Throughout this journal you would have noticed the little black and white square symbols [pictured right]. Ther QR codes allow you to view the multimedia content that has been developed by The Future Project on your smartphone. To use them, you must have a smartphone equipped with a camera and a QR code reader/scanner application. Visit your phone’s app store (examples include the Android Market, Apple App Store, BlackBerry App World, etc.) and download a QR code reader/scanner app. Activate the app to focus the phone's camera on the QR codes shown, and the mutimedia content should present itself for your viewing pleasure. Alternatively, visit www.thefutureproject.com.
Foreword
In last year’s foreword, Professor Ian Chubb, Australia’s Chief Scientist, referred to the success of the European Space Agency’s Rosetta Mission: the first spacecraft to successfully orbit and land on a comet. This astounding achievement was a testament to the application of scientific method and engineering processes to meet nearly impossible challenges, turning science fiction into science fact. was an ESA Avionics Systems Engineer who was lucky enough to have worked on this incredible program for a period of four and a half years more than 15 years ago.
At The King’s School, I was not an exceptional student, but many different opportunities and experiences I had in those important years taught me to tenaciously pursue my wish to become a space engineer. The students of The Future Project are also lucky to have similar opportunities and experiences provided to them by this program.
This year, I had several opportunities to see the students engaged in science and engineering projects and I was staggered to see the quality, level of understanding, and enthusiasm displayed. One of the most impressive aspects for me was seeing the open and honest reporting of failures, mistakes and setbacks for some of the experiments. Often such results are more important than an outright success.
The success of the ESA Rosetta mission was because it was designed not to fail, rather than to succeed. There is a very subtle, yet very important difference in this strategy. Rosetta implemented autonomous hardware redundancy management, and failure detection isolation and recovery algorithms. Fancy words for one fundamental principal: recognise a failure and correct it. The students and interns of The Future Project have understood this important principle. While some of their articles have not resulted in the expected outcome they desired, they have identified avenues for further research, narrowing their focus for future success.
wish to pay tribute to the teachers and communicators of The Future Project who have educated the students to such a high standard. In particular, the videos made to explain the research of colleagues, and the forums and exhibitions that enthralled the public, most notably "The Zoo of You".
You will find in the following pages the results of this unique collaboration between scientists, engineers and high school students; tabulated, graphed and analysed, these are impressive results indeed. Between these pages, I see The Future Project providing a path to create the next Howard Florey, Mark Oliphant or Ian Frazer. This may take decades to become reality, but hope readers will also see the beginning of this process embedded in this, The Journal of the Future Project.
thoroughly look forward to hearing of the future endeavours of all these students and I hope you enjoy reading this Journal as much as have.
Former ESA Spacecraft Avionics Systems Engineer (Rosetta Mission, et al.); and former TKS student, class of 1979.
Warwick Holmes
Introduction to The Future Project
In the dark and cold recesses of our solar system, a tiny lump of brilliantly engineered science woke and landed itself on a comet. With its scientific eyes open, it revealed a treasure chest of information about life that predates the sun. In a room in Sydney, a son was set free from the debilitating effects of a digestive disease and was able for the first time to enjoy a day at work. Each of these vignettes has a connection to the activities of The Future Project in 2015.
Science is an adventure into the wilds of the unknown. It is the most powerful tool humans have ever fashioned, and with it have engineered the marvels that allow us to reach out for the comets and inside to the tiny cells in our bodies.
The Future Project has one goal: to light the spark of this adventure in the hearts of the next generation. Of course we Australians need a new generation of thinkers and tinkerers – that much is obvious to all willing to consider our future. However, it is unlikely we will get there by impressing this need on our children or cramming facts into their heads and hoping that leads to creative thinking. Rather, we have to bring them with us, into the lab, into the world, along the great journey of ideas, and help them smell the salt in the air as the ocean of possibility calls their name.
am humbled to work alongside the wonderful scientists and engineers who have partnered with us again this year and especially my fellow teachers, Tom Riley and Matt Purser, without whose energy this venture would surely have failed.
This journal appears to mark the end of a productive year of investigating and communicating by all those incredible young people in the Project, and to some extent it does. However, what I truly wish is for these pages to be thumbed in 20, 30 years by one of our students reminiscing how their great adventure can be traced back to this very journal.
Roger Kennett
www.thefutureproject.com
In The Future Project’s Intern Strand, students from The King’s School and other schools work alongside researchers conducting authentic scientific research. This strand has gone from strength to strength in 2015. New ground was broken with the introduction of six girls from Baulkham Hills High School and Tara Anglican School as external students and the establishment of the Junior Interns rotation for Year 10 students. Furthermore, the return of three students as Senior Interns has permitted continuity with their researchers and also the further development of their exciting projects from last year. For two of these boys, Gerry Feng and Thomas Dickinson, this culminated in the life changing opportunity to fly to Singapore to present and compete at an nternational conference. Plans are also in motion for the first Senior Intern scientific article to be published in a peer-reviewed journal in the coming year.
The Mechatronics Strand has also achieved similar success, with David Gailey in particular carrying on his fantastic work from the previous year. David, together with his researcher Daniel Simmons, has refined his Heliprobe Print Bridge into a commercial product that will shortly be sold as a medical device by Vitramed. The Junior Mechatronics strand was also established in 2015 in the hope of producing suitable candidates for Year 11, through developing their use and programming of microcontrollers.
Tom Riley
The role of the group of Year 10 students in The Future Project’s Communication Strand is to effectively communicate the nature of the interns’ research and the broader scientific issues to the wider community. This strand is divided into two parts: the Public Presenters produce video documentaries describing the work of the researchers and their associated interns; and the School Presenters run engaging science programs for preparatory students of other schools, with an emphasis on dynamic, hands-on experiences. Over the course of the year, the students of the Communication Strand also assist with the staging of public forums or exhibitions examining topical scientific issues. This year, the Public and School Presenters worked together to put on the fantastic "The Zoo of You" exhibition, about which you can read in this journal.
Matt Purser
Members of The Future Project
Arian Bhatia
Dr Yanling Lu
Thomas Denny
Jock Mitton
Edward Dwyer
Eddie Poolman
Jordan Gao
Tim Sampson
Justin He Adesh Soni
Dr Kailing Wang
Dr Michelle Bull Angus Macdonald
Isha Deshmukh
Mike Morgan
Vannessa Edwards
Mr Matt Purser
Mark George Nik Sasic
Archer Holz
Tarun Tandon
Vithushan Lingam
Nisali Wellege
Lisa Cao
Mr John Manusu
Thomas Dickinson
Dr Hari Nair
Dr Ali Fathi
Markus Rangan
William Giang
Mr Daniel Simmons
Dr John Kavanagh
Yang Tao
Oli Lock Lian Yang
Dr Belinda Chapman
Dominic McAuley
Dinesh Ratnam
Mr Willow Norton
Gerry Feng
Mr Tom Riley
James Groutsis
Dr Julie Simmons
Nina Kettle
Dr Roslyn Tedja
Geoffrey Luo Marcus Yuen
David Galley
Ryan McShane
Alexander Dunlop
Razeen Parvez
Kalvin Fernandez
Mr Roger Kennett
Samuel Harrison Ted Simpson
Mr Eric Lin
Ms Darcii Terre
Casper Lu
Interactions between
Dietzia
C79793-74 and
gastrointestinal
microbiota in vitro
N.Wellege1, I.Deshmukh1, V. Edwards1, B.Chapman2, M. Bull2
THE FUTURE PROJECT1 AND VITRAMED BIOSCIENCE2
The King’s School, NSW 2151, Australia
The Battle of the Bugs
Microorganisms often have to compete in their environment for space, nutrients, and oxygen. Some microbes may produce chemicals to inhibit others, and this is often the case for microbes in the gastrointestinal (GI) tract. Our experiments examined how Dietzia, a probiotic bacteria, interacted with various other bacteria found in the GI tract. Some of these bugs cause disease, such as E. coli, whilst others are helpful, such as Lactobacillus
Being part of The Future Project has been an amazing experience. This unique opportunity has been a highlight of our senior year, being both
rewarding and enjoyable. We walk away with a greater insight into the field of ‘real world’ science and important life skills such as effective teamwork. We cherish all the memorable experiences we have had during our internship and would highly encourage others to be a part of this program.
ABSTRACT
Dietzia C79793-74 is currently being trialled as a potential probiotic in treatments for people with Crohn’s disease (personal communication, B. Chapman). The aim of this study was to investigate the potential for interactions between Dietzia C7979374 and a suite of microbes naturally found in the gastrointestinal (GI) tract. The suite of organisms were; Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus plantarum, Saccharomyces boulardii, Candida albicans, Escherichia coli, and Enterococcus faecalis. The first four organisms are generally regarded as beneficial (probiotic) bacteria, while the following two organisms are yeasts. S. boulardii is also used as a probiotic. In contrast, C. albicans, E. coli and E. faecalis, while normal members of the GI tract, are generally known as pathogens.
The potential for interactions with Dietzia C79793-74 was investigated in vitro using cross streak and well plate assays performed on Iso-Sensitest and Mueller Hinton agars. For all organisms except the lactobacilli, competition for iron was explicitly tested by supplementing the base media with an iron chelator. There were no observed zones of inhibition present under any of the conditions tested for any of the organisms. These results indicate that Dietzia C79793-74 has no substantial effect on the growth of these organisms in vitro. Conversely, the results suggest that growth of Dietzia C79793-74 is not easily disrupted by the growth of other GI microorganisms. However the lack of interaction between Dietzia C79793-74 and the test organisms in vitro may not be representative of interactions in the GI tract, and warrants further study.
INTRODUCTION
Dietzia C79793-74 is a bacterium that has been proposed as a potential probiotic to treat Crohn’s disease in the human body (Click, 2012), and which is currently the subject of a Phase 1 / Phase 2 clinical trial (Chapman, 2015). The genus Dietzia has cells that are Gram-positive, aerobic, short rod-and coccoid-like, non-motile, nonendospore-forming, non-acid fast, oxidase-positive, and catalase positive (Koerner, Goodfellow & Jones, 2009). Crohn’s disease is an Inflammatory Bowel Disease (IBD) which currently lacks effective preventative and curative therapies. IBD describes a group of conditions in which the intestines become inflamed (Sadadinejad, Asgari, Molavi, Kalantari & Adibi, 2012). Although Crohn’s disease is regarded as an idiopathic condition, studies reveal an interplay between overpopulating pathogens and genetic factors that can exacerbate symptoms. Crohn’s disease is commonly diagnosed in immunocompromised or antibiotic treated individuals. Pathogenic bacteria readily take advantage of hosts with a compromised immunity and this is a potential cofactor in inflammatory diseases (Huffnagle & Noverr, 2013).
The present study examined the interactions between Dietzia C79793-74 and a suite of organisms commonly found in the human gastrointestinal (GI) tract. The suite of microorganisms examined were Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus plantarum, Saccharomyces boulardii, Candida albicans, Escherichia coli and Enterococcus faecalis. All of the lactobacilli naturally occur in the GI tract, and are available as commercial probiotics. S. boulardii and C. albicans are yeasts; S. boulardii is used as a commercial probiotic and, in contrast, C. albicans
is an opportunistic pathogen, which means it can invade the body of compromised hosts. E. coli and E. faecalis are facultatively anaerobic pathogenic bacteria that also naturally occur in the GI tract.
The interactions of the organisms were tested in vitro. Iso-Sensitest and Mueller Hinton agars were the basis of the interaction assay as they are non-selective media and allow a majority of organisms to grow. These media are typically employed for testing antibiotic inhibition of bacteria in vitro. Mueller Hinton agar contains a starch component that absorbs any interfering toxins produced by other organisms. Most importantly, Iso-Sensitest and Mueller Hinton agars have a loose structure which allows for the better diffusion of organic, inhibitory compounds during a well plate assay.
Inhibition of L. rhamnosus, L. reuteri, L. acidophilus, and L. plantarum by Dietzia C79793-74 was tested on Iso-Sensitest and Mueller Hinton agars supplemented with de Man, Rogosa, Sharpe (MRS) agar. MRS agar is routinely employed as a growth medium for Lactobacillus, but is generally unsuitable as a testing medium for interactions between microorganisms. However, as lactobacilli can be fastidious, supplementation with MRS was undertaken to ensure that the test organisms received adequate nutrients for growth. A mixed formulation of Iso-Sensitest broth (90%) and MRS broth has previously been shown optimal for the growth of lactobacilli (Klare, Konstabel, MuellerBertling... Witte, 2005).
Isha, Nisali & Vanessa
Inhibition of S. boulardii, C. albicans, E. coli, and E. faecalis by Dietzia C79793-74 was also tested on Iso-Sensitest and Mueller Hinton agar, with or without the addition of an iron chelator. Iron serves as an essential growth factor for a majority of bacterium species (Robins-Browne & Prpic, 1984) and can be particularly important in pathogenesis. Pathogens must actively acquire iron from the host to establish themselves and invade bodily tissues. Pathogens and bacteria can source iron through the production of siderophores, which serve as iron chelating compounds. S. boulardii, C. albicans, E. coli, and E. faecalis all produce siderophores, enabling them to compete well with other microorganisms in low iron environments such as those encountered in the human body.
METHOD
Microorganisms
Dietzia C79793-74 was made available from Vitramed Pty Ltd, courtesy of Robert Click. L. rhamnosus, L. reuteri, L. acidophilus, L. plantarum and S. boulardii were sourced from commercial probiotic preparations. C. albicans, E. coli, and E. faecalis were obtained from the Vitramed culture collection.
Well plate assay
Interactions between Dietzia C79793-74 and S. boulardii, C. albicans, E. coli, and E. faecalis were tested on Iso-Sensitest and Mueller Hinton agars (Oxoid, ThermoScientific, USA), with or without the addition of an iron chelator, 2,2’-dipyridil (Sigma, USA). Interactions between Dietzia C79793-74 and L. rhamnosus, L. reuteri, L. acidophilus, and L. plantarum were tested on Iso-Sensitest agar (Oxoid) with a concentration of 10% or 20% MRS (Oxoid). All media were adjusted to a pH of 6.7 prior to autoclaving.
Test organisms were separately inoculated onto each type of medium, as relevant. Test organisms were inoculated by preparing heavy suspensions in Maximum Recovery Diluent (MRD; Oxoid). Three millilitres of suspension was inoculated per plate to entirely cover the surface of the plate, and then the residual fluid was removed using a transfer-pipette. This technique resulted in a confluent lawn of the test organism. At least six replicate plates were prepared per test organism per medium type.
After inoculation with the test organism, five wells were made per plate, using a sterile transfer-pipette as a puncturing instrument to create wells in the agar gel. The suction mechanism of the pipette resulted in uniform wells. Wells were inoculated with the following preparations of Dietzia C79793-74; 108 cfu/mL, 106 cfu/mL, and 104 cfu/mL diluted in MRD, as well as heat killed and filtered supernatant (Figure 1). The final two preparations were negative controls. Heat killed Dietzia C7979374 was produced by heating a water bath to 80°C and waiting for the
temperature to stabilise. Masking tape was then wrapped around the lid of the eppendorf vial to ensure water did not leak into the vial. More masking tape was then used to secure the vial on to the wall, so that only half the vial would be submerged into the water. The vial stayed in the water bath for 15 minutes. The second negative control was prepared by centrifuging Dietzia C79793-74 stock to remove the bulk of cells, and then filtering the supernatant through a 0.2 µm filter to remove remaining cells. All plates were incubated at 37°C for 48 hours and then observations were made.
Cross streak assay
Test media were prepared as for the well plate assay. A heavy suspension of Dietzia C79793-74 in MRD was streaked onto the centre of each test medium; replicate plates were prepared. The streaked plates were incubated at 37℃ for 24 hours to permit pre-growth of Dietzia C79793-74.
Heavy suspensions of each of the test organisms were then prepared in MRD. Suspensions were streaked at right angles to the Dietzia C79793-74 streak line. Multiple test organisms were streaked on each plate, as relevant. All plates were re-incubated at 37℃ for a further 48 hours and observations were made.
RESULTS AND DISCUSSION
Well plate assay
There were no zones of inhibition observed on any of the well plate assays. This suggests that Dietzia C79793-74 did not inhibit any of the test microorganisms; similarly, the growth of Dietzia was not restricted to a large extent by the presence of the other microorganisms.
Cross streak assay
As observed for the well plate assays, no distinct zones of inhibition by Dietzia C79793-74 were observed on any of the cross streak plate assays, for any of the test organisms. However, among the lactobacilli, L. acidophilus and L. reuteri exhibited confluent growth on the 10%
MRS streak plates (Figure 2a), but only isolated colony growth on 20% MRS streak plates (Figure 2b). As lactobacilli are well suited to growth at high MRS concentrations, it could be expected that better growth of these organisms would occur in the presence of 20% MRS. However, since our experiment produced results which suggest otherwise, it is possible that Dietzia C79793-74 at high MRS concentrations has a greater inhibitory effect on the growth of L. acidophilus and L. reuteri.
Interestingly, it was noted that C. albicans formed pseudo-hyphae on the cross streak plates, suggesting that conditions provided in this test approached those that stimulate C. albicans to adopt a pathogenic growth habit. However, C. albicans was also observed to have more confluent growth under high iron conditions (regardless of the base media), suggesting that the organism may have struggled to chelate iron under low iron conditions. This result in turn suggests that C. albicans did not function in full pathogen mode during the test.
Dietzia C79793-74, by contrast, was observed to thrive in a low iron environment, as evidenced by its vivid pink colour and mucoid growth.
REFERENCES
Huffnagle, G.B. & Noverr, M.C. (2013). The emerging world of the fungal microbiome. Trends in Microbiology. 21(7), 334-341
Klare, I., Konstabel, C., Mueller-Bertling, S... Witte, W. (2005). Evaluation of new broth media for microdilution antibiotic susceptibility testing of Lactobacilli, Pediococci, Lactococci, and Bifidobacteria. Applied Environmental Microbiology. 71(12), 8982-8986.
CONCLUSION
This study has provided no conclusive evidence regarding Dietzia C79793-74’s ability to inhibit the growth of any of a range of GI microbiota in vitro. Conversely, the results of this study suggest that growth of Dietzia C79793-74 is not easily disrupted by the growth of other microorganisms. In particular, Dietzia C79793-74 appears to compete well under low iron conditions, suggesting that it is capable of producing siderophores. Siderophore production by Dietzia C79793-74 has not previously been confirmed, although recent genomic studies suggest that other Dietzia strains may be capable of siderophore production (Procópio, Alvarez, Jurelevicius… Dirk van Elsas, 2011). Further testing of Dietzia C79793-74 under conditions that more closely simulate the GI tract is warranted, to provide conclusive results, particularly with regards to the potential for its interaction with L. acidophilus and L. reuteri Investigation of an alternative test system for exploring interactions between Dietzia C79793-74 and C. albicans is also warranted, given that C. albicans appeared to grow only in pseudo-pathogen mode in this study.
ACKNOWLEDGEMENTS
We would like to sincerely thank several people who were there to guide and inspire us along each step of our project.
Dr Belinda Chapman and Dr Michelle Bull for their guidance and knowledge, which made this journey possible.
Ms Darcii Terre, for assisting us with technical aspects of the experimentation process.
Mr Tom Riley, Mr Roger Kennett and all staff involved in The Future Project for providing us with this incredible opportunity.
Miss Moshkanbaryans, for assisting with the editing and writing process of our article.
Koerner, R.J., Goodfellow, M., & Jones, A.L. (2009). The genus Dietzia: a new home for some and emerging opportunist pathogens. FEMS Immunology and Medical Microbiology. 55(3), 296-305.
Procopio, L., Alvarez, V.M., Jurelevicius, D.A… Dirk van Elsas, J.D. (2012). Insight from the draft genome of Dietzia cinnamea P4 reveals mechanisms of survival in complex tropical soil habitats and biotechnology potential. Antonie Van Leeuwenhoek. 101(2) 289–302.
Robins- Browne, R.M. & Prpic, J.K. (1985). Effects of iron and desferrioxamine on infections with Yersinia enterocolitica Infection and Immunity. 47(3), 774–779.
Sadadinejad, M.S., Asgari, K., Molavi, H., Kalantari, M. & Adibi, P. (2012). Psychological issues in inflammatory bowel disease: an overview Gastroenterology Research & Practice. Volume 2012
Figure 2a 10% MRS: 90% Isosensitest
Figure 1
Figure 2b 20% MRS: 80% Isosensitest
THE
Separation of Fibronectin from human plasma
Blood Nectar
Our project involved isolating a protein found in human blood plasma called Fibronectin. In doing so, we can use the protein as the therapeutic drug to help those in medical need across the world. For our experiment, we used a machine which separated protein mixtures based on size and charge – a process known as electrophoresis. Our aim was to extract as much of the protein Fibronectin as possible and to investigate the necessary conditions for this to take place.
If there is one thing I have learned from The Future Project, it is the need for good organisational skills. I have discovered that in any science or medical career, the need to be on top of things is crucial. Not only do you have to know what you are doing, but you have to know what your partner is doing so you can work together and get the job done. I have enjoyed working with scientists and meeting likeminded people who are passionate about science. The Future Project has really opened me up to what a career in science would be like and it has made me even more determined to follow such a path.
Nina
The Future Project has taught me a lot about the importance of a good work ethic. When you work in a real laboratory environment, you really understand the need for good motivation, team skills, attendance, reliability and enthusiasm. I have learnt that when working as part of a team or partnership, you both work better and perform at a significantly greater level when at least one of you is showing good work ethic and encouraging the other to do the same. I have loved working as part of a team and applying science to real world issues; it has been a true eye opener.
Gerry
The King’s School, NSW 2151, Australia
ABSTRACT
Fibronectin is a high-molecular weight glycoprotein found in blood plasma that is composed of two nearly identical bound polypeptides, each of molecular weight 220 kilo Daltons (kDa). Both Fibronectin and Fibrinogen have an approximate isoelectric point (pI) of pH 5.5 to 6.0. They are found in lower abundance compared to Albumin and Immunoglobulin and therefore are generally more complicated to isolate from the other proteins in plasma. The Preparative Isolation by Membrane Electrophoresis (PrIME) Separation process is a plasma fractionation process that separates molecules on the basis of their size and charge through membrane electrophoresis. This study is a preliminary investigation of Fibronectin separation by PrIME. Results have shown some degree of separation of Fibronectin from Fibrinogen, however, further work is needed to establish and optimise process parameters and fine-tune membrane pore-size in order to achieve complete separation of Fibronectin from Fibrinogen.
INTRODUCTION
Plasma fractionation refers to the separation of different protein components from blood plasma. The main purpose for performing plasma fractionation is to isolate a variety of proteins that are present in plasma. These proteins, once purified to a high standard, can be then used as medicinal products that are in high demand in clinical practice on a global scale.
The tube was then centrifuged again at the same conditions and subsequently emptied. This was repeated until the cryo pellet had been washed three times with saline. After the final wash, 50mLs of citric saline buffer (tri-sodium citrate dehydrate and sodium chloride solution) was added and the four cryo pellets were emptied into a singular tube. The tube was then left overnight on a rocker at room temperature until all the cryoprecipitate had re-solubilised into a solution that can be then used for separation.
Fibronectin Separation by PrIME
The Fibronectin separation was completed using the PrIME B400 Instrument (NuSep Ltd). The central part of PrIME technology is the separation cartridge. This consists of a 1000 kDa separation membrane, bound by two restriction membranes (5 kDa). This separation membrane separates Stream 1 (S1) and Stream 2 (S2), making them fully independent from each other.
Fibronectin is a protein that has low abundance in human blood plasma with a concentration of 300μg/mL. Fibronectin can be found in two forms: insoluble cellular Fibronectin, which is found in the extracellular matrix (ECM) and soluble plasma Fibronectin, found in blood plasma. It has a double-bound monomer structure which results in Fibronectin having a comparatively high molecular weight of 440 kDa. The isoelectric point (pI) of Fibronectin is from 5.5 to 6.3. The pI, defined as the pH at which an amino acid contains no net electrical charge, is of particular importance as it can then be used to optimise the buffers that influence the movement of certain proteins across the separation membrane during electrophoresis. Therapeutic administrations of pure Fibronectin have been clinically tested to have significant benefits to corneal wound healing and critical multi-system organ failure, however the product remains expensive and scarcely available (Yoder, 1991).
METHOD
Cryoprecipitate Preparation
The re-solubilised cryoprecipitate was loaded into S2, and when an electrical current was applied, the proteins that were charged according to the buffer environment were transferred through the separation membrane into S1. This study investigated Fibronectin separation by PrIME under three major separation conditions:
1) Electrophoresis buffer MES Bis-Tris (50 mM MES and 6 mM Bis-Tris) pH 5.2,
2) Electrophoresis buffer Tris-Borate (46 mM Tris Base and 20 mM Boric Acid) pH 8.9, and
Plasma that is stored at -20°C is thawed at 4°C overnight, resulting in the cryo-precipitation of various proteins, most notably Fibronectin and Fibrinogen, leaving other proteins including Albumin and Immunoglobulin G in the cryo-supernatant. The plasma was then centrifuged at 15,000 RPM for 15 minutes at 4°C. Upon removal, the cryosupernatant was stored for other purposes, leaving the insoluble cryo pellets in the centrifuge tubes. 60mLs of normal saline solution was then poured into each tube and shaken vigorously to loosen the cryo pellet and to wash out any remaining soluble material.
3) Electrophoresis buffer Tris-Borate (46 mM Tris Base and 20 mM Boric Acid) adjusted to pH 8.0 with 1.0 mol hydrochloric acid.
Condition 1 was used for charged based separation whilst Conditions 2 and 3 were used for size based separation. The unit was cleaned and prepared with the running buffer prior to the separation process.
G.Feng1, N. Kettle1, K. Wang2, H. Nair2
FUTURE PROJECT1 AND PRIME BIOLOGICS2
The S1 and S2 streams were then conditioned with the appropriate running buffer for five minutes to test for leakage in the separation cartridge. The process was run at an electric potential of 250 V with the positive electrode configured at S2 and the negative electrode configured at S1. The product in S1 was collected at 30-minute intervals for at total of 360 minutes. One minute before each 30-minute interval ended, the depth volume and current displayed on the instrument was recorded onto a running sheet. At the end of each period, 500μL of S1 was pipetted out in a centrifugal tube as a sample. The remaining solution at the end of each fraction in S1 was emptied and collected in a pool. The stream tube then was replenished with a fresh 15mL of the appropriate running buffer. This sampling of each fraction continued for the entirety of the six-hour experiment. At the conclusion of all 12 30-minute fractions, a 500μL sample was also pipetted out the measuring cylinder that denoted the pooling of S1. If samples were not analysed immediately, they were stored at -20°C.
SDS-PAGE Analysis
Qualitative analysis was performed using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The samples were prepared by mixing 25μL of S1 stream sample with 25μL of dye solution (100μL DTT mixed with 400μL 2x Laemmli Buffer) then heated at a heating block for ten minutes at 95°C. The SDS-PAGE process was performed with Tris-Glycine 4-20% gradient gel that was inserted into a Bio-Rad gel running apparatus, with a Tris-Glycine SDS running buffer. The gel was run at 180 V for a 90-minute period. Upon the completion of SDS-PAGE, a Bio-Rad Gel Doc was then used to visualise protein bands. Once initial analysis had been completed and pictures taken, a Coomassie Blue Staining step was also applied to the gel.
RESULTS AND DISCUSSION
Figure 1 SDS-PAGE gel. A: pH 8.9 buffer B: pH 8.0 buffer C: pH 5.2 buffer (A) Lane 1: S2 0 Lane 2: S2 300 Lane 3: S2 END Lane 4: S1 30 Lane 5: S1 90 Lane 6: S1 150 Lane 7: S1 210 Lane 8: S1 POOL Lane 9: Fibronectin Standard Lane 10: Pre-stained SDS-PAGE Marker (B) Lane 11: S2 0 Lane 12: S2 300 Lane 13: S2 END Lane 14: S1 30 Lane 15: S1 90 Lane 16: S1 150 Lane 17: S1 210 Lane 18: S1 POOL Lane 19: Fibronectin Standard Lane 20: Pre-stained SDS-PAGE Marker (C) Lane 21: S2 0 Lane 22: S2 END Lane 23: S1 0 Lane 24: S1 30 Lane 25: S1 60 Lane 26: S1120 Lane 27: Fibronectin Standard Lane 28: S1 POOL Lane 29: Pre-stained SDS-PAGE Marker
Figure 1 are the gel doc images from the gels after the SDS-PAGE process was completed. The dark coloured protein bands signify the presence and approximate quantity of proteins in a particular lane. All three gels have a Fibronectin standard lane (Lane 9, Lane 19, Lane 27). The positioning of this particular band enables a comparison to be made to other lanes to determine the presence of Fibronectin.
Fibronectin is extremely difficult to handle due to its nature to cling and clump together, making it a ‘sticky’ protein. The challenge in this study largely existed in the molecular similarity and low abundance of Fibronectin, the target protein, in comparison to the major contaminant Fibrinogen.
The close proximity of both the pI and molecular weight of the two was the root of the problem due to the sole separation conditions of the PrIME process being size and charge. Furthermore, Fibronectin’s concentration is approximately 350μg/mL, while the average Fibrinogen concentration is 3mg/mL, which highlights the vast difference between the two. This low concentration of Fibronectin in plasma is the reason that the cryoprecipitation procedure was necessary.
All three gel pictures in Figure 1 shows that major protein contaminants such as Albumin and Immunoglobulin in plasma prior to cryoprecipitation were successfully removed in the procedure. This can be seen by the absence of any major protein bands aside from Fibrinogen, identified through the Pre-stained SDS-PAGE Standard Marker (Lane 10, Lane 20, Lane 29). These results indicate that both the cryo-precipitation procedure and the PrIME process were extremely effective in isolating both Fibrinogen and Fibronectin.
The first condition that was set in the investigation was Tris Borate buffer pH 8.9. As mentioned, the reasoning behind this initial specification was to separate on size, as pH 8.9 causes most proteins in the plasma to become negatively charged and therefore only move into a singular stream, S1. In Figure 1A the product streams in Lanes 4-8 have a distinguishable band denoting the presence of Fibronectin. This indicates a transfer of Fibronectin out of S2. However, high concentrations of contaminants such as Fibrinogen accompanied the Fibronectin and despite being noticeable, the separation condition was not optimal. A possible reason for this is the negligible difference in size of Fibrinogen and Fibronectin (approximately 340 kDa and 440 kDa respectively). However, the first condition was positive in showing the effectiveness of the cryoprecipitation procedure and the conditions under which both Fibronectin and Fibrinogen can be separated from plasma.
Following the analysis outcomes from the pH 8.9 specifications, an adjustment was made to the buffer in order to isolate Fibronectin from Fibrinogen. A pH 8.0 Tris-Borate buffer was created by adding 1.0mol hydrochloric acid to the original formula as necessary. The hypothesis was that a lower pH would result in a lower amount of proteins being negatively charged, thereby slowing down the movement of Fibrinogen and any additional contaminants. However, the pH 8.0 gel (Figure 1B) had no concentration of Fibronectin as can be seen by the lack of protein bands in Lanes 4-8. The conclusion that can be drawn from this set of results is that the molecular weights of Fibrinogen and Fibronectin are too similar, resulting in size-based separation being ineffective for the purification of Fibronectin.
REFERENCES
1. Yoder, M.C. (1991). Therapeutic administration of Fibronectin: current uses and potential applications. Clin Perinatol. 18(2) 325-341.
As such, a third parameter was created in order to separate based primarily on charge. Thus, the electrophoresis buffer was replaced with MES Bis-Tris pH 5.2 to attempt to isolate Fibronectin based on its approximate pI of 5.5-6.0. The gel picture in Figure 1C showed that there was also no transfer of Fibronectin with no protein band visible where Fibronectin was determined to be, mirroring the results of the Tris Borate pH 8.0 specification. There was also a significant decrease in the concentration of Fibrinogen, meaning there was overall less transfer of any proteins with pH 5.2 buffer. This can be attributed to the pI of Fibronectin with other proteins being again too similar. These results reveal that charge-based separation was not conclusive in isolating Fibronectin.
The outcomes were overall very encouraging, as preliminary investigation results suggest Fibronectin and Fibrinogen are able to be separated from plasma in high abundance as shown by the gel pictures. However, in order isolate Fibronectin alone, further investigation would look at a second separation step to remove Fibrinogen after the PrIME process. A number of different separation techniques could be implemented to further develop the downstream process in separating the Fibrinogen and Fibronectin mixture, such as chromatography.
CONCLUSION
The three variable running buffers that were used during the experiment (Tris-Borate pH 8.9, Tris-Borate pH 8.0 and MES Bis-Tris pH 5.2) were successful in separating Fibrinogen and Fibronectin from other contaminants. However, they were unable to isolate solely Fibronectin from the Fibrinogen. As the investigation was conducted as a preliminary investigation, the most promising electrophoresis buffer was Tris Borate pH 8.9. Further fine-tuning and experimentation could result in a successful separation of Fibronectin using either the PrIME process or other techniques, enabling this scarcely available protein to become more accessible to a wider market.
ACKNOWLEDGEMENTS
We would like to thank PrIME Biologics for making this paper possible and for their guidance throughout the year. Additionally, thanks go to The King’s School for providing the opportunity that is The Future Project. Special thanks go to H Nair, K Wang, R Tedja, R Sciberas, W Norton, T Riley and R Kennett.
2. Pantanowitz, L. (2003). Cryoprecipitate. American Journal of Clinical Pathology. 119(6), 874-881.
3. Lowe, G.D., Rumley, A. & Mackie, I.J. (2004). Plasma Fibrinogen. Ann Clin Biochem. 41(6), 430440.
C. Lu1, M. George
Testing Elastin and Albumin based hydrogels for use in cartilage regeneration and the treatment of diabetic wounds
Artificial Healing
Our research was aimed at creating a type of gel that could be injected into the knee as a way to stimulate the growth of cartilage in an area of damage. We also endeavoured to create a gel that would replace the standard treatment of diabetic wounds, namely antibiotics and bandages. These are wounds that cannot heal without intervention because the person suffering from diabetes cannot produce the platelets, clotting agents found in the blood, themselves. This results in an open wound that is easily infected.
This experience has opened our eyes to real world scientific research and its impact. During our work with Ali, we were given a glimpse into the imperative work that he is doing to help revolutionise the treatment of a wide range of diseases and
injuries. We also learnt how to design, conduct and deconstruct the results of experiments, an experience not usually undertaken by the average science student. This experience also provided us with the opportunity to access the exclusive Chemical Engineering Labs at The University of Sydney. This privilege is unknown to even the typical undergraduate and is limited to those conducting their PhD. Bearing this in mind, we were ecstatic to being invited on this journey. This research was extremely different to the typical inclass experiments and we learnt various research techniques throughout the process. To conclude, this experience has been extremely eye opening and we will be ever grateful for the opportunity.
Mark, Casper, Oli, Markus & Jordan
ABSTRACT
The University of Sydney, NSW 2006
Currently, diabetic wounds and cartilage disease are a rising issue in the world of public health in Australia. Over 80% of people over the age of 60 are suffering from cartilage disease such as osteoarthritis. A further 30% of Australians suffer from diabetes, and of this group, 4000 have died as a result of diabetic wounds that became inflamed and infected. Current treatments for these two debilitating medical conditions are ineffective, painful and invasive on a surgical level. This has resulted in the development of the two closely related polymers PNPHO and SPNPHO, which provide alternatives to these inefficient treatments. These polymers form injectable, thermo-responsive hydrogels that maintain similar properties to the body tissue they are designed to treat – cartilage growth and external diabetic wounds. The fact that the polymers respond to the temperature of the human body (37.4°C) will allow for easier handling and administration in medical clinics, and by bonding the polymers with various proteins (Albumin and Elastin) they can promote cell growth in the body, acting as a “scaffold” for the body to create cells and repair itself. Through experimentation, we can vary polymer and protein concentrations to optimise certain properties such as gelation efficiency, swelling ratio and degradation rate, to address clinical requirements. Based on the results, it was deemed that an Elastin-co-PNPHO polymer would be an effective mechanism for cartilage growth and an Albumin-co-SPNPHO polymer would be purposed as an alternative to the messy process of using basic dressings for diabetic wounds. Further in vitro and in vivo studies are required to fully confirm the potential of these biomaterials for cartilage and wound dressing applications.
INTRODUCTION
METHOD
80% of people over the age of 60 are negatively influenced by cartilage disease in Australia every year. Furthermore, 85% of diabetics will experience a prolonged debilitating wound before the age of 25, and these wounds are untreatable with traditional dressings. Our research into the synthesised polymers PNPHO and SPNPHO is vital in the development of a less invasive, more efficient and cost effective treatment for both of these conditions.
Our research was focussed on the synthesised polymers SPNPHO and PNPHO. Both these polymers possess a number of engineered characteristics, which make them viable for treatment in the human body. These characteristics include: temperature responsiveness, adhesiveness, mechanical strength and water solubility. The two polymers are similar, but were paired with different proteins throughout the study. PNPHO was paired with Elastin and SPNPHO with Albumin. The aim of our research was to find an optimum concentration of the polymer and the protein. To test this we created hydrogels of varying concentrations of each and measured the gelation time, efficiency and swelling ratio. Once an optimum was found, we tested the polymer’s mechanical properties and compared these hydrogels with those produced with a control polymer with baseline mechanical properties: polyethylene glycol diacrylate (PEG-da).
MATERIALS
The materials utilised were as follows: Phosphate Buffered Saline (PBS), a self-sythnesised Poly (NIPAAm-co-NAS-co-(HEMA-PLA)-coPEG) copolymer denoted as PNPHO, and the SPNPHO variant that is identical to PNPHO apart from the addition of 5-GMA, Elastin, PEG-da, pigskins for mechanical testing, glue, Elastin extracted from bovine ligament, and Albumin.
In order to test and consequently further fine-tune the two key properties of Gelation Efficiency and Swelling of our polymer, we varied concentrations of PNPHO (50mg/mL-150mg/mL) and mixed it with an appropriate ratio of Phosphate Buffered Saline (PBS) solution which was pre-chilled to 4°C, preventing any premature gelation due to PNPHO’s thermo-responsive nature in forming elastic constructs known as hydrogels at 37ºC. Furthermore, the use of PBS is essential as it accurately replicates conditions of the human body, thereby facilitating the biomedical nature of our research. The PNPHO suspension was stored at 4 °C for the PNPHO to fully dissolve within the solution. After the solution became homogenous, we were able to extract several 0.5mL samples of the hydrogel solution through chilled syringes and immediately placed them within a vial in an incubator preheated to 37°C to further simulate the human body’s conditions. In this environment the thermo-responsive state of the hydrogel is activated, causing quick solidification from its liquid state. The exact time taken was measured, as this amount of time is imperative in the clinical use of these hydrogels. Moreover, the original PNPHO hydrogel solution was also bonded with proteins, either Elastin or Albumin, by adding varied concentrations of the proteins and thoroughly mixing, leaving it overnight in refrigeration for the polymers to bond completely. This achieved the purpose of modulating the durability of the hydrogel, increasing its structural integrity and leading to a decreased rate of degradation.
The solidified hydrogel that was yielded was then placed in an excess of PBS solution heated to 37°C in an incubator to test its swelling capabilities. The swelling property was accounted for in two aspects by measuring the swelling in terms of area and the gravimetric swelling.
1, M. Rangan1, O. Lock1, J. Gao1, A. Fathi2, J. Kavanagh2 THE FUTURE PROJECT1, The King’s School, NSW 2151, Australia SCHOOL OF CHEMICAL ENGINEERING2,
Photos of the expanding hydrogels were taken periodically every five minutes in order to detect the rate of change in area. Comparison of the changes in area in the different concentrations of protein was seen through the image analysing software “ImageJ” that used a clear scale within the picture to calculate the area of the hydrogel at each interval of time.
The dry weight of the constructs (calculated from the added amount of PNPHO/SPNPHO and protein, WD) and wet weight (the weight of the swollen hydrogels, WS) were measured to calculate the gravimetric swelling ratio of the hydrogels.
Hydrogel solutions created with controlled concentrations of SPNPHO/ PNPHO bonded with either Albumin or Elastin respectively, and a hydrogel of PEG-da, L-ascorbic acid (Vitamin C), and Ammonium Persulphate, were all compared, with the PEG-da acting as a clinical gold standard. Their tensile strength was measured with an Instron 5943, equipped with a 100N load cell, through the application of the different hydrogels between the controlled variable of pigskin, which attempted to imitate human skin. The strain was applied on the two pigskins at a rate of 0.05cm/s, and the force was measured to calculate the shear stress. The calculated shear stress was used as an indicator for the adhesiveness of the hydrogels on pigskin, and by extension, human skin.
DISCUSSION
Nuclear Magnetic Resonance Spectroscopy (NMR)
PNPHO and SPNPHO polymers with different molecular structures were used in this study. To study the molecular structure of the PNPHO and SPNPHO polymers, we used Nuclear Magnetic Resonance Spectroscopy (NMR). NMR is a technique that reveals the magnetic properties of certain atomic nuclei, allowing us to determine the physical and chemical properties of atoms, or the molecules in which they are contained, as seen in Figure 1.
Both polymers PNPHO and SPNPHO are a combination of monomers that have been bonded together in order to produce a final substance that contains the properties for a suitable hydrogel that can be used in medical applications.
PNPHO’s main components are, N acryloxysuccinimide ester (NAS), polylactic acid derivatives (PLA/HEMA), and polyethylene (NIPAAm), all of which give numerous unique properties to the final product, making it suitable for use inside the human body. PNPHO attains properties such as thermo setting, which allows the formation of hydrogels upon the increase of temperature to 37°C (Fathi et al, 2014).
Other properties include: water solubility, to eradicate the usage of organic solvents to reduce the toxicity risk inside the body, thus allowing for safe internal use; protein reactivity, to bond with proteins to make the final product biologically favourable; and mechanical strength, to ensure its suitability in the environment inside a synovial joint. In total, these combined properties mean that it outperforms other, more basic polymers such as PEG-da in these aspects.
SPNPHO is created through a similar process, whereby the polymer possesses the properties mentioned above. However there are some minor changes to the process in order to make the final polymer more suitable for applications such as diabetic and other external wounds. Using the same polymer chain, with the addition of 5-GMA, we changed the nature of the polymer, making it more suitable for uses in external wounds and making the polymer noticeably more viscous, resulting in a faster gelation time. Moreover, it adopts antiseptic properties, preventing the growth of bacteria in areas where this may be an issue (i.e. open skin).
Protein-co-Polymer hydrogel formation and Thermo-gelation
Both PNPHO and SPNPHO have properties that make them suitable for their intended applications, but what makes them unique in their field, is their thermo-responsive properties. With this property, the polymer forms a hydrogel in PBS, at a tuned temperature of 37ºC (Fathi et al, 2014).
Conjugation of polymers and proteins
Conjugating the polymers, PNPHO and SPNPHO, with proteins Elastin and Albumin respectively, is pertinent to our research, providing the hydrogel with strength and a uniformed microstructure. This uniformed microstructure will ensure that the hydrogel remains in the desired form once injected into the human body.
The physical mixture of natural and synthetic polymers may lead to the formation of a scaffold with a non-uniform microstructure. To address this problem, polymers can be chemically conjugated to proteins to form homogenous hybrid hydrogels.
The hydrogel is formed through the conjugation of succinamide ester (NAS) groups and Amine (NH2) protein groups. The integration of Elastin through covalent bondage with PNPHO promotes the structural stability, mechanical properties, and live cell proliferation within the structure of hydrogels.
Both PNPHO and SPNPHO hydrogels exhibit thermo-responsive behaviour when dissolved in PBS. This is due to their polymeric bonds (Figure 1) and the ability to tune the properties of the polymers themselves to our liking. Once the polymer reaches the temperature threshold (37°C) it will become of a similar viscosity to the cartilage tissue found in the human body. Due to the nature of the polymer, going above the temperature threshold does not have any effects on the speed of gelation, or the final product.
By being thermo-responsive, the administrator of the polymer is able to use a much less invasive and more precise method of injection into the body, dramatically increasing the success rate of surgeries aiming to cure cartilage diseases.
By starting with a high viscosity liquid, it can be easily manipulated to fit the desired area, such as a diabetic wound or bone cartilage crevice. Upon contact with the body, the surrounding temperature will heat up the polymer, allowing it to take on its gelled form. Surgeries such as bone cartilage transplants can be done with less detrimental effects on the surrounding area, due to a smaller incision and more careful means of operation, such as the use of a fine syringe to transport the polymer (Hardingham, Tew & Murdoch, 2002).
By being thermo-responsive, we can ensure that the polymer does not form a hydrogel unless inside the body, and it is also not affected by catalysts or other triggers which could cause a mistreatment.
Figure 3: Conjugation of polymers and proteins
Our results demonstrated that the hydrogel with the highest concentration of Elastin with PNPHO promoted the most beneficial structure. Samples of 75mg and 100mg of PNPHO per millilitre of PBS were extremely fragile and degraded extremely quickly. However a test sample of 150mg of PNPHO per millilitre of PBS proved successful, providing a flexible and strengthened hydrogel.
Fourier transform infrared spectroscopy
Fourier transform infrared spectroscopy (FTIR) is a technique used to observe molecular interaction. This is done through a simultaneous collection of high spectral resolution data over a wide spectral range. Confirming that the polymers PNPHO and SPNPHO have conjugated with the protein to produce a hydrogel or conjugated system is imperative to ensuring accuracy and validity in our study. An FTIR was used to confirm the chemical conjugation between the protein and polymer. Analysis of the changed peaks between the graph produced by the PNPHO and the graphs produced by the protein-co-polymers confirmed the formation of a covalent bond between the polymers and proteins.
Figure 1
Figure 2: Protein-polymer hydrogel formation and its thermo-gelation behaviour.
Note that the solution in the syringe is a liquid and solidifies instantaneously at 37ºC
Figure 4: FTIR spectra of thermo-responsive polymer, Albumin-co-polymer and Elastin-co-polymer hydrogels
As shown in Figure 4, the polymers all have a characteristic peak at 1730cm-1 signifying the molecular interaction with the protein. The FTIR results showed that there was a decrease in succinimide groups in both the Elastin-co-polymer and Albumin-co-polymer, although the decrease in succinimide groups with Albumin was less than with Elastin. This is due to the chemical properties of Elastin, which is more reactive than Albumin at a molecular level. This decrease confirmed the chemical interaction of protein and polymer and ensured that the protein was integral to the structural strength of hydrogels in future experiments.
Gelation efficiency of hydrogels
Gelation efficiency of the polymers is a ratio which indicates how much of the polymer has reacted with PBS to form the hydrogel. Poor gelation efficiency means that the entirety of the hydrogel has not been formed, and structurally, may not be stable within the body.
Having high gelation efficiency has two main advantages, which affect the success of PNPHO and SPNPHO. Firstly, in terms of production, it means that a greater amount of the polymer created reacts with PBS to form the hydrogel, meaning less is wasted. This is particularly important, considering that the synthesis of the polymer takes a minimum of 24 hours. Secondly, greater gelation efficiency ensures that the polymer has dissolved and formed its corresponding hydrogel correctly. Poor gelation efficiency can indicate that the polymer has not bonded with the PBS, resulting in a malformed gel.
Varying concentrations of Elastin and Albumin were combined with fixed concentrations of PNPHO and SPNPHO respectively. The protein concentrations used were 3.75mg/mL, 7.5mg/mL and 15mg/mL.
In our experiments with PNPHO, the gelation efficiency was found to be 30%, 23% and 0% at 3.75mg/mL, 7.5mg/mL and 15mg/mL concentrations of Elastin respectively.
Figure 5: The effect of varying concentrations of Elastin on the swelling ratio of hydrogels fabricated with 150mg/mL of PNPHO.
For SPNPHO the gelation efficiency was found to be 40%, 19% and 36% at 3.75mg/mL, 7.5mg/mL and 15mg/mL concentrations of Elastin respectively.
Figure 6: The effect of varying concentrations of Albumin on the gelation efficiency of hydrogels fabricated with 150mg/mL of SPNPHO.
It was found that 3.75mg/mL of protein resulted in the highest gelation efficiency for both PNPHO and SPNPHO, as can be seen in Figures 5 and 6.
Swelling ratio of hydrogels
The swelling ratio of the hydrogel in the human body is of vital importance. Ensuring the hydrogel does not expand profusely will prevent chances of inflammation and pain around the area of injection. The optimum result is to reduce the swelling ratio as much as possible; this is done through changing the concentrations of protein in the hydrogel. To calculate the swelling ratio we used the equation:
formation of the PEG-da hydrogel. Similar to the methods used in previous experiments, the hydrogels were prepared in PBS.
As seen in Figure 9, the resulting hydrogels were burnt and were thus extremely fragile, and shattered under very slight compression due the intrinsic properties of the PEG-da hydrogels. In addition, it was also discovered that changing the concentration of catalyst had very little effect in mitigating the burning of the hydrogel. This shows the necessity of the elasticity of the polymer; the fragile polymers produced are not practically useful in the human body, considering that the hydrogel needs to maintain a uniform, homogenous structure under external pressure in order to function.
Adhesiveness
7: The effect of varying concentrations of Elastin on the
of hydrogels fabricated with 150mg/mL of PNPHO.
For PNPHO the swelling ratio was altered through the use of different concentrations of Elastin in the hydrogel. Elastin concentrations of 3.75mg/mL, 7.5mg/mL and 15mg/mL were used. The concentration of 3.75mg/ml yielded no results, whilst 7.5mg/mL and 15mg/mL had swelling ratios of 5.4 and 4.29 respectively.
In the hydrogels, the protein concentration of 15mg/mL in a solution of 150mg/mL of PNPHO proved to have a low swelling ratio, and thus was useful in our study. Due to the similarity of the two polymers, we saw a similar pattern in the swelling efficiencies of SPNPHO hydrogels.
Using a redox polymerisation technique, hydrogels of PEG-da, a polymer that is used clinically as a control experiment, were produced (Fathi et al, 2013). The hydrogels consisted of 200mg/mL and 100mg/ mL concentrations of PEG-da, combined with L-ascorbic acid and used varying concentrations of Ammonium Persulphate ((NH4)S2O8) as a catalyst. The role of the L-ascorbic acid was to redox the polymer chain, with the catalyst speeding up the crosslinking, and thus the
In order for the final product to be circulated in medical clinics, it needs to able to adhere to the tissue that it is attempting to heal. Using pigskin to simulate human skin, the adhesiveness and strength of the hydrogels were examined. Three hydrogels, one made with PEG-da, one with SPNPHO, and one with PNPHO, were spread on pigskin and the skins were pushed together.
As can be seen in Figure 9, the PEG-da hydrogels were fragile and crumbled under compression, and thus, as seen in Figure 10, failed to adhere the skins to each other. However, the other two hydrogels succeeded in sticking the skins together, and in light of this, these skins were tested through mechanical testing, whereby the skins were placed into a Universal Instron Mechanical Testing Machine to test tensile strength.
Figure
swelling ratio
PEG-da
Figure 9: (Left) Burnt PEG-da hydrogels; (Right) State of PEG-da hydrogels after slight application of pressure.
Figure 10: (Left) PEG-da failing to adhere skins; (Middle) Adhering pigskins by placing hydrogel between them; (Right) Placing pigskins into Instron 5943 for tensile testing.
This machine produced a strain/stress curve by applying forces in opposite directions in order to separate the skins, and measuring the resistance encountered by the hydrogels. The shear stress on the two pieces of the skin was measured and translated to the skin adhesiveness of the material based on universal standard method ASTMD3164-03.
Strain vs. Shear Stress Graphs
The results showed that the Albumin-based hydrogel required less strain to separate the pigskins, whilst the Elastin-based hydrogel held more strongly, and it took more force to separate the skins. We concluded that Elastin was the more effective protein in terms of adhesion strength, as it could cope with higher shear stress prior to separation, as seen in Figure 11.
CONCLUSION
Our research was aimed at improving the treatment of superficial diabetic wounds and osteoarthritis within the knee. These thermoresponsive, injectable hydrogels possess similar properties to that of human tissue and are thus designed to treat both internal and external injuries. We can therefore justifiably conclude that PNPHO and SPNPHO will provide a more efficient alternative to the painful and invasive surgical options. Both polymers are designed to form at the optimum temperature of the human body (37°C), which allows for ease of handling within medical practices and ensures the safety of the patient by preventing further damage of the knee, which can be caused by formation of the gel in unwanted areas of the joint capsule. Bonding the PNPHO and SPNPHO with Albumin and Elastin respectively, they can act as a scaffold on which the body can grow and repair healthy tissue and cartridge. Through extensive experimentation, we optimised the concentrations at which the properties of the polymer, such as gelation time, swelling ratio and degradation rate, were the most efficient in terms of the medical application of the polymer, coming to the conclusion that concentrations of 150mg/mL of polymer with 15mg/mL of protein yielded the most efficient hydrogel. The next step is to further refine the qualities of the polymer and to ensure that the final product meets the required medical standards for clinical applications, whilst having optimised structural and chemical qualities.
ACKNOWLEDGEMENTS
We would like to thank Dr A Fathi, who has been our guide and mentor throughout the year, along with Mr N Hurst and Dr J Kavanagh. We must also commend Mr T Riley and Mr R Kennett for their work and dedication to making The Future Project possible for us this year.
Noting that the higher the leading coefficient of the trend line in the graph, the more resistance in the hydrogel, it is clear that Elastin-coPNPHO forms the stronger hydrogel, likely due to the fact that Elastin has more primary amine groups to bond with the synthetic polymer in comparison to Albumin. This result shows that the Elastin-co-PNPHO produces the optimal hydrogel for use inside the body, due to its ability to withstand larger strain. NB: The graph for the PEG-da hydrogel has been omitted due to lack of adhesion as seen in Figure 10.
REFERENCES
1. Fathi, A., Mithieux, S.M., Wei, H., Chrzanowski, W., Valtchev, P., Weiss, A.S. & Dehghani, F. (2014). Elastin based cell-laden injectable hydrogels with tuneable gelation, mechanical and biodegradation properties. Biomaterials. 35(21), 5425-5435.
2. Hardingham, T., Tew, S. & Murdoch, A. (2002). Tissue engineering : chondrocytes and cartilage. Arthritis Res. 4(3), 63–68.
3. Fathi, A., Lee, S., Zhong, X., Hon, N., Valtchev, P. & Dehghani, F. (2013). Fabrication of interpenetrating polymer network to enhance the biological activity of synthetic hydrogels. Polymer. 54(21), 5534–5542.
Figure 11: Strain/Stress graph produced by Instron 5943 showing tensile strength of each hydrogel.
Singapore
Takecarefor bestdecision
On the 27th of May 2015, two Year 11 boys, Gerry Feng and Thomas Dickinson, flew to Singapore in order to present their scientific research at a global student poster competition, held at the International Society for Pharmaceutical Engineering (ISPE) Conference.
The boys presented a scientific poster that outlined their work as Senior Interns in The Future Project. Tom and Gerry’s project was to test the effect of the solvent detergent treatment on the PrIME Plasma Fractionation Process for the purification of human albumin and Immunoglobulin G. In layman’s terms, the boys tested the compatibility of the established viral inactivation technique to separate during these two prominent protein seperations from human blood plasma. These purified proteins can then be used in the manufacturing of life saving products for developing nations. The boys did fantastically well, placing third overall in the competition. This result is even more impressive when considering that the top two awards were taken out by PhD students, and all but two of the presentations were made by university undergraduates. The boys did brilliantly just to be selected to present, given the vast number of applicants.
Tom and Gerry have been working with PrIME Biologics, a plasma fractionation company that manufactures life saving therapeutic products in Singapore. The company generously sponsored the boys’ visit to the conference, and following their presentation they were able to visit PrIME’s main processing plant. This was an incredibly unique experience that the boys will never forget. Overall, the trip was an extremely enriching experience and the boys represented the school with great distinction. We cannot thank PrIME Biologics enough for their wonderful and generous tutelage of these budding young scientists.
In 2015, the Junior Interns Rotation was introduced to The Future Project. The aim was to provide an authentic research experience to a group of 26 students selected through an interview process, and to provide them with the necessary skills and information required to be a successful Senior Intern. It also provided an opportunity for the researchers to meet and inspire the potential Senior Interns about their research, while also playing a part in the selection process.
The students were placed in The Future Project Intern science class and instead of undertaking their usual Biology rotation, they enjoyed an intensive eight-week block of research science. The block centred on the key topics of DNA and Protein in the context of the research being conducted by the specific companies to which the students were assigned. The practical focus meant that the boys were exposed to equipment and techniques associated with two- or three-year university courses, rather than your usual Year 10 Science class. Below is a synopsis of each element of the rotation written by some of the Junior Interns.
DR JORDAN NGUYEN
A fascinating and inspirational speaker, Dr Jordan Nguyen showcased the unforeseen nature of science and its ability to employ the elegance of creativity to enhance our lives. His candid recount of his experiences and the extent of his determination to pursue a goal that could potentially change the lives of the disabled was truly captivating.
Dr Nguyen commenced his recount by addressing his initial dislike and frustration during his study of mathematics in high school – something he thought held little practicality in the ‘real world’. He stated that the discipline of Robotics and Science within UTS however, changed his perception of mathematics and gave him the incentive to critically evaluate, understand and appreciate its elegance. His fascination with robotics spurred his PhD, in which he designed a mind-controlled wheelchair to assist and facilitate the needs of the disabled. In a challenging course not lacking in obstacles and impediments, his logical and intuitive process in addressing the problems with the synthesis of mathematical reasoning and scientific knowledge were indeed thought provoking. Dr Nguyen went on to explain that he has since been planning the marketing aspects of his products and has shared his interests in robotics and science with all on his YouTube channel. The underlying regard of scientific innovation as a catalyst to allow a better understanding of prior knowledge was key to his fantastic presentation. Dr Nguyen undoubtedly intrigued us with what the future of science has to offer – a journey worth undergoing.
Eugene Wang
USYD – FACULTY OF IT AND ENGINEERING
In the final week of Term 2 The Junior Interns were visited by Dr John Kavanagh from The University of Sydney who is a Senior Lecturer specialising in Chemical Engineering. Dr Kavanagh gave the boys a number of research papers in the week prior to his visit, and asked that each group of students read and note a particular article. These articles ranged from the use of polymers to enhance bone density, to the development of improved insulin pumps. During the interactive lecture, the students had to present their chosen article to their peers and provide suggestions for future areas of research. The visit highlighted the thought provoking nature of research science and gave the students a valuable insight into university life.
PRIME BIOLOGICS
During the first few weeks of the Junior Interns rotation, we worked with PrIME Biologics, under the guidance of Dr Kailing Wang. We were taught basic pipetting techniques, as well as two modern methods used to determine the quality and quantity of proteins.
The Bradford assay is a quantitative technique performed by dying the protein with Coomassie Blue, a special protein-binding dye. The protein is then scanned in a machine where Ultraviolet (UV) light of a certain wavelength (595nm) is used to record the amount of UV absorbance by the stained protein. The absorbance of UV light is proportional to the amount of protein in the solution, thus allowing us to deduce the concentration of a certain protein in a sample.
The SDS-Page is a qualitative procedure that is used to distinguish the purity of a protein in a sample. By first applying Sodium Dodecyl Sulfate (SDS) to proteins, the proteins are denatured and linearised. These proteins are then placed into an acrylamide gel. Electrophoresis is performed on the gel, forcing the negatively charged proteins to ‘run’ towards the positive electrode. After staining the proteins, we can see the proteins as ‘bands’, which can be compared to control samples in the same gel to identify the type and purity of protein.
Our time with PrIME was thoroughly enjoyable and interesting. We learnt a considerable amount about proteins, in particular their practical uses and how to identify them, giving us a taste for what we might potentially be researching next year.
Jason Wan & Henry Roth
VITRAMED BIOSCIENCE
Vitramed, a company on the forefront of exploring new ways to improve gastrointestinal (GI) health, spent the majority of the four-week course with us. With their help, we were able to partake in a mock clinical trial using new techniques and knowledge that we accumulated as we progressed.
We started the internship as microbiologists learning the basic skills needed to conduct the experiments. This included learning how to correctly pipette miniscule amounts of liquid into eppendorf tubes, together with growing and isolating colonies of bacteria or fungi on specific agar plates. During our time with Vitramed Bioscience, we were involved in a small participant clinical trial. This was to test which probiotic could cure our patients if they were infected with a fungus called Candida albicans, a yeast that grows within the body.
Our aim was to discern if Lactobacillus acidophilus or any other forms of Lactobacillus would be able to help patients with this infection. Thus, we began our clinical trial, trying to determine if the L. acidophilus they were taking was able to improve their condition. Throughout this process, we acquired a plethora of new skills. Among these were spread plating, streak plating, DNA extraction and Polymerase Chain Reaction (PCR). We were also fortunate enough to utilise an array of new equipment such as pipettes, agar plates, centrifuges, vortexes, and DNA electrophoresis gel tanks.
The practical nature of the course meant that we were on our feet for the duration. Due to the countless new experiences in which we were able to partake, it was without a doubt one of the most interesting scientific experiences we have ever encountered.
Joseph Rylance, Allen Guo & Owen Mak
Heliprobe Print Bridge: Production Prototype [Senior Mechatronics]
This year in The
on the
and
David taught himself to use professional PCB design and 3D modelling software packages and then created a two layer PCB and plastic enclosure for the PCB and components. These designs were good enough that we were able to send them
I’d be impressed to get that kind of result from a university graduate, so I’m extremely impressed by what a self-taught high school student, working in his spare time, has been able to achieve with minimal guidance from me.
and
Junior Mechatronics
BACKGROUND INFORMATION
The Future Project’s Junior Mechatronics Program, run by Vitramed Mechatronics is overseen by owner, Daniel Simmons. This year, Daniel Marsh and Jack Taylor were assigned to the program, for which they worked three to four hours each week during their timetabled Engineering classes. When the program began in February, Daniel and Jack were given intensive lessons in C# programming language and the use of microcontrollers. Once these skills were mastered, the boys were given various project ideas from which to choose, including: a golf course robot that distinguishes between weeds and fairway grass; an automated sparring partner; and their chosen project, a device that measures punch force, similar to the popular carnival game High Striker.
PROGRESSION
From the genesis of the project the ultimate goal was to design a device that could measure the force exerted onto an object through a punch; a goal inspired by Daniel Simmons’ interest in boxing. In order to choose the appropriate method, the boys worked using different sensors for their microcontrollers to discover which would be most effective in measuring force output. They first tried an accelerometer sensor which measures acceleration in three planes (x, y, z), but the readings did not make sense and it seemed that they would need to rewrite the underlying program that converts the sensor data to something usable, which was too big a task for their project. In parallel, they worked with a pressure sensor that measures air pressure. They placed the sensor in a bladder from a soccer ball and partially inflated it. When they hit the bladder, the pressure in the ball would increase and they could measure the change in voltage on the sensor. At some point, the sensor stopped working, possibly because the punches were too strong and overloaded it. The last sensor the boys used was a Force Sensing Resistor that converts force into resistance. By adding some more electronic components they were able to use the sensor resistance to provide a changing voltage that could be measured with the microcontroller.
This works well but is very sensitive and more work is needed to work to make it less sensitive. Jack and Daniel also had to design the additional components of the overall model, such as the camera, SD card, screen, power source and punching bag.
FINAL MODEL
As seen, the final model consists of numerous interrelated components. The screen is programmed to display: the current punch value, measured on an arbitrary scale from 0-100; and the highest recorded score. In order for this to be possible the boys had to write a code that determines what data constitutes a punch. An SD card stores the maximum value for each punch, along with a photo, taken by the associated camera, of the boxer mid-punch once a threshold value is exceeded. The battery pack and the internal microcontroller, which contain the specific C# code written by the boys, are also contained within the custom built housing.
CONCLUSION
All in all, the Junior Mechatronics Program was an incredibly rewarding learning experience for Jack and Daniel, as they obtained a skill-set in a short period of time that would have been difficult to learn in a mainstream classroom setting. Furthermore, they look on it as work experience, where they learned the value of independence through problem solving and discovery-based learning. This project would not have been possible without the tireless commitment and invaluable tutelage of Daniel Simmons. Thanks must also go to the Industrial Arts Department who so kindly released the two boys from their usual classes. Jack and Daniel will now continue with this priceless opportunity as Senior Mechatronics Interns in 2016, picking individual projects that have a closer relationship to scientific engineering.
GLOSSARY
In electronics, a sensor is a device that measures a real-world property (such as temperature, force or acceleration) and converts it into an electrical signal.
A microcontroller is a tiny computer that is typically connected to sensors, motors, and small displays. Where a larger, normal computer is designed to run many programs, a microcontroller is far smaller, cheaper, requires less power, and provides purposedesigned programming for one job. For example, microcontrollers are found in most modern household appliances, from dishwashers to DVD players.
Daniel Marsh & Jack Taylor
The survival of Dietzia C79793-74 during simulated gastrointestinal transit
Dietzia: a tale of survival
Our work this year has been to test how well a probiotic bug survives the gastrointestinal transit. We used a simulated stomach and intestinal model, mixed into it a desired concentration of the bug, and took it out at varying time periods. We then diluted it down so that we reached a concentration number that we could spread plate to see how well it survived. We tested this again with varying pH levels within the stomach, and plated onto different salt concentrations to reveal how injured cells are affected by the presence of salt when being recovered on agar plates.
Our experiences in The Future Project have allowed us to further our understanding of real-world applications of science, which cannot be replicated
in a classroom. By undertaking a research project with The Future Project, we now have some idea whether we wish to pursue a career in science. The Future Project is a worthwhile experience because no other school in Australia gives a student the chance to work in a state-of-the-art laboratory with scientists and help them in their line of research. Thanks to The Future Project we have been exposed to more real-world science in a one-year period than we have in our whole schooling, and working first hand with bacteria has been quite a memorable and valuable experience for us all.
Ted & Vithushan
ABSTRACT
Dietzia C79793-74 has been shown effective in the treatment of Johne’s disease in cattle, and is being screened for its potential as a probiotic for treatment of Crohn’s disease in humans. Ideally, for delivery via the oral route, Dietzia C79793-74 should show some tolerance to gastrointestinal transit, including the low pH environment encountered in the stomach, as well as the digestive enzymes encountered in the small intestine, from the gallbladder and pancreas. To model gastric transit, Dietzia C79793-74 was inoculated into simulated gastric juice at different pH levels (pH 2, 3 or 4). To model gastrointestinal transit, after three hours the gastric model was neutralised with sodium hydroxide, and placed in a small intestine simulation containing bile salt and pancreatin; survival over time was evaluated by spread plating. After 120-minute (min) exposure to the gastric model at pH 2 there was a 3.22 log10 CFU/mL reduction in the number of viable cells of Dietzia C79793-74, then exposure for 180 min in the small intestine model resulted in an additional 1.55 log10 CFU/mL reduction in the number of viable cells. Dietzia C79793-74 survived very well at pH 3 and 4 in a gastric-only model, indicating that it has some natural acid resistance and may survive gastric transit if patients are pre-treated with an acid inhibitor. Furthermore, Dietzia samples from a pH 2 and pH 4 gastriconly simulation were plated on agar with different concentrations of salt to investigate cell injury. Possibly due to its naturally high salt tolerance, differential plating on agar with salt did not reveal cell injury for Dietzia C79793-74 at either pH.
INTRODUCTION
Crohn’s disease is a chronic form of Irritable Bowel Syndrome, generating inflammation of the small and large intestines, and in some cases leading to extreme rectal bleeding (Duerr et al, 2006). Importantly, Crohn’s disease has been likened to Johne’s disease, which causes similar chronic inflammation of the small and large intestine. The probiotic microorganism Dietzia C79793-74 has shown promising results in cattle with Johne’s disease as it supresses the Mycobacterium avium paraturbucerlosis (MAP) microorganism that is its cause (Click & Van Kampen, 2010).
Our experiments this year build upon the previous year’s work (Fraser et al, 2014) where we tested the viability of Dietzia C79793-74 through a simulated gastric model only, including in the presence of milk, added as a pH buffer. By including a simulated small intestine, we were able to get an understanding of the survival of Dietzia C79793-74 as it moves through the whole gastrointestinal tract.
METHOD
Preparation of Dietzia C79793-74
The probiotic Escherichia coli Nissle has shown promising results in patients suffering from ulcerative colitis (Kruis et al, 2004). The safety and efficacy of Dietzia C79793-74 for the treatment of Crohn’s disease is currently being evaluated by a clinical trial in which Vitramed is a co-investigator.
Through our experimentation with Dietzia C79793-74 in a simulated gastrointestinal model, we were able to test how an oral dosage of Dietzia C79793-74 would survive gastrointestinal transit, so that in the future this work can be used to help patients suffering from Crohn's disease. The pH of the stomach can reach as low as 1.5, and Dietzia is known to grow at alkaline and neutral pH (Yumoto et al, 2002), but its survival in a more acidic environment potentially limits the amount of viable cells transiting through to the intestinal environment. By simulating a whole gastrointestinal transit and incubating Dietzia C79793-74 in the mixture, we were able to take out a sample at designated time points and conduct a dilution series to then spread plate a suitable concentration. This showed us how many colonies developed at that particular time, in turn telling us how many of the inoculated cells survived the simulated gastrointestinal transit.
Dietzia C79793-74 was cultured in tryptic soya broth, with fructose (without dextrose; TSBF) for 96 hours at 29°C following the method of Click (2011). It was then harvested and concentrated ~25-fold by centrifugation (3,800 g, 10 min), resuspended in 0.9% saline, washed twice with 0.15% skim milk (reconstituted in water), and finally resuspended in 0.15% skim milk and glycerol. Concentrated cell suspensions were aliquoted into 1 mL vials and stored at -80°C until use. Dietzia C79793-74 aliquots were thawed at room temperature immediately prior to use.
Preparation of simulated gastric/gastrointestinal models
To re-create the stomach environment we prepared simulated gastric juices at pH 2, 3 or 4, as described by Huang & Adams (2004). Simulated gastric juice was prepared by adding pepsin (0.045 g) and NaCl (0.015 g) to 15 mL of deionised water, and adjusting to the required pH with diluted HCl. For experiments simulating the entire gastrointestinal transit, after 120 min of exposure to the gastric juice, bile salt (0.054 g/L) and pancreatin (0.04 g/L) (Huang et al, 2004) was added, and NaOH used to neutralise acidity (i.e. bring to pH 7) to simulate transition to the small intestine. The new solution was further incubated at 37˚C for 180 min, giving a total gastrointestinal transit time of 300 min (Proano, Camilleri, Phillips, Brown & Thomforde, 1990).
T.Simpson1, V. Lingam1, M. Bull2, B. Chapman2, D. Terre2
THE FUTURE PROJECT1 AND VITRAMED BIOSCIENCE2
The King’s School, NSW 2151, Australia
Inoculation of gastric/gastrointestinal models and sampling Dietzia C79793-74 inoculum, 0.3 mL, was inoculated into 1.2 mL of simulated gastric juices and incubated at 37°C. The inoculum was then diluted in Buffered Peptone Water (BPW) and appropriate dilutions spread-plated onto Tryptone Soya Agar with fructose (without dextrose; TSAF) agar plates to check the concentration. The gastric model was sampled at 20 or 30 min intervals (dependent on pH), and (as appropriate) the small intestine part of the simulated transit at 30 min intervals. All samples were diluted into BPW and spread plated onto TSAF. All experiments were performed in duplicate.
Differential plating
As a secondary experiment, we also plated some samples from pH 2 and pH 4 gastric-only models onto TSAF with an additional 3%, 5% and 7% salt to determine if non-lethal cell injury was occurring during gastric transit, even when plate counts indicated a high proportion of cells were surviving. In the presence of high levels of salt, injured cells with damaged membranes might find it harder to tolerate the higher osmotic potential and therefore result in fewer colonies (Wyber, Andrews & Gilbert, 1994).
Incubation
All agar plates were incubated at 29˚C for seven days, before the colonies were counted and recorded to compare survival at different pH in the gastric model or survival through the gastrointestinal model.
RESULTS
Simulated gastric model
In the pH 2 simulated gastric model, a 3.5 log10 colony forming unit (CFU)/mL (Figure 1) reduction in viable cell counts over 120 min was seen. A considerable decline in cells was noted between the 60 and 80 min samples, with a 1.8 log10 CFU/mL reduction, which was over half of all decline.
Throughout exposure to the pH 3 gastric model, Dietzia C79793-74 did not decrease in viable cell counts during the 210 min period (Figure 1). Further, it showed no signs of stress even when plated with colony sizes on exposed samples showing no difference from the unexposed inoculum. The lack of reduction in cell numbers signified some level of natural acid resistance of this microorganism.
Figure 1: The effect of pH on the tolerance of Dietzia C79793-74 to a simulated gastric model. Results are from duplicate experiments. Error bars are standard error of the mean.
2: The effect of pH
Simulated gastrointestinal model
In the full gastrointestinal model commencing at pH 2, after a small shoulder for 30 min, there was a steady decline in viable cells until plateauing at 180 min at 5.36 log10 CFU/mL (Figure 2). Interestingly, after exposure for 120 min in the pH 2 gastric model, the viability of Dietzia C79793-74 continued to decline after it was introduced into the small intestine environment at neutral pH. We observed a further 1.31 log10 reduction in the population of bacteria within the first 60 min in the small intestine model, before seeing next to no decline in cell counts after this initial decrease.
In the gastrointestinal model commencing at pH 4, the initial population of Dietzia C79793-74 was maintained for the experiment period, with only a 0.62 log10 CFU/mL decrease in cell count throughout the whole 300 min simulation.
Differential salt plating
Differential plating on TSAF agar with different concentrations of salt (0, 3, 5 and 7% NaCl) was evaluated to test if this method could distinguish between uninjured and injured cells after exposure to the gastric-only model (pH 2 and 4). Injured cells were expected to be more sensitive to high NaCl levels and therefore inhibited from growing on agar with higher salt concentrations. The results (Figure 3) indicated no significant difference between colony counts from a particular pH gastric-model on each concentration of salt agar. Notably, colony size on agar diminished as salt levels increased indicating that Dietzia C79793-74 was under some form of stress at higher salt concentrations, resulting in slower growth and therefore smaller colonies.
DISCUSSION
The survival of the probiotic microorganism Dietzia C79793-74 throughout the gastrointestinal transit was largely governed by the acidity of the initial simulated gastric environment. Dietzia C79793-74 was shown to have natural resistance to high levels of salt, low pH, and small intestine enzymes, thus enabling it to survive short-term exposure in environments above pH 2.
Simulated gastric transit
The pH 3 and 4 simulated gastric models had minimal effect on Dietzia C79793-74 viability as assessed by cell counts, with counts maintained around the inoculum level for at least 200 min (Figures 1 and 2). At pH 3 (Figure 1), counts indeed appeared to increase slightly. One reason the count may have increased slightly is due to a separation of clumped cells during the pH 3 exposure. Cell clumping may have occurred when the Dietzia C79793-74 was initially being concentrated by centrifugation.
In contrast, the pH 2 gastric model had very drastic effects on the survival of Dietzia C79793-74 (Figure 1). Cell counts dropped from 9.89 to 5.36 log10 CFU/mL in the simulated gastric environment at pH 2. A reduction in colony size was observed when recovering cells from the pH 2 environment, indicating that cells sustained injuries and recovered at a slower rate but the injuries were not sufficient enough to prevent cells from growing at all, even when recovered in a high salt environment (Figure 3a).
Combined, the results indicate that the pepsin enzyme added to the gastric model had minimal effect on the viability of Dietzia C79793-74.
Simulated gastrointestinal transit
The small intestine environment did little to affect the survival of Dietzia C79793-74 as it passed through with no significant loss in cell counts. The intestinal environment at pH 7 was more favourable towards the survival of Dietzia C79793-74 as it was neutral pH, although it did introduce exposure to bile salts and pancreatic enzymes. Interestingly, when transferred from an initial pH 2 gastric transit, the cell counts continued to decline between 120 min and 180 min before plateauing. This result suggested that enzymes in the small intestine must have negatively affected injured cells (Ramalheira et al, 2010).
Differential plating
The aim of using differential plating with different salt concentrations was to determine how many injured cells there were once they had completed the gastric transit. This was because the cells were exposed to a highly acidic environment and injured cells commonly have an injured cell membrane which makes it much harder for cells to maintain osmotic pressure in the presence of high levels of NaCl. By plating them on low and high salt TSAF, we were able to determine the presence of sub-lethally injured cells versus uninjured cells. However, it has been shown that Dietzia C79793-74 is highly salt tolerant; Hobbs, Bull & Chapman (2014) were successful in enumerating Dietzia C79793-74 samples from faecal matter using recovery on TSAF with 5-7% NaCl which suppressed the growth of background bacteria while allowing the Dietzia to grow. The results from our experiment show that Dietzia C79793-74 was able to grow, without loss of cell numbers, on agar of up to 7% NaCl, even after exposure to a low pH environment. However, colony sizes decreased with an increase in salt concentration, indicating there was some degree of growth inhibition (Wyber et al, 1994), but not enough to stop cell growth.
Figure
on the tolerance of Dietzia C79793-74 to a simulated gastrointestinal model. Results are from duplicate experiments. Error bars are standard error of the mean. Dashed vertical line (120 min) signifies change from stomach to small intestine model.
Figure 3: The effect of salt concentration in agar on the recovery of injured Dietzia C79793-74 from simulated gastric juices at pH 2 (A) and pH 4 (B).
CONCLUSION
Dietzia C79793-74 is able to survive transit through a simulated gastrointestinal model, although its optimum survival rate was exhibited when the initial gastric exposure was at pH 3 or higher. We can also conclude that Dietzia C79793-74 has a naturally high tolerance to salt even after exposure to a simulated stomach environment. This information is helpful when Dietzia C79793-74 is required to be recovered from stool samples, as its high resistance can be used to isolate the microorganism from other bacteria in samples; we have confidence that this method does not inhibit the recovery of potentially injured cells.
REFERENCES
1. Click, R.E. & Van Kampen, C.L. (2010). Assessment of Dietzia subsp. C79793-74 for treatment of cattle with evidence of paratuberculosis. Virulence. 1(3) 145–55.
2. Click, R.E. (2011). A 60-day probiotic protocol with Dietzia C79793-74 prevents development of Johne’s disease parameters after in utero and/or neonatal MAP infection. Virulence. 2(4) 337-347.
3. Duerr, R.H., Taylor, K.D., Brant, S.R… Cho, J.H. (2006). A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science. 314(5804) 1461–1463.
4. Fraser, B., Hariharan, S., Miller, S., Simpson, T, Bull, M., Chapman, B. (2014). Testing and improving the survival of Dietzia during simulated gastric transit: a first step in its screening as a probiotic for Crohn’s Disease. The Journal Of The Future Project. 1, 2-5.
ACKNOWLEDGEMENTS
We wish to thank all The Future Project staff involved, in particular: Mr T. Riley for tireless support throughout; Mr R. Kennett, for opportunities throughout; and a massive thanks to our researchers Michelle Bull, Belinda Chapman and Darcii Terre for their continual guidance, time and effort put into our project.
5. Hobbs, K., Bull, M. & Chapman, B. (2014). Testing the salt tolerance of Dietzia and its recovery from faecal material. The Journal Of The Future Project. 1 6-9.
6. Huang, Y. & Adams, M.C. (2004). In vitro assessment of the upper gastrointestinal tolerance of potential probiotic dairy propionibacteria. International Journal of Food Microbiology. 91 (3), 253–60.
7. Kruis, W., Frič, P., Pokrotnieks, J… Schulze, J. (2004). Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine. Gut. 53, 1617-1623.
8. Proano M., Camilleri, M., Phillips, S.F., Brown, M.L. & Thomforde, G.M. (1990). Transit of solids through the human colon: regional quantification in the unprepared bowel. American Journal of Physiology. 258(6 Pt 1) G856-G862.
9. Ramalheira, R., Almeida, M., Azeredo, J., Brandão, T.R.S., Almeida, G., Silva, J. & Teixeira, P. (2010). Survival of clinical and food isolates of Listeria monocytogenes through simulated gastrointestinal tract conditions. Foodborne Pathogens and Disease. 7 121-128.
10. Wyber, J.A, J. Andrews and P. Gilbert. 1998. Loss of salt-tolerance and transformation efficiency in Escherichia coli associated with sub-lethal injury by centrifugation. Letters in Applied Microbiology 19, 312-316
11. Yumoto, I., Nakamura, A., Iwata, H., Kojima, K., Kusumoto, K., Nodasaka, Y. & Matsuyama, H. (2002). Dietzia psychralcaliphila sp. nov., a novel, facultatively psychrophilic alkaliphile that grows on hydrocarbons. International Journal of Systematic and Evolutionary Microbiology. 52 85–90.
Optimisation of buffer conditions for the separation of IgG from human plasma
Histidine - the New,
Cheaper, Buffer Machine
Immune systems: everyone has them. They are the trusty combatants that fight against disease. Unfortunately, some people’s immune systems are compromised and can turn against themselves, launching an attack on healthy organs and cells in the body. These diseases are called autoimmune diseases and are very hard to treat. A unique protein found in human blood called Immunoglobin G (IgG) has been proven to help patients recover from autoimmune diseases. Plenty of IgG can be found in healthy human blood, but it is very difficult to extract IgG from the blood for people who need it the most. This year we have been working with PrIME Biologics to unlock a way to purify IgG in a more cost effective way.
We have thoroughly enjoyed our experiences in The Future Project. The PC2 laboratory in the Science Centre is amazing, especially considering the fact that most university students do not have facilities such as these. Furthermore, we are able to work alongside world-renowned scientists, on the forefront of ground-breaking science. Having the opportunity to be involved in something that is different, practical and exciting, as well as giving us the opportunity to write our own thesis, has enriched us as young scientists and is an experience we will never forget.
Tom, Lian & Lisa
ABSTRACT
Immunoglobulin G (IgG) is a protein found in human blood plasma. Once purified, it can be used as a therapeutic product to treat patients with various immune disorders. The electrophoresis buffer currently used during the IgG separation process is MES Bis-Tris pH 5.2. This study investigates the use of a Histidine buffer as a cheaper alternative to replace MES Bis-Tris, whilst maintaining the process yield and product purity. If successful, there will be a reduction in the cost of the process, leading to greater affordability of life-saving treatments for patients in developing countries.
INTRODUCTION
Immunoglobulin G (IgG), found in blood plasma, has a Y-shaped structure similar to many other Immunoglobulins (Ig). It is the most abundant antibody isotype found in the human cardiovascular circulation, making up 75% of serum (plasma) Ig. Antibodies play a vital role in the immune system, with IgG being the most abundant antibody in plasma, preventing many of the body tissues from becoming infected. IgG is 150 kilo Daltons (kDa) in size. IgG can be used to prevent disease in general, and to treat immune deficiency disorders, autoimmune disorders and infections.
The technology involved in the IgG separation process is PrIME, which stands for Preparative Isolation Membrane through Electrophoresis. It separates proteins with regards to both charge and size through a porous separation membrane. The centrepiece of the technology is the membrane cartridge, which consists of three polyacrylamidebased hydrogel membranes. A separation membrane is sandwiched between two restriction membranes, and two independent streams flow between the separation membrane and restriction membranes on both sides. The separation cartridge operates within a PrIME BF-400 separation instrument (NuSep Ltd). Electrophoresis separation occurs when an electrical field is applied across the separation cartridge.
METHOD
IgG Separation by PrIME
The pH of the buffer used determines the charge of the protein, depending on whether it is higher or lower than the isoelectric point (pI) of the protein. The pI of a molecule is the point on the pH scale at which it carries no electrical charge. If the pl (e.g. 5.6 for IgG) is higher than the pH of the running buffer, then the proteins will become positively charged; the opposite is true if the pl is lower than the pH of the running buffer. Due to the polarity, positively charged ions will move towards the negatively charged electrode, and vice versa. Stream 1 (S1) and Stream 2 (S2) are set up in a way that proteins will have to travel through the separation membrane which also ensures that they are separated based on size. The advantages of this process are: higher protein recovery; greater efficiency; and higher purity. The aim of this study is to investigate the effect of three different buffer compositions of Histidine buffer on the separation of IgG from human plasma by PrIME.
The separation process was conducted over a 360-minute period using the PrIME BF-400 instrument. S1 and S2 were cleaned with buffer. The system ran for five minutes, then the buffer was drained. S1 was then filled with 15mL of human plasma solution and S2 was loaded with 15mL of buffer.
Both S1 and S2 continuously circulated within the system at speed of 10mL/min. The electric potential was subsequently applied with a constant voltage of 250 V. Data was recorded in a running sheet, including the voltage, current and volumes of both streams. At 30-minute intervals, aliquots of S2 were collected for further analysis. The remaining liquid was collected in a pool. Fresh buffer was then loaded into S2 every 30 minutes for a total of 360 minutes.
This process was repeated with a Histidine buffer of pH 5.2, a Histidine MES Bis-Tris buffer with a pH 5.26, and a Histidine Acetate buffer with a pH of 5.24
T. Dickinson1, L. Yang1, L. Cao1, K. Wang2, H. Nair2
THE FUTURE PROJECT1 AND PRIME BIOLOGICS2 The King’s School, NSW 2151, Australia
Figure 1: Diagram of IgG separation membrane
SDS-PAGE Analysis
Samples collected from the separation process described above were then qualitatively analysed using SDS-PAGE. The samples were prepared by mixing 25μL of SDS-DTT sample buffer and 25μL of protein samples. Alongside 10μL of Bio-Rad pre-stained SDS-PAGE standards to act as a marker, the samples were heated up to 95ᵒC and loaded into the 4-20% SDS-Glycine (SDS-PAGE, NuSep). SDSPAGE gel was run with SDS-Tris-Glycine running buffer at 180 V for 90 minutes. Upon completion, the gels were visualised by BioRad Gel Doc and then stained with Coomassie Blue stain.
Bradford Assay
IgG concentration in the collected fractions was quantified by the Bradford Assay. This was conducted in the 96-well microplate. A standard curve was generated with a series of dilutions of a standard human IgG concentration (sigma) at 5mg/mL. All protein samples were diluted in a ratio of 10:1 (180μL of buffer and 20μL of IgG). 10μL of each diluted sample was loaded with a micropipette and placed into the microplate, along with 10μL of standard and stream samples. This was repeated three times to maximise accuracy. Next, 250μL of Bradford reagent was added with a multi-channel pipette (eight channels). The microplate was incubated for colour development at room temperature for five minutes. Following this, the microplate reader was used to receive readings at a wavelength of 595nm.
RESULTS AND DISCUSSION
Analysis of the data was achieved through a qualitative SDS-PAGE analysis and a quantitative protein Bradford Assay analysis. A comparison was made between the Histidine, Histidine MES Bis-Tris, Histidine Acetate and the MES Bis-Tris control buffer with regard to the quality and purity of IgG after PrIME separation.
Figure 2 illustrates a qualitative SDS-PAGE analysis of the specific proteins separated by each of the three aforementioned buffers together with the control buffer, MES Bis-Tris. Lane 1 is the standard molecular weight marker, Lanes 2-5 correspond to the MES Bis-Tris control buffer, Lanes 6-9 correspond to the Histidine MES Bis-Tris Buffer, Lane 10 is the Human IgG used as a control for comparison, Lanes 11-14 correspond to the Histidine Acetate buffer, and Lanes 1518 correspond to the pure Histidine buffer.
The SDS-PAGE provides a protein separation profile from which the quality and purity of IgG can be analysed with respect to each separate buffer. IgG has a known molecular weight of 150 kDa and thus the samples can be compared with the known molecular weight marker shown in Lane 1.
Lane 12 illustrates the amount of IgG extracted when using the Histidine Acetate buffer. In addition to IgG that has a molecular weight of 150 kDa, multiple protein bands are visible in the gel, indicating the presence of a number of protein contaminants. This is similarly the case in Lane 16 when using the pure Histidine buffer. The protein band at the molecular weight of IgG in Lane 16 is the least dense of the entire protein separation profile. This indicates that pure Histidine buffer is the least effective of those buffers used when considering the overall quality and purity of the IgG separated.
The qualitative SDS-PAGE analysis indicates that the MES Bis-Tris control buffer in Lane 3 was effective in purifying IgG. This corresponds to the very dense protein bands indicating the presence of purified IgG. The lack of other protein bands in Lane 3 illustrates the high purity of the IgG protein with the use of the MES Bis-Tris as separation buffer.
Lane 7 of the SDS-PAGE analysis is the IgG extraction from human plasma using the Histidine MES Bis-Tris buffer. This buffer was similarly effective in separating out IgG, as shown by the high-density band at about 150 kDa. However, a number of contaminants are present further down the gel indicating a lower degree of purity.
Lane 1: Marker Lane 10 Human IgG 10 μL
Lane 2: M S1 0 Lane 11: HA S10
Lane 3: M S1 end Lane 12: HA S1 end
Lane 4: M S20 iLane 13: HA S20
Lane 5: M S2 pool iLane 14: HA S2 pool
Lane 6: HM Lane 15: H S10
Lane 7: HM S1 end Lane 16: H S1 end
Lane 8: HM S20 Lane 17: H S20
Lane 9: HM S2 pool Lane 18: H S2 pool
These qualitative results suggest that the Histidine MES Bis-Tris buffer was as effective in separating IgG from human plasma as the MES Bis-Tris control buffer in terms of the quantity of protein extracted, although the purity was lower due to the presence of other protein contaminants. However, Figure 3, depicting the quantitative Bradford Assay analysis, indicates that both the Histidine Acetate and Histadine MES Bis-Tris buffers produced a significantly higher yield of IgG. Both aforementioned buffers increased the yield of IgG when compared with the control MES Bis-Tris buffer (Figure 3). Indeed they are also far more effective in terms of the yield of IgG than a pure Histidine buffer. This is a very important result as it indicates that mixed Histidine buffers may be used as effective alternatives, as they increase the separation process efficiency in purification of IgG from human plasma. However, as observed in the qualitative SDS-PAGE Analysis, the purity of the IgG extracted using the Histidine mixed buffers is reduced when compared with that of the MES Bis-Tris buffer.
The results of this experiment are valid as the starting material and separation conditions were kept identical, except the type of buffer used for separation. Other variables were also kept identical throughout the separation process, such as the volume of human blood plasma used in S1 constant and the volume of buffer used in S2, so that the measurements detected for the dependent variable, the yield and purity of extracted IgG, were as a result of the independent variable. Additionally, a control buffer (MES Bis-Tris) was used as a baseline for comparison. All three mixed buffers were kept at the same pH to ensure the experiment was fair and valid.
CONCLUSION
Overall, the use of Histidine in the buffer composition results in the separation of more IgG from human plasma than the original MES Bis-Tris buffer. Out of the three Histidine buffers tested, Histidine MES Bis-Tris performed the best in terms of IgG yield, even though its purity was less than that of the control. Considering that Histidine buffer is significantly cheaper than the standard buffer MES Bis-Tris, it is recommended that Histidine be used in the buffer composition to maximise IgG separation efficiency, with further purification steps to be undertaken in order to remove contaminants. If the membrane pore size was further fine-tuned, the target protein may also be purified with less contaminants. Should Histidine buffer composition be adopted for the industrial scale manufacturing process, there will be a significant reduction in the cost of the process, leading to greater affordability of life-saving treatments for patients in developing countries.
ACKNOWLEDGEMENTS
With thanks to the PrIME Biologics team, including Dr Kailing Wang, Dr Roslyn Tedja, Willow Norton and Rebecca Sciberras, for assisting in the completion and write-up of this experiment.
REFERENCES
1. Cheung, G.L., Thomas, T.M. & Rylatt, D.B. (2003). Purification of antibody Fab and F(ab’)2, fragments using Gradiflow technology. Protein Expr Purif. 32(1), 135-140.
2. Hellstern, P. & Solheim, B.G. (2010). The use of solvent/detergent treatment in pathogen reduction of plasma. Transfusion Medicine and Hemotherapy. 38(1), 65-70.
3. Sigma Aldrich. (2014). Human Albumin. Retrieved from http://www.sigmaaldrich.com/life-science/ metabolomics/enzyme-explorer/enzyme-reagents/ human-albumin.html#PP.
Figure 2: Separation of IgG from human plasma, shown by SDS-Page analysis
Key: M – MES Bis-Tris; H – Histidine; HM – Histidine MES Bis-Tris; HA – Histidine Acetate
Figure 3: Comparison of IgG quantity from separations using different buffer composition.
Communicators: Public Presenters
“Effective communication of science gives people accurate information upon which to base decisions. By making science accessible, science communicators help counter the misinformation and misconceptions which clutter public debate.”
Jesse Shore, President of Australian Science Communicators (2010-2012).
The Future Project is very fortunate to have access to several dedicated teams of researchers in The King’s School’s Science Centre. Speak with any of these researchers and you will quickly discover their passion for improving their understanding in their chosen field with a view to helping improve the quality of life of people around the world.
The Year 10 students of The Future Project’s Communication Strand were charged with the responsibility of communicating this important work to the wider community, and facilitating discussion about the accompanying scientific issues.
Early in 2015, The Future Project’s Communication Strand was approached by one of the research partners, Vitramed Bioscience, with an idea for a public exhibition. Vitramed Bioscience is examining the role of microorganisms in a range of gastrointestinal diseases, and is developing new diagnostic tests and treatments for diseases such as Crohn’s Disease. It was agreed that the Communication Strand would collaborate with Vitramed Bioscience and The Australian Society For Microbiology on a new exhibition in the atrium of the Science Centre, entitled “The Zoo of You”. The goal of this exhibition was to communicate the important role that microbes play both on and inside the human body.
“The Zoo of You” opened in the Science Centre atrium in August of this year. Descriptions of the students' contributions to this exhibition, including its associated installations and workshops, can be found in this journal.
In the second half of this year, the Communicators focussed on the task of producing video reports on the activities of The Future Project’s research partners and interns. This research has involved many different areas of scientific endeavour, from extracting specific proteins out of blood, to developing polymers to help with the healing of diabetic wounds. To help explain the science and the significance of this research to a lay audience, the Communication Strand has created a series of short documentary-style videos. First the students filmed the interns at work in the Science Centre, and then interviewed them about the nature of their research. This footage was then edited together with other video elements such as music, banners and titles. It is hoped that these videos can help raise community awareness about some of the health issues affecting the lives of everyday Australians. This content can be viewed using the QR codes that can be found throughout this journal.
It has been a very rewarding year for The Future Project’s Communication team, and all of the students involved have enjoyed the opportunity to hone their communication and digital media skills. We are looking forward to exploring other ways of informing and inspiring people about the wonders of science in next year’s program.
Edward Dwyer, William Giang, James Groutsis, Ryan McShane, Mike Morgan, Razeen Parvez, Eddie Poolman, Dinesh Ratnam, Tim Sampson, Yang Tao, Marcus Yuen
As communicators, it is impossible to express our gratitude for the opportunity to learn and develop in such an engaging environment and in a way which helps to inspire the next generation of young scientists to ask questions and learn more about the incredibly complex world we inhabit.
During my time as a Future Project Communicator, I have had the amazing opportunity to not only expand the knowledge of other students, but also my own.
The experiences I have engaged in and the knowledge I have gained have been incomparable to any other program. I hope to continue working with The Future Project in the coming years.
Outside the Science Centre lies a quote from Albert Einstein: “Imagination is more important than knowledge”. We hope that our work as School Presenters has not only helped children to learn more about science, but also to imagine where the adventure of science could take them.
Communicators: School Presenters
Over the course of the year, the School Presenters created teaching modules designed to stir up excitement about science in children, whilst also teaching important and interesting topics in ways children both enjoy and understand. Our target audience were Year 5 students from a variety of local schools who visited us for an action packed half-day. Working in teams, we developed our learning modules from concept to execution. Each team had a specific set of learning outcomes based around the broad theme of flight and had to be hands-on, inspire a love of scientific inquiry, and be scientifically accurate.
FLIGHT OF THE QUADS
We thought: “What could be more fun than flying a quad copter drone?” so we built this module to help our students understand the complex flight of a quad copter. While this module was one of the most popular with the younger children due to its practical nature, it was one of the most difficult concepts for them to grasp. We were able to successfully explain how the four rotors move in different directions and how this motion achieves both flight and control by varying the relative speeds of the four rotor blades. Our students then enjoyed putting into action what they had learned by flying the quad copters themselves. Despite the students being first-time pilots, we did not have one significant crash over the whole year!
FALLING SCIENCE
We wanted to build a module designed to teach how science is a method of thinking and experimenting that allows us to discover answers to questions. While building and testing mini-paper helicopters, our students learned about posing a hypothesis and how to distinguish between the independent, dependent and controlled variables. Each group created their unique paper helicopter with a different independent variable, such as the number of rotors, the length of the rotors and the mass of the helicopter. Whilst the students prepared for their test run, we explained the reason why the helicopters spin on descent: that air has mass that takes up space and when a helicopter is dropped the air provides resistance. We also demonstrated how the helicopter pushes down on the air so the air reacts and pushes upwards on the helicopter slowing its descent. Each group then independently timed and recorded their results in a table. From their data, they could then answer the questions they had posed.
HOW A HOLE MATTERS
Some Frisbees have holes in the middle, but what effect does this actually have? How does a Frisbee fly anyway? We designed this module to explore the science behind a Frisbee by engineering identical Frisbees with differently sized holes. Launching a Frisbee in a controlled manner proved a significant challenge and the students learned first hand of the challenges of experimental work and the creativity required to imagine clever ways to ensure valid results.
FLOATING ON
Lift is often achieved with wings, but another approach to flight is to build a craft that floats because it is lighter than air. Archimedes worked out why things float and it was our challenge to bring that ‘eureka’ moment to our students. The lesson began with a quick game aimed at sparking the students’ interest. The students were asked to decide whether a certain object would float or sink, which resulted in mixed responses and a few laughs to kick off the module. By the end of our demonstration, the students had learnt one of the most important elements to buoyancy: in order for an object to float, it must weigh less than the water it displaces. With this knowledge in hand, the students were given a block of plasticine and five minutes to create the most buoyant boat they could. The ideas were varied with lots of great discussion. Following the same principle, the students were taught that in order for an object to float in the air, the object must weigh less than the air it displaces. Whilst some struggled with the concept of an object floating in ‘thin’ air, the simple demonstration of a hot air balloon saw many eyes widen with excitement. The module showed that science in practice is a priceless learning experience.
DIRTY HANDS DETECTIVES
In Term 3, The Future Project held an interactive public exhibition called “The Zoo of You” which explored the world of microbes. It attracted around 200 visitors of all ages at our opening on Sunday of Science Week. We ran one of the sessions aimed towards infants, called “Dirty Hands Detective”. This was our youngest audience yet, so making the session fun but still teaching science was a great challenge. This module aimed at teaching children about some diseases such as Cholera and Chicken Pox, and to emphasise the importance of washing your hands. We first taught them about a few common microbes, and the diseases they cause using plush toy versions of the microbes. Secretly, one of these toys was covered in a dust, which would glow under Ultraviolet (UV) light, meant to simulate a disease. After playing a game (which spread the dust) one of the School Presenters dressed in a HAZMAT suit arrived, and told the children that a disease had spread. We then used the UV light to show the children how the disease had spread from the one child who had touched the contaminated toy to everyone. Finally, we taught them that getting rid of the disease was as simple as washing their hands correctly and gave them a fun way of remembering this technique. We ran this session six times at “The Zoo of You”, and although the work was tiring, seeing the delight on the children’s faces throughout the day made it worthwhile.
Angus Macdonald, Jock Mitton, Archer Holz, Thomas Denny, Samuel Harrison, Tarun Tandon, Geoffrey Luo, Adesh Soni, Nik Sasic, Arian Bhatia, Kalvin Fernandez, Alexander Dunlop & Justin He
"The Zoo of You" Interactive Exhibition
Microscopic organisms, or microbes, are very small living organisms; so small that we need to use a microscope to see their individual cells. Microbes make up more than 60% of the Earth’s living matter and scientists estimate that some two to three billion species share the planet with us.
Of the number of cells that make up our bodies, only about 10% are human, which are the building blocks of organs like the brain, skin and heart. The other 90% belongs to trillions of microbes, mainly bacteria, living on or inside you. It has also been estimated that for the 23,000 genes that make up our body, there are more than 50 times this number contained within the collective bodies of the microbes that inhabit us. We are only just beginning to understand the roles, both positive and negative, that these microbes play in our daily existence.
In August of 2015, an interactive exhibition entitled "The Zoo of You" was opened in the Science Centre of The King's School. This exhibition was a collaboration between The Future Project's Communication Strand, Vitramed Bioscience and the Australian Society for Microbiology, with a goal to communicate the important role that microbes play on and inside the human body.
"The Zoo of You" featured many different exhibits and installations. The students of the Communication Strand researched and supplied the information included in these exhibits, and collaborated on the design of the exhibition space itself.
In a section of the exhibition entitled Meet Your Microbes, visitors were invited to study the latest Transmission and Scanning Electron Microscope poster images of common microbes found in the human body. Learning From Light explored the different examples of microscope technology used to produce such images, with several microscopes set up with real specimens to examine. Multiplying Microbes looked at the science behind the culturing and study of microbes on agar plates, and included examples of cultures taken from various locations around the school. In Microbes and Human Health, posters described some of the latest scientific studies that are giving us valuable insights into how the microbial world impacts on our health. The centrepiece of the exhibition was a homage to Leonardo da Vinci's "Vitruvian Man", comprised of around 200 individual petri dishes on which live microbes had been cultured, making this a truly living exhibit.
In preparation for the official gala opening of "The Zoo of You" exhibition, the students of the Communication team helped create content that was used to advertise the event, including posters, flyers and content on social media.
The exhibition was officially launched on Sunday the 16th of August to mark the beginning of National Science Week for 2015. It was very pleasing to see a wide range of people from the School community at this event: current students and their extended families; past students; and other interested parties from other schools and the scientific community. Microbiologists from all over the country volunteered their time to run hands-on activities and workshops for visitors in the ground level classrooms, and these were filled with enthusiastic participants all afternoon. Student volunteers from the Communication Strand helped to run some of these workshops, which included a detective-style story exploring the way that microbes are transferred from person to person, and using jets of water to blast away aggregations of microbes known as "biofilms". Other students manned the displays in the Science Centre atrium, keen to engage visitors with their knowledge of some of the most important microbes that have a significant bearing on our daily health. A colouring contest, lucky door prizes and guessing competitions all helped to ensure that visitors to the exhibition came away smiling.
Many of the exhibits and installations remained in place until late 2015, allowing the school community to continue to come and gain some insight into the world of microbes that exists on and inside every one of us.
Special thanks must be given to Dr Belinda Chapman of Vitramed Bioscience for her inspiration and dedication to this seminal exhibition and event.
Collaborators and Interns Under the Microscope
DR BELINDA CHAPMAN
BSc(Hons)
Syd, PhD Tas, GradDip (Science Management) UTS
Dr Belinda Chapman was interested in science from an early age, being particularly captivated by the written accounts of naturalist Gerald Durrell, and the wonders of space as seen through the classics of science fiction.
But it was largely due to her inclusion in a high school program that toured many of the significant scientific research institutions in the Sydney area that she was inspired to pursue a career in science. An enthusiastic science student at Granville South High School, Belinda was one of two Year 10 students offered the opportunity to spend a day with the researchers at places such as the CSIRO Animal Health Laboratories at Prospect, the RAAF Base at Richmond, and the Physics Department of the University of Sydney.
During high school Belinda also undertook several weeks of work experience at Westmead Hospital at the Institute for Clinical Pathology and Medical Research, where she assisted in the running of a medical trial that was being held at the time. Belinda went on to study Physics and Chemistry for her HSC, unknowing that it would be in the field of Biology that she would discover her real passion.
Electing to study Science at The University of Sydney, Belinda went to a lunchtime lecture at the end of her first year of university to hear one of the tutors, Ilze Dalins speak about Second Year Microbiology. Ilze’s vivid descriptions of a microscopic world just beyond the limits of our own human senses brought a hitherto unknown field to Belinda’s attention. Under the mentorship of Ilze, Dr Peter New and Dr Trevor Duxbury, Belinda went on to study in microbiology and agricultural chemistry. Along the way, she was given further research opportunities which taught her the ‘process’ of microbiology: the cognitive tools required to explore the world of microbes.
After graduating, Belinda worked as a research microbiologist in the food industry for several years, before eventually joining the CSIRO. Joining the CSIRO had been a dream of Belinda’s since her initial exposure to the organisation while at high school. During her ten years with CSIRO’s Food Division, Belinda collaborated with many internationally-renowned companies in the food industry. Her main focus was on the development of new food processing technologies to deliver foods safe from harmful bacteria such as E. coli, Salmonella and diseases such as botulism. During her work with CSIRO she also completed her PhD part-time, under the mentorship of Dr Tom Ross.
Leaving CSIRO after five years as a research manager, Belinda joined Vitramed Pty Ltd in 2012. Together with her colleague Dr Michelle Bull, Belinda is currently engaged in research relating to the role of
microorganisms in a range of gastrointestinal (GI) diseases, and the development of new diagnostic tests and disease treatments. It is in this capacity that Belinda has joined The Future Project, with Vitramed Bioscience being one of the founding partners of the program in 2014. She counts herself very lucky to be doing what she truly loves, saying "it's all play to me; every time look down a microscope or publish a paper it feels like play." It was Belinda who provided the inspiration for "The Zoo of You" exhibition that was held in the Science Centre atrium this year.
A passionate advocate of scientific literacy and communication, Belinda likens The Future Project to a type of experiment in communicating science to the next generation of scientists: "We want to find the passion for science in these kids." Belinda is looking forward to continuing her partnership with The Future Project, and extending the benefits of the program into the community and with other less advantaged schools in the area. She says, "I would love to hear one student from this program say that they want to study science, and to return to the program as a PhD researcher to mentor other students in the future. That's why I view this initiative as a ten-year plan."
Belinda has always made the most of professional opportunities that have come her away, and it is extremely fortuitous that her path has led her to her current collaboration with The Future Project at The King's School. Not that she is letting her latest commitments slow her down: Belinda is currently studying part-time for a Masters of Biostatistics with Macquarie University.
TED SIMPSON
Year 11, The King's School
Ted Simpson is one of a select group of students who have elected to return to The Future Project as a Senior Intern after a successful year as a Junior Intern in Year 10. During this time, Ted has been working with Vitramed Bioscience under the guidance of Dr Belinda Chapman and Dr Michelle Bull.
Vitramed Bioscience has been working on a probiotic bacteria that might be used to treat Crohn's Disease, a type of inflammatory bowel disease. Ted's research group has been testing the survival of this probiotic through a gastric model, designed to simulate the conditions of the stomach and small intestine. When asked about why he has returned to the program in Year 11, Ted says, "basically I felt that it was such a good experience the first time around, I'd probably feel guilty if didn't take the opportunity again. This time around it has been even more enriching... we are adding to the work that we did last year and gaining a deeper understanding of how this probiotic survives."
Ted has found it easier to balance his time with The Future Project and his academic commitments this year, with most of the research completed during intensive sessions over the first and second end-ofterm breaks. He says, "I found it quite easy just to give up a little bit of my holidays to come in and do the research. It's also been a lot easier to do the writing for the papers this year, having come back with a better understanding of the work that we've been doing."
With his student intern partner, Vithushan Lingam, Ted has written an article on his research for The Journal of the Future Project, and is looking forward to collaborating on a paper that will be submitted to a peer-reviewed scientific journal in the coming months.
Having already worked with Vitramed Bioscience for a full year, Ted has been able to use the experience that he has gained in the laboratory and the subsequent reporting phase to assist his intern partner and the Junior Interns. He reflects on the process, "coming into The Future Project wouldn't have had a clue how real scientific research is conducted. Now I've had first-hand experience of how it works."
Ted has been enrolled at The King's School since 2009 when he began as a Year 5 student. He is currently studying Biology and Physics, and is considering a double degree in Science and Economics at the Australian National University.
Our Collaborators
PrIME Biologics (PrIME) is a Singapore based company commissioning a plasma fractionation facility in Singapore to address the US$1billion Asian Therapeutic Plasma products market. PrIME uses the PrIME Technology in association with GE Healthcare's chromatography in a process called PrIME +.
Currently much of the plasma collected in Asia and other parts of the world is discarded. If this plasma could be used to manufacture therapeutic plasma products, much of the world shortage of plasma products could be addressed and many, possibly millions, lives could be saved. At PrIME Biologics, we value the potential and ability to save lives and to provide safe and affordable plasma based therapeutics. We aim to achieve this through the use of our revolutionary PrIME Technology to process the plasma that is being discarded by many emerging countries.
PrIME Biologics was established to manufacture therapeutic plasma products at the highest levels of quality and safety. This was achieved, while also nearly doubling the total amount of therapeutic products produced from each litre of plasma. PrIME biologics goal of ‘Safer Plasma Fractionation Through Innovation’ summarises not only the characteristics of this innovative technology, but also the ideology of the company.
Today PrIME Biologics is refurbishing and recommissioning our Singapore cGMP therapeutic plasma manufacturing facility in Science Park II. We expect this to be completed by the first quarter of 2016. From that time onwards we will be able to process plasma in an accredited cGMP facility. Specifically PrIME Biologics will be initially processing human plasma to produce Albumin and IVIG. We will also be able to produce other plasma products including FVIII and FIX for customers who require these additional products.
Disposability ensures the elimination of possible batch-to-batch contamination. This allows us to process plasma which might otherwise not be able to be processed. Further, the PrIME Technology provides an additional level of pathogen safety to the normal viral/ bacterial processes used by all the fractionators. It should be noted that only plasma that has been tested in accordance with EMA and HSA guidelines will be processed to ensure the therapeutic products produced using the PrIME Technology are as safe as possible.
Vitramed was founded to distribute medical devices with a focus on gastrointestinal (GI) health. This includes diagnostic devices, single use devices used during endoscopic procedures, and devices used for surgical and other procedures. From an original base in Sydney, Vitramed has grown to serve GI customers in Oceania and South East Asia, with offices currently in Sydney, Kuala Lumpur and Singapore, and moves underway to open offices in Thailand and Sri Lanka.
Vitramed has enjoyed rapid growth by maintaining a team of people who have knowledge and experience in the gastrointestinal health field and who are genuinely passionate about helping our customers serve their patients as well as possible. Vitramed maintains close relationships with thought leaders in the GI field and has been able to help guide the development of new medical devices, ensuring that manufacturers are making the most of the knowledge of experienced doctors, to ensure that new devices are meeting the changing needs of GI professionals.
The close relationship Vitramed enjoys with the GI health industry lead to the creation of Vitramed Bioscience, which was created to conduct GI-related microbiological research. Vitramed Bioscience operates a Physical Containment Level 2 (PC2) microbiology lab at The King’s School where some of Australia’s most senior microbiologists are working on projects at the leading edge of research to help with serious GI conditions. Vitramed Digestive Health develops and distributes specialist food products for individuals and health professionals. It is currently finalising a powdered shake drink that is aimed at people with serious GI conditions such as Crohn’s Disease and will be released under the new Food For Special Medical Purpose category.
Vitramed has always maintained a high level of technical knowledge in order to support the medical devices on which our customers rely. Vitramed Mechatronics was created to allow this capability to flourish. Vitramed Mechatronics supports all of the technical needs of the Vitramed group and conducts research and commercial projects related to health, sports and agriculture. The projects utilise a mixture of programming, robotics, and mechanical engineering knowledge.
A feature of the culture of Vitramed is an obvious passion for science and engineering and an enthusiasm for exposing young people to the potential of science and engineering to provide enjoyable, rewarding careers. The opportunity to be part of The Future Project has been an excellent outlet for this passion and both Vitramed Bioscience and Mechatronics enjoy an excellent relationship with staff and students at The King’s School who share this vision.
The Faculty of Engineering and Information Technology is ranked among the world's top 50 engineering and information technology faculties in the 2014-2015 Times Higher Education Rankings, and achieved an overall five-star rating from the Australian Government’s Excellence in Research Australia. The Faculty has research clusters in the areas of Biomedical Engineering and Technology, Clean Energy, Complex Systems, Food Processing, Human-Centred Technology, Materials and Structures, Robotics, and Water and the Environment. The University of Sydney began teaching Engineering in 1883, and its alumni have made significant contributions to the development of Australia. The faculty offers a diverse range of degree offerings across Engineering, Information Technology and Project Management including:
• And combined degree programs with Commerce, Law, Medical Science, Project Management and Science.
What all our students have in common is a thirst for knowledge. They reach beyond our campus to think through issues that affect the wider world. This might be done by spending part of their degree overseas, working with a local community or as a volunteer with one of our outreach programs, such as The Future Project.
Our graduates have excellent employment prospects as well as workready qualifications that are recognised in Australia and worldwide: our engineering degrees are accredited by Engineers Australia; our Chemical and Biomolecular Engineering degrees by the Institution of Chemical Engineers; our Project Management degrees by the Project Management Institute; and our IT degrees by the Australian Computer Society. The faculty works with hundreds of companies to support students through scholarships, vacation work and industry-sponsored projects.
New Staff
DR YANLING LU
Yanling is a Senior Development Scientist at PrIME Biologics. Initially trained as an Applied Chemist in China, Yanling moved to the United Kingdom in 2002 and completed her MSc degree in Applied Biomolecular Technology at the University of Nottingham. In 2006, she completed her PhD in Physical Biochemistry working on solution structures of engineered antibodies. She then joined the world leading biopharmaceutical company MedImmune Ltd (Cambridge, UK) as a Senior Scientist in Bioprocess Development where she led the characterisation and formulation development of monoclonal antibodies. In 2009, she moved to Australia and took up the Postdoctoral Fellow position at the University of Sydney to lead a biomedical research project to study cardiac muscle proteins using structural biology tools. Yanling is the lead author of over a dozen of research articles and she has regularly presented at international conferences.
DR ALI FATHI
Ali is Industrial Research Manager at the University of Sydney having graduated in Biomedical Engineering. Ali has published numerous research articles and has been engaged with Research and Development of numerous bio-tech and bioengineering companies. Ali is the inventor of five national and international patents about a revolutionary platform technology that can minimise the need for open surgeries for the treatment of different tissues from bone and cartilage, to skin and muscle.
MS DARCII TERRE
Darcii Terre is a Medical Science student in her third year at the University of Western Sydney. Throughout the past year, Darcii has undertaken periodic work experience with Vitramed Bioscience, including assisting with students in The Future Project. Darcii has a passion for medical research and microbiology, which she intends to pursue through further study and her career.
MR ERIC LIN
Eric has joined PrIME Biologics as a Development Scientist/Quality Control specialist. He is a graduate with a major in biochemistry from the University of Sydney. Eric has worked for several years in public pathology laboratories and has a broad knowledge of the operations of pathology laboratories across the local health district. During his employment in the Royal Prince Alfred Hospital as a hospital scientist, Eric participated in correlational studies for numerous pathology tests such as Aldosterone, Plasma Renin Activity, Urine Drug Screening and Methotrexate.
DR ROSLYN TEDJA
In June, Roslyn sadly left The Future Project to take up a position as an Associate Research Scientist at The Yale School of Medicine in America. Roslyn’s enthusiasm and passion for developing the students will be sorely missed but we wish her every success in her fantastic new role.
Acknowledgements
We would like to take this opportunity to thank the many who have helped make The Future Project such a success this year.
The School Executive for their unwavering support
The Headmaster, Dr Timothy Hawkes, for his educational leadership in developing this extraordinary learning opportunity
The Bursar and his team for their continued help, vision and fiscal support of The Future Project
Karl Sebire for his brilliant graphic design of this Journal
Tina Moshkanbaryans for her tutelage of the Interns and countless hours proof reading the Journal
Jenny Tan for all her work as the Bursarial representative for The Future Project
The entire Science Faculty for their generosity in time and willingness to be involved
The Industrial Arts Department for their ongoing support of the Mechatronics Strand and their assistance with “The Zoo of You”
Baulkham Hills High School and Tara Anglican College for their willingness to participate in The Future Project
Warwick Holmes for his fervent and passionate support of Science at The King’s School
Julie Saad for her endless help with Future Project matters
All of the microbiologists who kindly gave up their time to make “The Zoo of You” such a great success
