatunder the skin – specifically in the cheeks,causing hollow and sunken facial features. It iscaused by aging and diseases and is oftenassociated with Aids sufferers. “Treatments forHIV/Aids will continue to progress, and thenthe focus will shift to improving the associatedconditions, such as lipoatrophy,” says Kotzé.These types of applications for the microparticleplatform are achievable in the short termand the team will pursue this objective.But soft tissue fillers also hold promise as aremedy for medical conditions, such asvelopharyngeal inadequacy, often caused bycongenital defects such as a cleft palate. Bulkingof the soft palate with a soft tissue fillercan help correct this condition, enabling betterspeech and swallowing. Similarly, Kotzé saysit could play a role in correcting gastroesophogealreflux disease (chronic heartburn)and bulking of the urinary sphincter in urinaryincontinence. Finalising a product for theseapplications is a longer-term aim.Using an emulsion process, the Dermapearlmicroparticles are made of polycaprolactone,a polymer commonly used in implant applications.Hyaluronic acid (a substance naturallyoccurring in the human body) is used as carriermedium.The Dermapearl business plan was a runnerupin the 2008 SA BioPlan BiotechnologyBusiness Plan Competition. The team outlinedplans to produce the microparticles in SouthAfrica and then partnering with an establishedcompany in the dermal filler market. There arecurrently no producers of dermal fillers inSouth Africa, and “with a new industry comesnew job opportunities,” says Kotzé. She saysthe review panel – comprising internationalventure capitalists – all commented extremelyfavourably on the potential of the invention.The new dermal filler stems from earlier CSIRresearch in which a ceramic biomaterial wasdeveloped to encourage the regenerationof bone. Trials showed that bone cells regeneratedrapidly when using this product with itssimilar geometric shape to the shape nowused in the dermal filler.With patent applications in place; an upcominginternship in the United States forSA Bio Plan semi-finalists; and CSIR fundingfor preclinical studies, Kotzé is optimistic aboutthe future of their invention, even though it willtake several years to bring it to market.– Alida BritzA microscopic view of a Dermapearlmicrosphere, with the portand pores covering its surface clearlyvisible. To the right, cells can be seengrowing on and in the microparticleafter 72 hours.Enquiries:Lara KotzéLKotze@csir.co.zaS C I E N C E S C O P E N O V E M B E R 2 0 0 973


E N A B L I N G T E C H N O L O G I E S F O R H E A LT HWhen engineering meets biologySYNTHETIC BIOLOGY EMPLOYED TO FINDSOLUTIONS TOSOME OF AFRICA’SKILLER DISEASESBY DR MUSA MHLANGAFIFT Y YEARS of tremendous progress inmolecular biology, genomics and biochemistryhas identified many key cellularcomponents and processes common to all life.In parallel, rapid advances in informationtechnology, optics, physics and other enablingtechnologies have permitted the manipulationand monitoring of biological systems. Thecombining of the two fields of progress havegiven birth to synthetic biology, an emergingdiscipline that exploits advances in our biologicalknowledge with enabling technologiesto construct – from the molecular level upwards– new biological systems and devices.Dr Musa Mhlanga, research leaderof the synthetic biology emergingresearch area, pictured with a newsuper-resolution microscope thatenables researchers to see detailedstructures at the level of singlemolecules within cells. This investmentis one of several investments by theDepartment of Science and Technologyin synthetic biology over thepast two years.S C I E N C E S C O P E N O V E M B E R 2 0 0 974


Key to this, is the application of principlesnormally found in engineering, to biology.To better understand the unique intellectualand strategic ambitions of the CSIR’s syntheticbiology emerging research area, one needsnot look further than a few projects in thenewly established research groups.The molecular biomaterials group, under thedirection of Dr Justin Jordaan, focuses onthe bottom-up construction of bio-molecularsystems from a combination of biological andpolymer precursor materials.The group aims to be a leading developerof synthetic biological systems, using abottom-up manufacturing approach. This canbe achieved by using the superior technicaladvantages of their technology enabling andproprietary ReSyn technology platform.They have developed a novel protein immobilisationmatrix consisting of cross-linkedpolyethyleneimine strands that enable theconstruction of advanced biological systems.These matrix particles are produced in anemulsion, enabling size control, an importantcharacteristic for their intended applications.The advantages of this technology are the extraordinarilyhigh biological-binding capacityand high enzyme activity maintenance afterimmobilisation. These properties allow for theimmobilisation of combinations of proteins tothe particles and have uses in areas as diverseas industrial enzymes and clinical diagnostics.One of the broad aims of the gene expressionand biophysics research group is to enhancethe study of synthetic biology by developingsingle-molecule technologies that will revolutionisethe study, modelling, engineering andimaging of gene expression in vivo. Led byDr Musa Mhlanga, who is also the leader ofthe synthetic biology emerging research area,they intend to use a number of model systemsto achieve this goal. One important focus ison developing advanced optical imagingapproaches to achieve super resolution ofbiological objects. Such studies can beused to examine infectious diseases and hostpathogen-induced transcription, to screen fornatural compounds, drugs or small moleculesthan can ‘engineer’ transcription and to studystochasticity and ‘noise’ in gene expression.Decrypting gene expression, and thusenabling its fine control, is one of the fundamentalaims of synthetic biology’s bottom-upapproach.This group works very closely with the highthroughput biology group lead by Dr NeilEmans. High-content screening (an extensionof high-throughput biology techniques) allowsfor the evaluation of multiple biochemical andmorphological parameters in cellular systems,if biological readouts in the system areamenable to quantitative data collection invivo. Using automated imaging and microscopytechniques, as well as cell-basedassays developed with the gene expressionand biophysics group, the high-throughputbiology group aims to identify genes andchemical compounds which are implicated ininfectious chronic diseases. By combining theimaging of cells with image analysis algorithms,individual components of the biologicalsystem are assigned quantitative properties.Recently the CSIR acquired an siRNAgenome-wide library that will enable theability to screen the entire human genomefor genes implicated in HIV infection, forexample. With innovative technology developedwithin this CSIR group, several genomewidescreens are planned.The CSIR synthetic biology group has setitself the goal of developing the foundationtechnologies and research developmentcapacities that cover the entire syntheticbiology workflow. Through a new humancapacity development programme, and bytraining graduate students and postdoctoralfellows, it aims to be the leading syntheticbiology centre in South Africa and among theleaders worldwide. In doing so, it also hopesto play an influential role in developing thefield in South Africa through local and internationalpartnerships and contribute to findingbasic research solutions to the diseasesaffecting South Africa and Africa today.Enquiries:Dr Musa Mhlangammmhlanga@csir.co.zaTreating internal bleeding onthe spot with ultrasound wavesCSIR SCIENTISTS PL AYED A PART in the development of a transportable device forthe treatment of internal bleeding. Immediate treatment after sustaining an injury and beforean equipped medical facility can be reached, can make the difference between life anddeath. ScienceScope reported in March 2009 that high intensity focused ultrasound (HIFU)is at the heart of an American-developed device in which ultrasound waves are directed towardsa specific point within the body, resulting in rapid heating of diseased or injured tissues.When heated, the biological tissues shrink and fuse, stopping bleeding in deep tissueand within organs such as the liver, spleen, pancreas, etc. One currently approved applicationof HIFU in the medical field is for the treatment of prostate cancer, while approval is alsobeing sought for the treatment of pancreatic cancer and uterine fibroids.The CSIR’s contribution related to the development of transducers, in collaboration with theUniversity of Washington in Seattle, USA. Transducers convert electric pulses to ultrasoundwaves. For the full story, See ScienceScope, March 2009, page 53.S C I E N C E S C O P E N O V E M B E R 2 0 0 975


The CSIR is confident thatan optimum method willsoon be found for highthroughputculturing ofcells with minimal celldamage in a laboratory.Hepatocyte cells growing on the 3Dtemperature-responsive polymerPOLYMERSCIENCECALLED UPONIN CELL-CULTURINGBREAKTHROUGHHepatocyte cell clusters releasedspontaneously from the temperatureresponsivescaffold at 25°CA breakthrough in the culturing of human cells on a mass scale with use of atemperature-responsive polymer has led to the filing of an international patentapplication.ScienceScope reported in March 2009 that researchers at the CSIR have developed a cell-culturing device that would greatly reduce the humanfactor in cell culturing, while allowing cells to grow in an environment that closely resembles conditions within the human body.One aspect of the innovation featured a temperature-responsive polymer used as a coating on 3D fibre scaffolds. This means that cells can grow ina 3D, natural state – rather than on a flat surface. The scaffolds are porous, allowing for oxygen and nutrients to reach the cells, yet they containenough surface area for the cells to bind to and proliferate.When the temperature is changed, the cells are released without harming them. In this way one can harvest and release cells by merely changingthe temperature of the media, and without damaging the cells’ surface proteins. Since March 2009, the project team has made substantialprogress and has now demonstrated proof-of-concept for non-invasive thermal cell culturing.Enquiries:Avashnee Chettyachetty@csir.co.zaS C I E N C E S C O P E N O V E M B E R 2 0 0 976

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