The Journal of the Future Project - 2016

How do I use the QR codes in this journal?

Throughout this journal you will notice 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

Nothing inspires an understanding of science more than ‘doing’ science. It allows for posing questions. Testing. Learning from failure. Trying again. ‘Learning by doing’ is immensely powerful. And it is for this reason I was thrilled to hear about The Future Project and its collaborations with industry and the research community.

To me, one of the most valuable things about studying science and engineering is the ability for students to learn how to formulate and tackle problems using symbolic reasoning.

It is about developing a healthy scepticism and keeping an open-mind about the problems at hand. Most importantly, it is about developing sharp questioning skills to build wider reasoning.

Through studying science and engineering, ultimately we want confident students who love solving tough, abstract problems (or at least who are not put off by them) and who ask “why is it so?”

Facilitating pathways into science and engineering careers is one of the most challenging pieces of the STEM (Science, Technology, Engineering and Mathematics) puzzle which The Future Project is helping students tackle. Enabling students to get hands-on experience and exposure to what a career in science and engineering looks like in practice is invaluable. So too are the relationships and connections with industry that students in The Future Project are able to form. In turn, students will then be able to use these partnerships as stepping stones for their future career choices.

New areas of science and engineering are constantly emerging and so too are the ways in which science and engineering are overlapping. Thirty years ago, nanotechnology was an area that was not specifically designated within physical chemistry. This is also the case with big data, data analytics, massive sensors and genomics. There are fields that don’t yet exist, that students involved in The Future Project will be a part of. That is an incredibly exciting prospect.

Professor Mary O’Kane NSW Chief Scientist & Engineer

Introduction to The Future Project

For those new to The Future Project, a quick overview of its aims: to motivate and engage the next generation of scientists and engineers by giving students from local primary and secondary schools the opportunity to collaborate with scientists and engineers, solve realworld problems and communicate this innovation to the broader public. This is achieved in two ways: firstly, by providing opportunities for students to witness researchers in action or to work alongside researchers as interns on research projects; and secondly, providing opportunities for students to communicate this innovation to other students or the broader public through various forms of public, print and electronic media.

Why? Because early and positive interest in science and engineering is likely to lead to later interest, engagement and study of science and engineering. A more scientifically literate society is one which is better informed to make decisions. It is also the hope of those involved in The Future Project that it will promote more favourable pathways for young people into science and engineering studies and vocations in the future. Given the federal government’s current National Science and Innovation Agenda, The Future Project is already a wonderful opportunity to develop the social capital in young people, through partnerships and collaboration with universities and industry, showcasing research and entrepreneurial skills, and provides an exemplar for other STEM programs to follow.

For many students nowadays, uncertainty about the correct answer causes hesitation and fear of failure. Each of the students involved in The Future Project has shown commitment to a journey of investigation, one which at times was uncertain, but one which yielded new knowledge to themselves and can now be communicated to others. As a result, students in The Future Project are willing to take risks, pose hypotheses, experiment, fail, seek answers, experiment again, think critically, and synthesise various information and data to create something new.

What is also distinctive about The Future Project is that it provides an incubator for biomedical companies to conduct some exciting research. For example, PrIME Biologics has developed and commercialised blood fractionating technology to manufacture therapeutic plasma products which are safe and affordable. Quantal Bioscience has a diverse interest in microbes, particularly gut health, food-related health, and equine health. They have also extended the outreach of the program by mentoring students from Doonside Technology High

School. Sangui Bio was founded in 2015 and, while based at the Kolling Institute, researchers spend time at The Future Project studying inflammatory signalling in disease and its effect on immune function. Vitramed Mechatronics main objectives are to design and develop medical devices, as well as to find solutions through robotics and image processing. The University of Sydney makes a rich contribution to the program through its biomedical engineering research. As this outline describes, students involved as Senior Interns have access to cuttingedge research and innovation.

The Intern program is unique because it allows students from a range of schools – Baulkham Hills High School, Cherrybrook Technology High School, Mamre Anglican School, and The King’s School – to work together alongside leading scientists and engineers. Not just for a few hours, or for a few days, but several hours per week for three school terms and eight days of intensive laboratory research in the school holidays. Nothing they do is prescribed. Each research project is authentic and organic, co-designed with their research supervisors depending on the collaborating organisations’ research interests. Each group has advanced the knowledge, in a small but significant way, of their area of research by proposing investigations, conducting ongoing research, analysing data, and forming conclusions about what this means.

Their research has included: optimising the quality control of proteins separated from blood using specialised membrane technology; testing of capsule material to be used in a clinical trial; the effects of supercooling on the shelf-life of raspberries; the role of anti-oxidant proteins during stress; and growing algae to explore the commercial viability of extracting lutein and omega-3 fatty acids used in health supplements. Students involved in Senior Mechatronics have prototyped an autonomous vehicle with potential applications in agriculture. Junior Mechatronics students have developed a device to monitor and notify the temperature in medical equipment and a device to aid sports training which monitors and records basketball shots scored or missed. All of this research has had a focus on providing new information to the research organisations involved as well as the broader community.

This year, 21 Year 11 students participated as Senior Interns and since 2012 there have been a total of 67 Senior Interns. For students from The King’s School, a Junior Interns program has allowed 48 students to be involved as Junior Interns and Communicators; 101

since these programs began. Through our School Presenter program, approximately 110 students have visited The Future Project from local primary schools to learn a little more about the science of flight.

Whilst research is an important aspect of The Future Project, the other important aspect is communication. To some extent, this research would be pointless if it was not shared and made available to others. Students involved as science Communicators have worked on a number of projects with a focus of sharing an understanding of science, engineering, and research with the broader public. A significant achievement for them was a curated exhibition of artefacts from the Temora Aviation Museum which explained the science of flight. This was further built on by School Presenters who conducted workshops with local primary students investigating concepts of lift, thrust, and drag. Finally, another group of students interviewed Senior Interns about their involvement in The Future Project, creating short documentaries of each research project.

I am ambitious – for the program and for the students involved. The program is at capacity; only limited by the number of researchers and research benches available. But the program is fortunate in that there are regular enquiries from new research groups and new schools. The next step is to provide greater access to schools further afield and students of greater need, not necessarily coming to The King’s School facility, but possibly to industry research laboratories more local to them. This will be the challenge for 2017.

I am very grateful for the ongoing support of the fabulous scientists and engineers who have partnered with the program again this year. They are dedicated to our mission of motivating and engaging the next generation of scientists and engineers. The program was first piloted with Associate Prof Ben Herbert in 2012 as a proof of concept. The contribution from Prime Biologics, Vitramed, and the University of Sydney since these early days, and Sangui Bio and Quantal Bioscience more recently, has been crucial. Together we share a passion to bridge the gap between formal learning of science and engineering with informal learning in an industry setting. This is one of the most valuable aspects of the program – students engaged with scientists and engineers, conducting purposeful research, and sharing this knowledge with others.

/TheFutureProject.au

www.thefutureproject.com

Of course, the success of the program, in part, is due to the dedication of a number of teachers who oversee the Intern and Communication students. Their role is to provide an important link between education and industry partners and to manage the students’ completion of tasks throughout the year. I thank Christian Eatough, Roger Kennett, William Pope, Matt Purser, and Vera Munro-Smith for their energy and commitment. It is their passion for enriching the lives of young people, and in helping them see a future which makes a valuable contribution to society, that assists The Future Project achieve its goals.

This journal records the many achievements of the students involved. It bares testimony to their hard work and inquiry, their dedication in seeking answers, and their passion for sharing what they have learnt through their involvement in The Future Project. I congratulate all of the students involved, for those who have graduated from the program or had their work published should be proud of their achievements.

Brad Papworth Director – The Future Project

Members of The Future Project

Ben Herbert

Matthew Ghannoum

Riya Gaikaiwari

Rachel Chua

Hanson Chen

Michelle Bull

Shaeel Hassan

Oliver George

Christian Eatough

Jack Chu

Belinda Chapman

Liam Brown

Oscar Han

Bradley Gardner

James Dugdale

Jason Chhoeu

Pravin Chanmugam

Ben Billyard

Allen Guo

Zane Gale

Anthony Dingwall

Keith Cheng

Matthew Chamoun

Sami Al-Hiffi

Michael Goh

Christopher Gailey

Shanith de Mel

Hu Chen

Eliza Chalik

Immanuel Abosh

Alex Noh

Jamie Nicholas

Gordon Nguyen

Hari Nair

Vera Munro-Smith

Tina Moshkanbaryans

Alistair Moon

Nicholas Molnar

Keshav Mohan

Jacques Millichamp

Zoe Marshall

Daniel Marsh

John Manusu

Owen Mak

Charlie Lowe

Eric Lin

Marcus Lim

Alan Liddle

Jui-Ko Li

Olivier Laczka

Matthew Kolibac

Zain Khan

Roger Kennett

John Kavanagh

Elizabeth Karsten

Tony Jerkovic

Meryem Jefferies

Mohan Huang

Ben Hines

Janish Hettigama

Members of The Future Project

NOT PICTURED

DUGDALE, Nathan

GANTASALA, Navneet

GRAY, Ben

HARRIS, Jacob

KEOGH, Darcy

PENMAN, Darcy

SAAD, Christian

SASIC, Aleks

MANTON, Aaron

Michael Wu

Emma Winley

Joshua White

Charlie Webster Kailing Wang

Thomas Walsh

Jack Taylor

Tehilan Tampoe

Kamil Syed

Benjamin Stewart

Alexander Siu

Daniel Simmons

Rohan Shankar

Tej Shah

Joseph Rylance

Henry Roth

Samuel Rider

Naida Rasheed

Andrew Quattrocchi

Matt Purser

William Pope

Steven Pham

Brad Papworth

Angus Padley

Willow Norton

Leo Zhang

Jay Wyndham

Protocol development for testing the tolerance of Dietzia-filled capsules during simulated gastric transit

Capsules - can they deliver?

The probiotic Dietzia has been used by Quantal Bioscience in their development of a cure for Crohn’s disease, a long term inflammatory disease of the intestines. Quantal Bioscience is in their second phase of the clinical trial to test the use of Dietzia as a viable form of medication. Since this second phase includes a larger number of patients (from six people in the first phase to thirty people in the second), Quantal is attempting to administer Dietzia to patients via capsules. As a result, this paper concerns the development of a protocol to test the suitability of capsules in containing Dietzia, particularly to test whether these capsules will survive gastric transit.

The protocol was developed by first testing numerous conditions that could affect capsule integrity on surrogate capsules not containing Dietzia. Once such conditions were narrowed down, capsules containing Dietzia were tested to assess the viability of the protocol. The capsule protocol that was designed in this trial was effective in determining whether the capsule would survive gastric transit. However, it was unable to pinpoint causes of capsule failure when it occurred. If further research is undertaken to enhance this protocol, this aspect should be taken into account.

Naida Rasheed

N. Rasheed1, R. Gaikaiwari1, Z. Marshall1, B. Gardner1, J. Rylance1, M. Bull2, E. Winley2 and B. Chapman2 THE FUTURE PROJECT1 and QUANTAL BIOSCIENCE2

ABSTRACT

The following paper concerns the development of a capsule testing protocol for the encapsulation of Dietzia C79793-74. This protocol was developed as a result of the advancement of a Phase 1/Phase 2 clinical trial concerning the administration of Dietzia C79793-74 as a treatment for Crohn’s disease. The larger scale of the next Phase 2 clinical trial requires a practical method of administering a dosage combined with ease of transportation of the probiotic to a larger number of trial participants over a longer time period. The use of capsules would satisfy these criteria if they could be shown to survive gastric transit predominantly intact. The protocol developed in this paper was designed to model gastric transit in order to test the period of time for which the capsule remained intact. Qualitative measurements such as visual turbidity and swelling, as well as quantitative measurements (optical density) were taken in order to test numerous aspects of capsule integrity. The protocol put in place overall was successful in determining whether a capsule would survive gastric transit. However, it was largely unable to pinpoint the causes of capsule failure, a component that might need to be addressed in order to select appropriate capsules for the next Dietzia C79793-74 clinical trial.

INTRODUCTION

Crohn’s Disease is an inflammatory bowel disease and has no current cure, with minimal treatment being available. Dietzia C79793-74 has been proven effective in the treatment of Johne's disease in cattle (Click & Van Kampen, 2010), and in a recently completed Phase 1/ Phase 2 clinical trial has shown promising results as a probiotic treatment for Crohn’s disease in humans. Johne’s disease and Crohn’s disease are very similar chronic wasting diseases. Human studies are now moving onto a larger scaled Phase 2 clinical trial. In the first trial, the Dietzia C79793-74 was personally delivered to six patients in frozen liquid form in vials using small cooling containers to maintain the frozen state, and therefore maintain viability of the probiotic. On a larger scale, this delivery form is less technically feasible due to the increase in size of this phase, in which at least 30 patients will be undergoing this treatment for 12 weeks, twice a day. Freeze drying the Dietzia C7979374 into a crystallised powder and putting it into a capsule, is a suitable option to allow ambient temperature or refrigeration (not frozen) transportation and storage.

The use of capsules does present some issues. The capsule must be able to hold the correct dosage of Dietzia C79793-74 and also have some tolerance to gastric transit in order for it to deliver an adequate viable dose of the probiotic. In the first clinical trial, participants were all taking an acid inhibitor to increase stomach pH and the liquid Dietzia C79793-74 dose was ingested in milk. These measures were previously shown to enhance the survival of Dietzia C79793-74 through gastric transit (Fraser et al., 2014; Simpson et al., 2015). Delayed release capsules made of vegetable material were chosen to experiment with, as they are produced to last longer through gastric transit compared to standard, non-delayed capsules. This is critical for Dietzia C7979374 as it is of most use the further it travels through the gastric transit. An appropriate protocol for encapsulating Dietzia C79793-74 and testing of gastric transit tolerance, although influenced by the gastric transit simulations conducted by past Future Project interns (Fraser et

al., 2014; Simpson et al., 2015), has not previously been developed. Consequently, there were numerous variables to consider including in the protocol, such as the presence of water when sealing, the positioning of the capsule, and the requirement of gyroscopic motion to simulate movement of the capsule during gastric transit.

METHOD

Skim milk powder-filled capsule testing

An initial trial was conducted to narrow down the conditions that would finally be utilised to test Dietzia-filled capsules. Skim milk powder was used instead of Dietzia C79793-74 to test the designed protocol (due to lower costs). The first variable tested was capsule sealing with and without water. For water sealed capsules, the open edge of the capsule top was dipped lightly in a shallow tray of water and the capsule was then capped and pushed closed using the locking mechanism inbuilt within the cap. Capsules sealed without water were simply capped and closed with the inbuilt locking mechanism.

The second variable tested was the pH of the gastric model solution. To mimic gastric transit, a 0.9% (w/w) saline solution was prepared, adjusted to either pH 2.0 or pH 4.0 using HCl, and 6 mL of solution was added to 15 mL centrifuge tubes. A skim milk powder capsule was then placed into each tube simultaneously and submerged into the gastric model solution using a modified transfer pipette (Figure 1). Sufficient capsules were filled to examine in duplicate every 30 min of gastric model exposure. The tubes were placed in an incubator at 37°C (as this replicates the human body’s temperature) and tubes were shaken in a gyroscopic motion every 30 min. A sample was removed every 30 min, after an initial 60 min incubation, for optical density (OD) measurements at 600 nm (these measurements recorded the concentration of bacteria in a solution). Qualitative assessments of all remaining capsules in gastric model solution were undertaken every 30 min to assess the following characteristics: turbidity, swelling, stress points, internal material appearance and gross failure.

Dietzia-filled capsule testing

Dietzia-filled capsules were used for the final design protocol. In order to fill capsules with Dietzia C79793-74, a transfer pipette was modified to work as a scoop (Figure 1). Empty capsules were weighed and the base was filled with Dietzia C79793-74. All capsules were watersealed as described above. The capsules were then weighed again to determine the maximum weight of Dietzia C79793-74 that could be filled into each capsule, in order to deduce whether this quantity was sufficient for a dosage of the probiotic for the next clinical trial. The method of capsule submersion was slightly modified to prevent capsules from moving into the modified transfer pipette, holding them in place in the centrifuge tube. Furthermore, the gyroscopic motion during capsule incubation was not used, based on the results of the surrogate capsule trial. Quantitative measurements were taken at 0 min, and then every 30 min after the first 60 min of incubation, since the previous trial suggested that no significant change occurred between these two time points.

RESULTS

As shown in Table 1, capsules incubated at either pH 2 or pH 4 were recorded as having failed between 2 – 2.5 h. It is therefore possible the capsules would have survived actual gastric transit through the human gut. From the above qualitative assessments, it is interesting to note that although turbidity did not have a significant visible change, capsule swelling continued to increase. Capsule swelling therefore, may have been a significant contributor to gross failure.

Figure 2 details the optical density (OD600nm) measurements of the gastric model solutions following incubation of the skim milk powderfilled capsules, at both pH 4.0 and pH 2.0. Comparing the qualitative and quantitative assessments, the optical density measurements were greatest at the time-points where capsules were observed to be breached and/or grossly failed. However, the optical density measurements further indicate there was considerable variation in the time-to-failure for the capsules.

The results presented in Table 2 illustrate that capsules encasing Dietzia C79793-74 failed significantly earlier than capsules containing skim milk (Table 1). Interestingly, the pH 2.0 saline solution seemed to enter the Dietzia-filled capsules within 30 min of incubation, suggesting a fault within capsule lining. As for the skim milk powder-filled capsules, swelling seemed to be a major hindrance to the survival of the capsule in gastric transit.

As can be seen in Figure 3, although gross capsule failure of Dietzia-filled capsules occurred at 60 min, optical density increased dramatically at 90 min rather than at 60 min. The results were similar, however, to the surrogate capsules, in that optical density measurements, after increasing rapidly for one batch of samples, remained lower for the remaining batches.

DISCUSSION

The focus of these experiments was to develop a reliable and valid protocol for encapsulating Dietzia in order for Quantal Bioscience to conduct tests and choose an appropriate capsule for delivery of this probiotic to patients in a Phase 2 clinical trial. The trial involved the development of a protocol to test qualitative features such as gross failure and capsule swelling, as well as the quantitative measurement of optical density in order to determine capsule integrity. This protocol was then tested using a surrogate material and subsequently using Dietzia

Figure 1: Capsule filling and gastric transit tolerance apparatus. (A) Capsule filling using a modified transfer pipette. (B) Modified transfer pipette holding filled capsules below liquid level.

Figure 2 Optical density measurements of pH 4.0 (Batch A) and pH 2.0 (Batch B) saline solutions during simulated gastric transit of skim milk powder-filled capsules.

Figure 3 Optical density measurements of duplicate (Batch A and Batch B) saline solutions during simulated gastric transit of Dietzia-filled capsules

At this time, we are not able to explain the observation of gross failure of the capsules containing Dietzia within a much shorter time period (at 60 min) than the gross failure of surrogate capsules containing skim milk (150 min). Although qualitative observations suggest that the reaction of Dietzia with the inner capsule lining may have caused this accelerated gross failure, the short timeframe in which this investigation was conducted did not allow for further analysis of this fault. Since the capsule protocol was designed specifically for the encapsulation of Dietzia, further refinement of the method should include investigation into such causes of premature gross failure in order to maximise the likelihood of tested capsules surviving gastric transit.

Further refinement of the method could involve greater accuracy in the filling of the capsules with Dietzia. For example, the use of a pharmaceutical capsule filling machine would decrease the amount of dosage variation.

Time Point (min)

capsule failure

CONCLUSION

The capsule protocol designed in this project was successful in its ability to test gross failure of capsules, pinpointing the time that this failure occurred. However, it was unable to determine causes of such failure (particularly in reference to capsules containing Dietzia). As a result, to enhance the protocol, further research should be conducted to include features in the protocol which will identify such causes of failure. This will allow for a selection of more suitable capsules to encapsulate Dietzia, thereby assisting Quantal Bioscience in delivering adequate dosage of the probiotic to patients involved in the Phase 2 clinical trial for Crohn’s disease.

skim milk powder-filled capsules during simulated gastric transit*

of gastric model solution Capsule swelling

30 No Not observed All capsules swollen Mostly concentrated toward bottom of capsule

60 No Not observed All capsules swollen Concentrated toward bottom; one pH 4 capsule punctured

90 No Some liquid penetration into capsule; turbidity in pH 4 tubes All capsules increasingly swollen Separated to top and bottom of capsule; powder thickened where capsules were breached

120 2/4 failed Turbidity in pH 4 tubes No further change Milky sediments on side of capsule; clumped

150 Yes Some tubes slightly turbid Slight increase in swelling Concentrated mainly toward top and bottom; pH 4 capsules very cloudy inside

180 Yes No further change No further change Concentrated mainly toward top and bottom * Results from gastric model solutions at pH 2 and pH 4 were the same unless otherwise noted.

30 No Clear Around Seal Dietzia is wet but still in capsule - almost in liquid form

60 Yes Clear Around Seal, Capsule Dietzia still at bottom of capsule- wet

90 Yes Clear Increasing Swelling Dietzia still at bottom of capsule- wet

120 Yes Murky, Dietzia powder starting to emerge Increasing Swelling Dietzia at bottom of tube and floating in solution

150 Yes Murky, Dietzia powder released No further change Dietzia at bottom of tube and floating in solution

REFERENCES

Click, R. & Van Kampen, C. (2010). Assessment of Dietzia subsp. C79793-74 for treatment of cattle with evidence of paratuberculosis. Virulence. 1(3): 145-155.

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.

Simpson, T., Lingham, V., Bull, M., Chapman, B. & Terre, D. (2015). The survival of Dietzia C79793-74 during simulated gastrointestinal transit. The Journal of The Future Project. 2: 34-38.

Table 1: Qualitative assessment of

Table 2: Qualitative assessment of Dietzia-filled capsules during simulated gastric transity

Bradford

protein assay optimisation for the quality control of IgG separation membranes used in PrIME

Goodies, baddies and antibodies - the battle within

Globally, millions of people suffer from rare genetic blood disorders such as hemophilia A and B, anemia, autoimmune and infectious diseases. These patients require life-saving plasma derived therapeutics such as albumin, IgG, and factor VIII in order to live relatively normal and healthy lives. Producing these active biologics from blood plasma is expensive and time-consuming. Also, many patients from developing countries have far less access to these treatments than do patients from developed countries. PrIME is a membrane electrophoresis technology that manufactures safer and affordable plasma products.

Our project involved optimising a quality control (QC) test method for membranes used in the PrIME process to isolate Immunoglobulin G (IgG) from blood plasma so as to increase the manufacturing efficiency of these life-saving plasma therapeutics. For our experiment, we used a tangential flow electrophoresis system, which is used in the QC testing of membranes that are vital to the protein purification process. These membranes separate proteins from human blood plasma, based on their size and charge. Our aim was to establish the most accurate and reliable method for estimating protein concentration and thereby enable the analysis of protein transfer across these membranes.

The Future Project has opened my eyes to the application of Science in the real-world. Working on a research project which has real life application was an amazing opportunity that cannot be replicated in a classroom. The Future Project has helped me develop my interpersonal and time management skills. Working in such a diverse environment with so many different people has helped me understand the importance of these life skills and how to best apply them in the work place. Fostering relationships with colleagues was one of the best aspects of the experience. Thanks to the Future Project I have been exposed to a professional laboratory environment and having the opportunity to extract therapeutic proteins has been a truly memorable experience.

Liam

While a challenging experience, The Future Project has definitely been a life-changing opportunity. Working in a multidisciplinary environment alongside some of the most dedicated researchers has humbled me and transformed my perception of “lab work”. Applying scientific erudition to alleviate global issues such as disease has cultivated my scientific passion and proficiency, while teaching me the importance of patience, diligence and communication. Immersing myself in research beyond the Science classroom, I have gained unmatchable time-management and cooperation skills, enhancing my work ethic and determination toward future investigations. I will forever treasure the gamut of eye-opening discussions held, making The Future Project the most memorable and inspiring highlight of my senior year.

Eliza

E. Chalik1, L. Brown1, M. Jefferies2 , A. Jerkovic2, O. Laczka, K2. Wang2, H. Nair2 THE FUTURE PROJECT1 and PRIME BIOLOGICS2

ABSTRACT

Immunoglobulin G (IgG) is a high-molecular weight glycoprotein found in blood plasma. It is the second most abundant protein next to albumin. Plasma concentration of IgG is approximately 13 mg/mL. It is composed of four peptide chains and exists as a monomer, with a molecular weight ranging between 150 and 170 kilodaltons (kDa). The Preparative Isolation by Membrane Electrophoresis (PrIME) separation principally separates protein molecules from plasma according to their charge (pI) and size, through membrane electrophoresis. Using PrIME technology and protein estimation (Bradford protein assay), this study aimed at optimising the Bradford assay employed to measure the transfer rate of proteins across the 1000 kDa membranes (from four different membrane batches). These 1000 kDa membranes are used in IgG separation/purification. Preliminary data have shown some indications about the optimal dilutions to be applied for the Bradford assay in order to obtain a more accurate measurement of protein concentrations and transfer rates.

INTRODUCTION

Plasma fractionation refers to the process of isolating various proteins from blood plasma. It was originally developed by Dr Edwin Cohn to treat allies suffering from shock and burns in the Second World War (Figure 1). In the extraction process, target proteins are purified and concentrated. Their work is responsible for the development of blood serum products that are in high demand by clinical facilities around the world (Kilbourne, 2007). Plasma derived biologics include albumin, immunoglobulins (A, D, E, G and M), coagulation factors FVIII, FIX, FXI, and protease inhibitors, among others (Burnouf & Radosevich, 2000).

Immunoglobulins are a class of glycoproteins known as antibodies. They are present in the serum and in cells involved in the immune system. These glycoproteins attach themselves to foreign substances such as bacteria, viruses, and toxins, and assist during their destruction by immune cells. There are five classes of immunoglobulin: IgG, IgA, IgM, IgD, and IgE (Martin, 1969). Immunoglobulin G (IgG) is the major immunoglobulin in blood and has a plasma concentration ranging from 8 to 18 mg/mL. IgG is a complex protein weighing approximately 150 kDa and having a typical ‘Y” shaped structure (Figure 2). It is composed of two heavy chains, each weighing 50 kDa, and two light chains, each of 25 kDa. These heavy and light chains are joined together by disulfide bonds (Figure 2).

The very ends of the heavy and light chains form an antigen-binding region which binds to specific foreign molecules, toxins or cells, also called antigens (Biology Exams 4U, 2012). Human IgG is also glycosylated at various sites by sialic acid and N-glycosyl residues. These glycosylations are necessary for its function (PrIME, n.d.). IgG has been successfully used to prevent infectious and autoimmune diseases and treat immunodeficient patients (Jolles, Sewell & Misbah, 2005). IgG treatment has not only improved the quality of life, but has also saved the lives of millions worldwide.

During the Second World War, a number of viral removal steps were added to the original Cohn process, consequently creating a multi-step low yield process. Originally, the aim was to separate albumin, which represents 55-60% of the total protein volume in plasma.

Figure 1 Albumin used to treat battlefield injured during WWII (Kilbourne, 2009).

Figure 2 Immunoglobulin (IgG) structure (Biology Exams 4U, 2012).

This product was originally used to treat injured soldiers, but over time, additional plasma proteins were separated and used clinically. Since the early 1980s, chromatographic methods, in combination with ultrafiltration, have started to replace Cohn fractionation since they offer higher purity and yield, and are easier to automate (Graham & Rickwood, 1997).The removal or inactivation of pathogens such as viruses and prions is a vital aspect of plasma fractionation (Buchacher & Iberer, 2006). High purity ensures a lower risk of adverse reactions caused by contaminating substances. Viruses are inactivated in plasma products with solvents, detergents and heat treatment (Burnouf & Radosevich, 2000).

Currently, therapeutic plasma fractionation technologies only allow the recovery of approximately 50% of total proteins, destroying the remaining albumin and intravenous immunoglobulin in the process. The PrIME process consistently yields over 85% of the therapeutic plasma products from the same quantity of plasma. This high yield is achieved by eliminating steps taken to purify each of the therapeutic plasma products.

The global demand for blood plasma products is increasing by as much as 13% every year. However, plasma medicines (biologics) are currently being produced by a decreasing number of facilities, forcing populations to become dependent upon imports from other countries (Australian Government Department of Health, 2006). Becoming selfsufficient in plasma fractionation, especially in developing countries, can help meet domestic demand. Thus, an efficient IgG purification method from plasma that can also meet the strong safety regulations and limitations, remains in high demand today.

MATERIALS AND METHODS

Standard protein preparation

Bovine serum albumin (BSA) was prepared at a 25 mg/mL concentration in 54 mM Tris borate buffer (TB) (pH 8.9).

Cartridge preparation - membrane batches SMI201603330/30.1/30.2/04.1

A visual analysis was performed on the restriction membranes to ensure no impurities were present. The first of the two restriction membranes were then inserted into the PrIME cartridge and sealed with a diversionary filter of plastic. Another visual analysis was carried out on the test membrane to ensure the best quality results would be obtained from the experiment. The separation membranes being tested (each of four different batches: SMI201603330/30.1/30.2/04.1) were placed into the cartridge, locked in with another diversionary filter of plastic before another restriction membrane was placed into the cartridge (Figure 3). The PrIME cartridge was then sealed and inserted into the BF400 machine.

cartridge

Cartridge leakage test

and sample stream

To test the assembled cartridges for leakages, 20 mL of 54 mM tris borate (TB) buffer were flowed into Stream 1 (S1) and Stream 2 (S2) of the PrIME-BF400 system for 10 min. The principle of this test is to apply pressure, produced by both the stream and buffer pumps to the separation and restriction membranes, and the cartridge itself. This test indicates any leakage across the separation membrane between the two streams and the restriction membranes separating the buffer stream. Changes in volume in either stream after 10 min indicates a leakage. S1 and S2 need to be equal or no greater than 1 mL difference for the cartridge to pass the leakage test. Three separation cartridges were tested per membrane batch in order to carry out the QC procedure.

Protein transfer

Proteins were transferred across the 1000 kDa cartridge using a BF400 (Gradipore) PrIME machine. Fifteen mL of 25 mg/mL BSA were introduced in the S1 feed tube and 15 mL of TB Buffer (54 mM) were added to the S2 feed tube. Prior to starting the separation, the temperature of the running buffer was recorded and 1 mL from each of the feed tubes (S1 and S2) was collected. Once the separation process was started, the current (mA) was recorded. The run time was 30 min. At one minute before the end of the run time, the running buffer temperature and current were recorded and another 1 mL was sampled from both S1 and S2.

Bradford assay

The protein concentrations of the samples collected were quantified using the Bradford assay method. The assay was performed in a 96well plate. The samples were prepared at five different dilutions using 54 mM TB buffer and subsequently transferred into the plate. Table 1 describes the volumes of sample and buffer introduced depending on each dilution factor preparation.

Ten microlitres (µL) of each dilution ratio were first added into each well of the plate, followed by the addition of 250 µL of Bradford reagent, using a multichannel pipette. The plate was then covered and left to incubate for 5 min. The absorbance values were finally obtained using a microplate reader (BioRad, iMarkTM microplate reader) at a wavelength 595 nm. Each dilution was measured in triplicate in order to determine the standard deviations (SD).

Figure 3 PrIME

assembly

flow schematic: A. Restriction membrane, B. Separation membrane and C. Restriction membrane.

RESULTS

Bradford assay results for each dilution of the fractions collected from four different membrane batches are shown in Table 1. S1-30 retention represents the proportion of BSA measured in S1 after the 30 min cycle. The transfer rate S2-30 expresses the proportion of BSA that was displaced from S1 into S2, across the membrane. Mass balance represents the total amount of standard protein in both streams (retention S1 + transfer S2). These values are all expressed as percentages relative to the concentration of BSA initially present in the sample S1, at the start of the separation process (Table 2). Figure 4 illustrates the results obtained for each batch, depending on the dilution factor applied. The results obtained from batch SMI20160330 and SMI20160330.1 clearly show that a 1/20 dilution factor, resulting in mass balances above 125%, does not allow to obtain correct absorbance readings and consequently, accurate results from the Bradford assay.

The most consistent and effective dilution factors for the evaluation of IgG separation membranes were evaluated from the retention/ transfer rates and mass balances observed for each batch. Despite the differences observed between batches, the results show that a 1/50 dilution for the Bradford assay yields the most consistent and accurate results across all batches, with mass balances closer to 100%.

Table 1: Bradford assay dilutions

Table 2. Results obtained for each Batch applying various dilution factors

Figure 4 Retention rates, transfer rates and mass balances calculated using the Bradford assay, for each dilution of the samples using separation membranes from (a) batch SMI20160330, (b) batch SMI20160330.1, (c) batch SMI20160330.2, and (d) batch SMI20160304.1 (n=3, error bars are +/- Standard Deviation).

Further diluting seems to result in protein loss, and consequently, reduced mass balances. For batches SMI20160330 and SMI20160330.1, the 1/120 dilution yields good results in terms of mass balance. However, the SD obtained for SMI20160330 shows that the higher the dilution, the more likely it is to generate an experimental error. Figure 5 illustrates the results obtained applying a 1/50 dilution, for each batch. Interestingly, the results obtained from batches SMI20160330.2 and SMI20160304.1 diverged significantly from the ones yielded with the other two batches. This could be due to variations in operator and/or equipment.

DISCUSSION

As shown in Figures 4 and 5, most of the data obtained displayed variations in the transfer and retention rates obtained. The 1/20 dilution yielded high variability among the results and unrealistic values, indicating that this dilution is not suitable for the QC test method. Results of the 1/120 dilution lack consistency. The mass balances and transfer rates obtained applying a 1/50 dilution gave the most accurate and consistent results and could be used in QC testing. However, further testing needs to be carried out, such as evaluating the operatorto-operator variations and determining the discrepancies generated through the use of different machines. The readings obtained using the 1/100 dilution gave satisfactory results; however, reducing the dilution factor as much as possible should help limit the experimental error and therefore the 1/50 dilution appears to be the most appropriate.

BSA can be easily transferred through the membranes with large pore sizes used in this experiment. As IgG is 150 kDa in size (65 kDa for BSA), further testing with larger proteins could be beneficial in determining optimum dilution factors. Such experiments would better reflect the conditions created when using a larger protein such as IgG and thereby improve the reliability of the data obtained. In the future, additionally to the assay employed in this study, membrane pore sizes could also be tested using analytical techniques such as a scanning electron microscopy.

Figure 5 Transfer rates and mass balances calculated using the Bradford assay for the 1/50 dilution of samples from each of the four separation membrane batches (n=3, error bars are +/- Standard Deviation).

Due to the limited running time with the BF400 instrument and the potential operational errors affecting the results, further testing is required to obtain more consistent and reliable results. Finetuning additional process parameters such as incubation time and temperature, sample preparation and pipetting procedures could ensure the reliability of future investigations.

Overall, this collection of preliminary data across five different dilutions has shown that dilutions above 1/20 allow better conditions toward future QC processes of PrIME membranes.

CONCLUSION

Our results indicate that dilution factors including and above 1/50 are best suited for QC tests of membranes. Further testing and fine-tuning of the process parameters is required to ensure the most accurate and reliable QC test method conditions. Through additional investigations, dilutions above 1/50 could potentially be used to create optimal QC

REFERENCES

Australian Government Department of Health. (2006). Global demand for plasma products. In Review of Australia’s Plasma Fractionation Arrangements (pp 39-52). Retrieved from http://www.health.gov.au/ internet/main/publishing.nsf/

Biology exams 4U. (2012). How antibody works and inactive antigens? Antigen – Antibody interaction. Retreived from http://www.biologyexams4u. com/2012/11/

Buchacher, A. & Iberer, G. (2006). Purification of Intravenous immunoglobulin G from human plasma: aspects of yield and virus safety. Biotechnology Journal. 1: 148-163.

conditions for the IgG membranes. Combined with the data already collected to date, these preliminary results will be helpful for optimising a test method for the QC of membranes used in the PrIME process for the purification of IgG from human blood plasma.

ACKNOWLEDGEMENTS

We would like to thank Eric Lin and Vera Munro-Smith for their advice and guidance during our project and Brad Papworth and Roger Kennett for their support. The King’s School and PrIME Biologics are thanked for the opportunity provided to us in working in a professional scientific environment and for making The Future Project possible.

Burnouf, T. & Radosevich, M. (2000). Reducing the risk of infection from plasma products: specific preventative strategies. Blood Reviews. 14: 94-110.

Graham, J. M. & Rickwood, D. (1997). Subcellular fractionation: A practical approach. IRL Press at Oxford University Press: Oxford.

Jolles, S., Sewell, W.A.C. & Misbah, S. A. (2005). Clinical uses of intravenous immunoglobulin. Clinical and Experimental immunology. 142:1-11.

Kilbourne, K.H. (2009). WWII ‘Critical Care’ Battlefield Therapy Still Saving Lives. Retrieved from http:// www.politico.com/ppta/wwii-critical-care.html

Martin, N. H (1969). The immunoglobulins: A review. Journal of Clinical Pathology. 22: 117-131.

PrIME Biologics Technical Document. (n.d.) Purification of Human Serum albumin and Human Normal Immunoglobulin G by Integrating PrIME Plasma Fractionation and GE Plasma Technology.

Prototyping an algal bioreactor to harvest lutein

Healthy Products from Algae

Algae are complex organisms from which commercially valuable omega-3 fatty acids and carotenoids such as lutein can be derived. The first challenge was to build a bioreactor: a tank in which to grow the algae. This required some problemsolving in order to join clear acrylic tubing and PVC pipe fittings. The bioreactor was designed to be cheap, reliable and scalable on a commercial level. The second challenge was to grow small batches of algae which could be used to start the bioreactor. The third challenge was to monitor growing conditions and measure the growth of algae. Finally, once a sufficient amount of algae was cultivated, the challenge was to harvest this and extract valuable substances such as lutein and beta-carotene. This research was valuable for several reasons. Firstly, a number of substances in algae, in particular lutein, are suggested to have a variety of health benefits: lutein may help prevent macular degeneration; lower the risk of cataracts; and promote cardiovascular health. Secondly, omega-3 has been linked to reducing the risks of heart disease and stroke, as well as possibly alleviating various emotional and behavioural disorders such as depression and ADHD.

Using algae as the main source for these substances has the potential to lessen the burden of overfishing as a higher concentration of lutein and omega-3 can be extracted when using bioreactors. The health foods and supplements industry also sees these substances as commercially valuable.

Through our involvement in The Future Project this year, we have gained a valuable understanding of what ‘real’ science and engineering involves. Furthermore, we have learnt the importance of teamwork in order to achieve our goals. Although the project was at times challenging and difficult to balance with our other school commitments, we gained important experience in problem-solving, scientific investigation and engineering. Overall, all the hard work we put in was well worthwhile.

H. Roth1, M. Wu1, H. Chen1, A. Padley1, B. Papworth1, J. Kavanagh2 and D. McClure2

THE FUTURE PROJECT1 and THE UNIVERSITY OF SYDNEY2

ABSTRACT

Algae are polysynthetic organisms from which omega-3 fatty acids (omega-3s) and carotenoids, such as lutein, can be derived. Currently, lutein is sourced from marigold petals; however, this is ineffective as the yield from the flower is only 2%. Moreover, omega-3s are also extractable from fish and krill; however, these omega-3s are algal in origin. Thus, the initial purpose of this research was to establish sustainable growing conditions for a reasonable algal harvest. This was further extended to consider the processes by which these substances could be efficiently extracted using different ratios of ethanol. By designing a cheap and scalable bioreactor, the authors aimed to examine the effects of different conditions, such as pH and growing period, required for a significant yield in algae. It was hypothesised that an indoor bioreactor would create a more stable growing environment at a satisfactory growth rate for commercial production, rather than an outdoor bioreactor. The algal strain of Desmodesmus asymmetricus was grown in bioreactors maintained under stable environmental conditions to ascertain reliable data regarding biomass productivity. The findings indicate a growing period of three weeks or a pH below nine yielded a harvestable amount of algae with an approximate growth rate of 0.2 day–1. Moreover, the investigation discovered that 3.75% (w/v) of algae to ethanol as a solvent resulted in optimum extraction of lutein; an increase in mass of algae saw a decline in extraction efficiency. These results provide new insights regarding the growth of algae as a commercial product and are likely to impact future developments in the burgeoning health foods and supplements industry.

INTRODUCTION

Lutein is purported to assist in the prevention of macular degeneration by increasing the thickness of the macula (American Optometric Association, 2016). Other health benefits associated with lutein consumption include lowering the risk of cataracts, strengthening eye tissue and promoting cardiovascular health. In addition to being important in biological processes, omega-3 has been linked to reducing the risks of heart disease and stroke, while being used to alleviate various emotional and behavioural disorders such as depression and ADHD, along with other related diseases (Connor, 2000). Recently, the idea has emerged for algae to be harvested for lutein and omega-3, as they contain higher concentrations of these beneficial molecules than other sources (up to 70% lipids). Therefore, using algae as the main source for these components has many benefits. Not only does it lessen the burden of overfishing, but a higher concentration of lutein and omega-3s may be extracted when using bioreactors rather than large fishing vessels. However, to increase commercial feasibility of products from algae, the cultivation of algae and extraction process need to become more efficient, with attention paid to factors such as growth rate and overall productivity. In addition, a low cost, scalable, and simple bioreactor design is necessary. The overall objective of this study was thus to explore the optimum cultivation and harvest conditions for algae whilst optimising the extraction process. Using the strain Desmodesmus asymmetricus (CSIRO CS-899), pH and temperature conditions were monitored during the growing phase, with follow up investigation of solvent extraction using thin layer chromatography (TLC) and different ratios of ethanol.

METHODS AND MATERIALS

A key purpose of the design was to produce a low cost and scalable bioreactor. This consisted of a clear acrylic (Perspex) tube (2000 mm height, 94 mm diameter) with end caps of polyvinyl chloride (PVC) threaded stormwater fittings (90 mm diameter), secured by Soudal T-rex crystal sealant. An aquarium pump and flexible air delivery tubing were added to provide bubbles to the bottom of the bioreactor to aid circulation. The algal strain used was D. asymmetricus (obtained from the Australian National Algae Supply Service, CSIRO) and grown in a MLA media of macronutrients, micronutrients and distilled water (Table 1).

Table 1: Components of MLA media (table courtesy of Xuyu Yun, USYD)

16.9 CuSO4.5H2O 1.00

6.96 Na2MoO4.2H2O 1.00

2.47 NaHCO3 0.75 CoCl2.6H2O 0.60 MnCl2.4H2O 0.45

Operation and inoculation of algal flasks

To ensure that there was always access to fresh algae, a small batch was passaged every two weeks. For each batch, macronutrients, micronutrients and distilled water were added. The macronutrients and micronutrients were mixed in a ratio of 125:1 and 1000:1 respectively before being added to 400 mL of water. Once this media was properly mixed it was divided into eight 50 mL conical flasks and each plugged with cotton wool and covered with a loose sterile aluminium foil cap (Figure 1). The flasks were autoclaved for 20 min at 121oC prior to 1 mL samples of media being added to inoculation flasks.

Construction and conditions of bioreactor

Two bioreactors, A and B, were constructed and operated indoors (Figure 2) to allow replication and additional growth of algae. When designing the construction of the bioreactor, it was decided to make a simple and scalable device, using off-the-shelf supplies readily available at most hardware stores. Through a process of experimentation, it was found that regular PVC pipe joining solvent cement was ineffective in securing and sealing the acrylic tube to the PVC pipe fittings due to a 1.5 mm difference in internal/external diameter preventing effective bonding of these parts. Instead, this problem was resolved by using a particular Soudal T-rex crystal sealant, due to its ability to flexibly bond to both acrylic and PVC to create a watertight join. With the tube now fully watertight, each bioreactor was installed side-by-side in an upright position, using cable ties, under the same lighting conditions indoors. Air delivery tubes from the aquarium pumps were weighted down with stainless steel nuts and lowered to the bottom of each bioreactor. The main purpose of the air delivery tubing was to pump small bubbles from the bottom of the bioreactor in order to agitate and stir the algae as it was growing, thus allowing it to receive more light. An earlier prototype bioreactor was installed outside and preliminary results collected (Figure 3).

Determination of algae biomass concentration and growth

To determine biomass concentration, optical density (OD) at 685 nm was measured using a spectrophotometer every day for approximately three weeks. This provided an easy and relatively accurate method to monitor algal growth and determine biomass concentration. Growth rate was calculated and plotted as a function of OD versus time in days (Figure 4) and logarithm of OD versus time in days (Figure 5) for bioreactors A and B.

The period of consistent growth (between the lag and decline phase) was determined by plotting of In(X/X0) versus time in order to determine a line of best fit and gradient; the coefficient of this line being the growth rate. The algae concentration (X) was calculated after a period of time (t, days) based on initial algae concentration (X0) and specific growth rate (μ) using the following equations:

Figure 1 Fresh algae being passaged in inoculation flasks

Figure 2 Set up of indoor bioreactors A & B (L to R)

Figure 3 Installation of outdoor bioreactor

Lutein extraction process

Harvested algae were initially freeze-dried to remove all liquids (courtesy of D. McClure) prior to adding the dried algae to ethanol solvent. To determine which ratio of algae to solvent yielded the most efficient extraction of lutein and omega-3, various masses of algae (0.000 g, 0.025 g, 0.500 g, 0.075 g, 0.100 g) were each added to 2 mL of ethanol in separate 10 mL Eppendorf centrifuge tubes. These test samples were vortexed to thoroughly mix and dissolve components from the algae. Test samples were then centrifuged to separate the supernatant algae extract from discarded algal cells.TLC was used to separate the components under investigation (University of California, Los Angeles, 2015). This was achieved by spotting a small sample of the algae extract at a start line 2 cm from the bottom of the silica gel TLC plate, along with standards of lutein and beta-carotene using a pipette tip and allowing to dry. TLC plates were developed by standing vertically in a beaker containing 20 mL of ethanol, covered with a watch glass. Extraction was allowed to continue until the solvent front rose 7 cm up from the start line. After removing from the solvent, each spot of separated component and standard was then marked and photographed after the remaining solvent had evaporated (Figure 6).

RESULTS

Outside vs inside bioreactors

Initially, the investigation was to consider differences in algal growth between an outside bioreactor, under day-to-day weather and climatic conditions, versus an indoor bioreactor under relatively constant temperature (approximately 24oC) and artificial lighting conditions (standard fluorescent lighting parallel to the bioreactor). The results indicated a standard growth pattern (Figure 4) of a short phase of low algal population (lag phase), followed by a period of substantial growth. After approximately three weeks, there was a decline in growth before the population of algae reach a plateau (peak phase). Following this, the algae started to die as nutrients, particularly carbon dioxide, were likely being consumed faster during photosynthesis than they could be replenished through respiration and from the atmosphere, due to the large demand of the population during the peak phase. Preliminary results of the outside bioreactor indicated that the algal population grew at a much faster rate during autumn due to warmer conditions and greater daylight. However, a sudden heat wave of 35oC on 6 April (Australian Bureau of Meteorology, 2016) saw the population of algae collapse.

Figure 4 OD versus time for algal samples from 12 L bioreactors A & B

Figure 5 Log OD versus time for algal samples from 12 L bioreactors A & B

Figure 6 Separated spots for beta-carotene (C), lutein (L), and extract (E) for TLC plate D

It is surmised that the heat, and not other conditions, was a potential cause for this decline as higher temperatures are known to be harmful to algae. Similarly, when restarted in early winter, the outdoor bioreactor suffered much slower growth, possibly due to the colder conditions (average temperature of 22.5oC; Australian Bureau of Meteorology, 2016) and shorter daylight being less suitable for growth.

Inside bioreactors A & B

An initial indoor bioreactor of algae grew at a steady rate of approximately 0.2 day–1 over a period of three to four weeks. Subsequently, two further indoor bioreactors (A & B), were established to test the replicability of these results with samples taken daily and tested using spectrophotometry to calculate the growth rate of each. Growth trends for each bioreactor A and B show a correlation in algal growth, determined by graphing OD against time in days (Figure 4). That is, a slow growth lag phase, followed by a rapidly increasing growth phase. If the logarithm of OD is graphed against time in days (Figure 5), then the trend is relatively linear and shows a growth rate of approximately 0.2 day–1

Extraction of lutein

After determining which plate carried the best ratio of algae to solvent (Figure 6), the sample was run through a spectrophotometer scanning from 400-800 nm to analyse for the presence of lutein. This provided added proof, along with the TLC data, that ethanol could be used to extract lutein from the sample of algae. As seen in Figure 7, two peaks and one shoulder can be observed. The peaks at 425 nm and 670 nm appear to confirm the presence of chlorophyll, which is heavily present in the algae. However, the shoulder peak at 450-475 nm is highly likely to be lutein or another possible carotenoid, again providing evidence that this can be successfully extracted from the algae. Further analysis of this data using the Beer-Lambert law, reveals a yield of approximately 3% of lutein using ethanol as the solvent, after making assumptions for the molar mass of lutein, cell density of the solution (0.25 g/L), and knowing the mass of algae in the sample. Similarly, these results add further support for ethanol being a viable way to extract economically important components in algae such as lutein.

Improvements on experiment

The reliability of results could be further improved through greater repetitions or multiple bioreactors. This would increase sample size of data and allow comparison against known values. Moreover, as the sample size increases there would be more data to better graphically model the growth rate. The validity of spectrophotometric data could be improved by more closely aligning OD wavelengths with those of D. asymmetricus, thus improving the determination of the amount of algae in each sample. Better management of controls, such as temperature, light and agitation, would minimise discrepancies between bioreactors and would provide further validation of results.

DISCUSSION

Outside vs inside bioreactors

This research discovered that the outside temperatures and conditions during Autumn were relatively ideal for the growth of algae, where the temperature during this period ranged from 20-28oC. However, outdoor growing conditions are potentially more volatile, with unpredictable hot or cold weather, cloudy periods or days with greater ultraviolet radiation potentially impacting the growth of algae. Noticeably in the outdoor bioreactor, the pH level rose to 11, at the time when the growth collapsed. This may be attributed to the algae consuming carbon dioxide from the growing media faster than it could be replenished, thus making conditions unsustainable for algal growth. Furthermore, there is the potential for the colder and lower light conditions of Winter to significantly reduce growth rates, rendering the production not commercially viable. In contrast, the growing conditions of temperature and light for the inside bioreactor are much more consistent, and while slightly slower at around 0.2 per day, are more reliable in terms of commercial production. Hence, the decision was made after preliminary research to relocate the bioreactors indoors, thus allowing a sufficient supply of algae in preparation for extraction. Overall, it was found that indoor bioreactors are more reliable for cultivating algae due to more consistent conditions.

Extraction of lutein and omega-3

Investigation into extraction processes using TLC plates indicate an optimum amount of algae:ethanol solution. Too high or too low amounts of algae reduced the amount and purity of extracted components. This optimal ratio of algae:ethanol was found to be 0.075 g of algae in 2 mL of ethanol or 3.75% (w/v). This result is useful when considering how to economically process algae in order to extract important components such as lutein or omega-3

Overall viability as a commercial process

Commercial viability

The higher yields of algae, and therefore lutein, were promising and with further investigation, may prove to be commercially viable over extended periods of time. The simple, scalable design makes production and running of these bioreactors very accessible to most agricultural farmers, fish farmers, aquaponics operators or hobbyists.

The initial aim of this investigation was to construct and test a low cost, accessible and scalable bioreactor that could be used to grow algae, with prospective algal farmers in mind. Each bioreactor costs approximately $300 and can be constructed in a short period of time. The investigation demonstrated that it is viable to build and operate a bioreactor to produce sufficient algae with the potential to yield economically valuable products such as lutein, omega-3 or betacarotene.

Figure 7 Spectrophotometer readings of sample with 3.75% (w/v) algae to ethanol

Research questions that remain unanswered

There is significant potential for further research in this area; for example, effects of different nutrient levels, light conditions, temperature, pH and dissolved carbon dioxide on growth rate, add valuable information for commercial operations. Further investigations could measure retardation factor of different ratios of algae:ethanol, or other solvents, to optimise the TLC extraction process and make it more cost effective.

CONCLUSION

Overall, this research achieved its aim of providing insight into whether a low cost, scalable bioreactor was a commercially viable method of cultivating algae and extracting valuable components such as lutein.

A number of aspects of the investigation add to the limited body of knowledge in this area. For example, a satisfactory growth rate can be obtained under indoor conditions.

REFERENCES

American Optometric Association. (2016). Lutein & Zeaxanthin. Retrieved from http://www.aoa.org/. Australian Bureau of Meteorology. (2016). Parramatta, New South Wales April 2016 Daily Weather Observations. Retrieved from http://www.bom.gov. au/climate/.

While growth rates of the indoor bioreactors were slightly less than the outdoor bioreactor, the indoor conditions are much more reliable and easier to control, making it easier to predict the commercial viability of growing algae to extract chemicals sought after by the health food and supplements industry. Furthermore, the research discovered a highly efficient method of extracting valuable substances available in algae. This information can be used to create more efficient and cost effective extraction processes. There is also potential to further investigate yields and extraction of omega-3 as this may provide insight into the commercial viability of bioreactors as a means of generating products for an increasingly health-conscious society.

Connor, W. E. (2000). Importance of n−3 fatty acids in health and disease. The American Journal of Clinical Nutrition. 71(1): 171S-175S.

University of California, Los Angeles. (2015). Thin Layer Chromatography. Retrieved from http://www. chem.ucla.edu/.

Supercool science unzipped

DOONSIDE TECHNOLOGY HIGH SCHOOL

Our Science teacher, Ms. Shandil, came into our classroom excitedly announcing that our school had been selected to participate in The Future Project. All we knew was that we had the chance to collaborate with real scientists, about real research, in a real science laboratory. After meeting Dr Belinda Chapman and Dr Michelle Bull from Quantal Bioscience who were visiting our school, we were all excited to become a part of this program. There was instantly a fight within our class to be chosen for one of the four places originally on offer. After submitting expressions of interests, we were lucky enough to be offered eight places, with students working in pairs. We went to our first session with high hopes, and we were not disappointed.

The purpose of this program was to develop students with a passion for science and to expose them to facilities outside of their school, allowing them to develop their skills within the lab and out in the field.

Belinda Chapman and Michelle Bull educated us and ran the program, providing us with the chance to come to The King’s School on a regular basis and begin experimenting. With their charisma and knowledge, we were immediately comfortable and confident enough to jump straight into the learning and researching process.

After a discussion on what we wanted to get out of the program, Belinda and Michelle put us to the test, making sure we were ready for the lab. They expected us to learn and follow the correct safety procedures within the lab before we started our research.

SUPERCOOL RASPBERRIES

Our first taste of science research was on the topic of supercooling. Quantal Bioscience was working on a small industry project looking at the potential of a newly developed supercooling refrigerator for extending the shelf-life of perishable foods. With the help of Belinda and Michelle we brainstormed foods with which we could test the rate of perishability. We eventually decided on raspberries, as they begin to expire quickly, are expensive and are quite a popular item in our supermarkets.

The research began with making some pre-experiment measurements on fresh raspberries, including subjective visual appeal, colour (using a smart phone colour application) and texture. From there we sorted the raspberries into three groups for storage under three different conditions.

• Normal Refrigeration

• Supercooling Fridge

• Freezer

After setting up this experiment in our first session and making the first series of measurements, we passed this experiment over to a group of Senior Interns from The King’s School and Baulkham Hills High School to complete the storage trial. This research project ran for six weeks, and you can find out about the results of this research in one of the articles in this Journal. Having had a first taste of real science, we were ready to move onto our main research project – something very different from raspberries, but still supercool.

UNZIPPING “ZIPPER”

Our main research project for the year, working with Quantal Bioscience, has been in the pioneering area of research on the gut microbiome of horses. Horses notoriously suffer from the disease colitis, which inflames the bowels, and can be due to disturbances in the gut microbiome. When Belinda and Michelle learned that Doonside Technology High School had a pony called Zipper on the school farm, we decided to recruit Zipper into the Doonside research team. We began our new area of research by collecting samples of Zipper’s faecal matter into separate plastic containers for culturing, and also placed a sample into a special collection tube to stablilise the DNA in the sample. This special collection tube was then shipped to the Australian Genome Research Facility (AGRF) in Melbourne to analyse the 16S rRNA sequence of the DNA in the excrement. The remaining samples were taken back to the laboratory at The King’s School. In order to carry out the research on Zipper, we needed to learn some new techniques in the lab.

Spread Plating: The number of bacteria in suspension can be rapidly quantified by using the spread plate technique. In this technique, the sample is appropriately diluted and a small aliquot transferred to an agar plate. The bacteria are then distributed evenly over the surface by a special spreading technique. We learnt to use pipettes and aseptic techniques to prevent contamination within our agar plate.

Streak Plating: In microbiology, streaking is a technique used to isolate a pure colony from a single strain or mixed group of microorganisms. It is a plate that has been streaked showing the colonies thinning as the streaking moves clockwise.

We used streak plating to separate single isolates of bacteria from our initial spread plates for further study.

Microscopy: Microscopy is simply the use of a microscope. During The Future Project we have been given the opportunity to use microscopes with some of the highest magnifications, specifically 1000x.

On our third visit to The King’s School, we took further samples of Zipper’s faecal matter and diluted them through a dilution series and spread them onto agar dishes to isolate specific microbes. We did this so we could see which bacteria were present in Zipper’s microbiome and how much of each type of bacteria we could see. We were particularly interested in finding bacteria that are known, in the scientific literature, to be important in equine gut health. The results from AGRF came back in a long spreadsheet, which was narrowed down to the 16 most prevalent bacterial phyla. Bacteriodetes proved to be the most prevalent bacterial phyla at 46.8%, and is composed of three large classes of bacteria that are widely distributed in the environment in soil, sediments, seawater and in the guts and on the skin of animals. Members of the genus Bacteroides are opportunistic pathogens. These results provide us with more detailed knowledge of Zipper’s microbiome than the information we can get from agar plating.

Overall, we have taken another step forward in our understanding of the bacteria in the equine gut, which will help us in the future to understand and treat colitis and other equine diseases. We are working with Quantal Bioscience as pioneers at the forefront of equine microbiome research, developing our skills in all areas of the lab and research. A big thank you to Belinda, Michelle and Ms. Shandil who have made this experience an incredible learning opportunity for us.

Junior Mechatronics: Monitoring medical equipment

This semester we have been designing a device under the guidance and direction of Daniel Simmons, General Manager of Vitramed. The purpose of the device is to monitor the temperature in medical equipment and notify the owner if the temperature goes outside a certain range from the target via email or text message. A later addition to the device was a shake detector, which will likewise notify the owner if their machine stops shaking for longer than a certain period of time.

We coded using C# onto a Raspberry Pi. We were amazed by the capabilities of the Raspberry Pi; it is extremely powerful for such a small computer (size of a playing card) and we experimented with many different simple programs such as a ‘blinky’ code, which helped us learn the basics of programming. After this, we moved on to the development of the actual device. We started simply with the first part of the code we created which only displayed whether a button was switched on or off. This was then adapted to the shake detector, as it is essentially a switch that is turned on and off many times a second. However, we encountered problems when we began to experiment with the temperature detection component. We had multiple issues in our code, which were difficult to identify due to an often-unreliable piece of hardware. We overcame this problem through the acquisition of better hardware

At the end of our time in the Mechatronics room, our product consisted of a Raspberry Pi encased in a plastic box with two extensions and a large interactive display. The extensions detect shaking and temperature respectively, and this information is displayed on the screen. This final product is aimed at people that store valuable, heat sensitive goods in fridges and freezers. By alerting the user to conditions which potentially threaten their produce, they can take action that would prevent significant financial losses.

This experience has allowed us to develop our knowledge in a wide variety of areas. As we worked towards creating this device we developed our skills in writing and interpreting code, as well as general problem solving, skills that are vital to modern day society and the future of engineering. Another invaluable experience was the way in which we interacted with Vitramed. Working with Daniel as interns rather than students allowed us to gain experience in an office environment that is rare for boys our age. This allowed us to act predominately independently, with Daniel intervening only to help us when we got stuck. This experience allowed an in depth look at a work environment and provided us with valuable skills that we can use in the future.

Jacob Harris Harry Inwood

Junior Mechatronics: Building a basketball training system

ABSTRACT

Prior to the development of sporting training systems, players were not able to accurately measure their improvement. The widespread popularity of devices such as the Fitbit, demonstrate how measurement can motivate people to train and improve. Measurement of training gains is relatively easy in many sports and athletic events. However, Basketball has seen no such progress. Due to the physically complex nature of basketball, recording systems are difficult to develop. While some commercial systems do exist, they are overly complex, unreliable and well beyond the financial reach of amateur players. Our device acts as a bridge to enable amateur players or professionals to accurately measure their shooting accuracy and plot their personal improvement. We anticipate this will contribute to player motivation and has the potential to form the basis of a fully automated basketball scoring solution.

DEVELOPMENT

This semester, under the guidance of Daniel Simmons, General Manager of Vitramed, we built a Basketball training system. We started by producing a plan of what we wanted to create to suit the needs of the people who would be purchasing the product. Firstly, we needed to learn all about the little steps we would need to take in order to get closer to achieving our goal. It started by watching videos on C# coding and how to use Microsoft Studio. From learning the basics, like flashing a light, we were able to start coding our program. We had to start with little steps like how to register a shot, and from there we continued our development. Coding was not the only aspect we had to learn: we were also required to understand the hardware involved in the product and how construction and connection would be carried out. We created a prototype which we trialled and improved.

FUNCTIONALITY

The device accurately measures the basketball shots made or missed in a certain amount of time. An elongated mechanical switch registers when a ball has passed through the ring, indicating a successful shot. This switch is read by the Raspberry Pi computer which also manages the timing of the training session.

Detecting a missed shot proved more complex. Our solution was an accelerometer attached to the rear of the backboard. A ball hitting the backboard or attached ring causes it to shudder. With clever analysis, this shudder signal can be detected by the Raspberry Pi. If the shudder is not followed by a switch event (a basket) within a short period of time, it is counted as a miss.

Clever analysis of these signals can also differentiate a swish, where the ball goes through the ring without causing a shudder in the backboard. A coach might wish to award more points to these shots as further motivation for the players. Our system cannot detect an air ball: a shot that neither goes through the ring nor hits the backboard.

BENEFICIARIES

Our device will benefit those looking to get better at basketball by having accurate records of results and improvements. As the player can focus on shooting only and is free from the tedium of keeping count and timing, they can effectively use our system without the need for a second person. We expect to see similar motivational gains as the Fitbit has achieved for walking and jogging.

While our prototype is quite advanced, there are still some refinements required before we could mass produce. While it has a primary use as a training motivator, it could easily be turned into a fully automatic scoreboard system.

LESSONS LEARNED

During the build of our device we developed many theoretical and practical skills including:

• Coding in c#

• Learning about the components of computers, and the different ports and their related processes

• Soldering components together

• Assembling electrical components

• Learning practical skills on how to accurately test devices through the use of programming.

• Understanding project management and development

• Managing time

The skills we have learnt will prove useful in Year 11 and 12 when time management and project management are needed.

Joshua White

Jamie Nichols

Senior Mechatronics

ABSTRACT

This year, the Senior Mechatronics Interns have been working hard to achieve an autonomous rover that is able to roam fields and backyards to do things such as search for and efficiently exterminate weeds or measure soil moisture levels. Before specific problems, such as weed identification or determining drainage patterns can be addressed, the basic rover platform needs to be created so that it can move autonomously over an area and wirelessly transmit collected data back to a server computer where more powerful computations can take place and further instructions can be issued. The rover is programmed to be able to follow a set algorithm to create a height map of the area and take soil moisture content readings at regular intervals. This can provide significant help for farmers in the agricultural industry and the everyday lives of home gardeners. By taking moisture readings over time, we can use the height and moisture data to create a 3D map showing drainage after rain or irrigation. It provides a more efficient and cost effective method of maximising production. So far, we have been able to achieve many of our goals, which include functioning sensors, a moving vehicle and a modular platform that altered at any time. We are beginning to work on its autonomy, and algorithms to determine weeds. While airborne quadcopter drones are gaining a lot of attention, their limited flight time and susceptibility to adverse weather mean they are not well suited to many of the tasks our land rover will be able to achieve.

WIRELESS SIGNALS

One of the goals of Vitramed Mechatronics is to develop algorithms such as those used to identify weeds in a photograph of the ground under the rover. Ideally, such algorithms would be stored centrally on the internet for the following reasons:

• As algorithms evolve over time, they will be instantly available to units in the field, without having to update the unit with new software.

• The intellectual property associated with algorithms can be secured more effectively on a server. The server could receive an image and return information about the contents of the image without the remote unit having access to the algorithm.

• Remote computers can be more powerful with less concern about power usage.

To communicate with the rover, we decided to use a set of XBee radio frequency transceivers to send and receive data. We went with the XBee-Pro 900HP Module which has a line of sight range of over 1 km. These modules plugged in via USB to the Raspberry Pi 2, which is what the XBee requires. We had to create our own radio protocol to send data in byte packets. These worked by having a ‘header’ byte at the start which was the identification of the command. This was followed with the parameters for that command. This allowed us to send different commands over the single serial channel effectively.

CONTROL SYSTEMS

The rover is controlled by two main devices: The Raspberry Pi 2, the main computer on board the rover which controls everything including wheels, steering, signals and responses; and the Polulu servo motor controller, which is a second board off the Raspberry Pi 2 which controls the servo motors and main driving motor, so that we are able to have a smooth and consistent acceleration or deceleration as well as custom set steering. By incorporating a ‘heartbeat’ communication model, our rover is able to immediately detect a breakdown in communication with the base unit and stop immediately. As we increase the autonomy and collision avoidance capabilities, we intend to have the rover continue its mapping job, storing the data locally and uploading it to the cloud when communications are restored.

MECHANICS

We modified an off the shelf chassis and gearbox kit, adapting this for our purposes. We installed a tray on top of the rover which holds the fixing for all of the computer boards and wiring, but also other sensors that are hidden in its hull, such as the gyroscopic sensor. The centre of mass for this project had to be relatively central, as it could overbalance and flip if it came across a significant obstacle. To accommodate this, the placement of many chip boards and batteries were changed.

SENSING

Our rover needs to have a precise location as well as information about the angle of the current pitch, yaw and azimuth. We included a nine axis gyroscopic and compass unit which gives us real time information about the axes of orientation of the rover. This is essential as the rover needs to be able to map an area precisely. By integrating the angle sensing, we are able to create a high resolution height map of the terrain over which the rover travels. Of course, we included a GPS module, but this only gives fairly low resolution spatial information.

We included an optical flow sensor to give us mm spatial precision. These sensors are regularly used in computer mice to measure the flow in two dimensions of the surface under the mouse to position the cursor on the screen. We used this sensor to likewise get highly accurate information about the movement of the ground under the rover which could feed into the locational model in the on board computer. After an early incident we understood the need for some inbuilt collision avoidance capability. To achieve this, we installed ultrasonic detectors. These can build up a model of the surrounds in much the same way as bats do, but measuring the delay between a pulse and echo of ultrasound. Our rover still has some way to go in managing a complex environment, however it gave us a good appreciation of the challenges facing designers of self-driving cars.

INTERFACE

The program used to control the rover was first based on a program that was made to test servo controllers. The program was adapted so it sent its commands through the XBee wireless transceivers. However, it was quickly improved upon, and features were added such as a heartbeat, emergency stop, keyboard control and the capability to receive information. We also implemented our own protocol for servo control so as to save bandwidth and simplify our code. It went through a second major revision becoming a UWP as well as adding dedicated elements for pitch, yaw and sonar as well as many cosmetic improvements. Eventually we hope to move to a cloud based platform, where the rover is largely autonomous but would receive general instructions, and relay captured data to the cloud. The data could then be analysed by computers more powerful than a Raspberry Pi or consumer computer. This would free the rover from single base station and greatly expand its potential operational range.

AUTONOMY

Our intent is to make the rover completely autonomous. Virtual borders could be set by the user and the rover would complete a set task within the area with no further instructions. Depending on the use case, the rover could also relay data taken to a cloud interface, where it could be

I. Abosh1, O. Mak1, D. Marsh1, J. Taylor1 and D. Simmons2 THE FUTURE PROJECT1 and VITRAMED MECHATRONICS2

analysed and presented to the user. Or the rover could be used to apply fertiliser or herbicide on a smaller scale, directly where they are needed, reducing waste and environmental damage.

CONCLUSION

Throughout this year, all four of the Senior Mechatronics Interns have learnt many new skills and improved those skills we already had. It gave us an opportunity to work on a project that interested us all and where we did not know what the ultimate result would be. Some of the skills we learnt were: programming in C#; Serial Communication through the XBee; I2C protocol; and working with a Raspberry Pi. We ultimately achieved a robot that is able to return its gyroscopic readings, as well as its distance from objects around it. We also achieved communication to the rover through Serial Communication so we could control it from our laptops. We have developed a simple electronic toy into a sophisticated mechatronic, intelligent device and we are close to granting it autonomy. It will join the growing fleet of semi-autonomous devices which are quietly revolutionising the way humans interact with the physical world.

Autonomous Rover Platform

Project Overview

The goal of this project is to create an autonomous wheeled drone that can navigate various terrain. A key design feature is that the drone communicates wirelessly with a computer, which can handle moer intensive computations (such as image detection algorithms) and can also pass data to cloud-based processes or databases. Where possible, the project aims to allow cloud based algorithms to do as much processing as possible. This allows algorithms to be centrally optimised and deployed from a location where the IP associated with the algorithms is secure. The first goal for the platfor is to create a 3-D map of the area outside The Science Centre. Following on form this, the drone will take soil moisture readings at regular intervals and help analyse drainage patterns after rainfall. Future uses for the Rover platform include image based detection of weeds in grassed areas and soil levelling operations.

Challenges

• Computer control of servos for motion control.

• Learning to connect sensors to microcontrollers using various data formats.

• Creating a protocol for sending sensor and control information over the wireless connection.

• Ensuring safe operation of the rover if/when wireless communication is interrupted.

• Creating algorithms to allow the rover to autonomously navigate terrain.

• Developing computer software to act as a user interface and to manage communications.

CLOUD DATAPROCESSING APPLICATIONS

CLOUD DATABASE

RASPBERRY PI COMPUTER

A Raspberry Pi is a full computer shrunk to the size of a credit card. We are using it with a stripped-down version of Windows 10 which allows the rover to perform basic functions onboard. The Pi will communicate with a more powerful base computer, which will aggregate the data gathered by the sensors, and perform complex computations. A human operator will also be able to control the rover remotely from the base computer through the Pi.

XBEE WIRELESS TRANSCEIVER

The ZigBee is a small wireless module capable of sending and receiving binary data in the 900 MHz spectrum. It shares the spectrum cellular networks and has a range of ~2km. It will be the primary way the rover and the Pi communicate with the base computer. A pair of XBee wireless modules allow data to be sent both ways between the Rover and the computer. We have developed our own protocol for sending sensor data and control signals. We also send a ‘regular heartbeat’ message. If the hearbeat message is not received, the rover pauses operation.

OPTICAL FLOW SENSOR

This sensor provides accurate distance data using the same type of sensor used in optical computer mice. The sensor takes continuous low resolution photos and looks at how the pixels move from frame to frame.

GYROSCOPE / ACCELLEROMETER

One of the sensors collects gyroscopic and accellerometer data. The gyroscope data is combined with distance data from the optical flow sensor to generate a 3D map of the terrain.

ULTRASONIC DISTANCE SENSORS

These sensors pulse ultrasonic waves and measure the time taken for the waves to be detected again after bouncing off objects. The time taken to detect the return wave can be used to calculate the distance to the object the wave bounced off.

SERVO CONTROLLER

The Raspberry Pi sends control signals to this controller which sends servo signals to the steering servo and motor controller in the chassis.

4WD CHASSIS

A 4WD RC Car chassis was selected but could be substituted if required. This chassis is rugged, cost effective and easy to maintain.

The effect of supercooling on the shelf-life of fresh raspberries

Fresh or Frozen… Or somewhere in between?

All fruit is subject to a slow process of rotting, breaking down the produce until we as humans deem it inedible. Over the years, there have been many improvements in food preservation, from fridges, freezers, drying out food and many more, but what is the next step forward? The concept of supercooling has come to the forefront of this research.

Supercooling is the process of chilling a liquid below its actual freezing point without the structures that make up that substance on a molecular level becoming solid. In order to test this on food, we assessed the effect of supercooling on the microbial and aesthetic appeal of fresh raspberries by putting some in a normal refrigerator, a freezer and a supercooling freezer. We then took samples from all three storage types and tested them for their levels of mould, bacteria and yeast growth, as well as their visual attraction.

The results from this found that supercooling may have an advantage over refrigerated storage when comparing the number of spoiled fruits as a whole, but no significant changes were found in the microbial data. There were some changes in the aesthetic quality, texture and colour when testing between the storages, but they were only subtle. At this stage, it does not seem a realistic goal to increase the shelf life of fresh raspberries to six weeks with supercooling. However, a three to four week shelf life may be achievable with improved temperature control and/or improvements in packaging.

N. Rasheed1, R. Gaikaiwari1, Z. Marshall1, B. Gardner1, J. Rylance1, M. Bull2, E. Winley2 and B. Chapman2 THE FUTURE PROJECT1 and QUANTAL BIOSCIENCE2

ABSTRACT

Supercooling is a process that has the potential to preserve raspberries for an extended period of time, compared with conventional refrigeration. Supercooling has previously been successfully applied in preservation of other perishable foods, assisting in preserving textural integrity, microbial quality and general appeal. We assessed the effect of supercooling on the microbial and non-microbial quality of raspberries over a storage period of six weeks. Over the entire storage period the results indicated that supercooling may have an advantage over refrigerated storage in terms of reducing numbers of grossly spoiled individual fruit. However, no significant differences in microbial counts were observed among non-grossly spoiled fruit under different storage conditions. Differences in non-microbial quality (texture and colour) were subtle when comparing fruit stored supercooled or under standard refrigeration, but refrigerated fruit were generally firmer at the end of the storage period. It does not appear feasible to achieve a six-week shelf-life for fresh raspberries with supercooling. However, a three- to four-week shelf-life may be achievable with improved temperature control and/or improvements in packaging.

INTRODUCTION

Supercooling is the process of chilling a liquid substance below its freezing point without the crystalline structures becoming solid. A substance below its freezing point will crystallise in the presence of a seed crystal, or nucleus, around which the crystal structure can form. However, liquid substances can be trapped in a metastable state well below their freezing point (Schülli et al, 2010), maintained through the application of accurate temperature and atmospheric pressure control. Nucleation of the ice crystals may occur though, if materials are subjected to vibration or temperature fluctuation. Some of the ways products can be supercooled is through the substance being exposed to near static air, via immersion in a water bath with brine or ice slurry, or through immersion in alcohol.

Supercooling has the potential to simultaneously improve the quality and shelf life of food. The supercooling process also has the potential to reduce food wastage by improving the consumer appeal of fresh foods towards the end of their shelf-life. Supercooling can be used to lower the temperature of stored food to sub-zero temperatures (typically between -1°C and -15°C - varying with the type of product), hence reducing microbial growth (Stonehouse & Evans, 2015). The process is different to freezing, in that it specifically aims to avoid the formation of ice crystals during the cooling process. If this is successfully achieved, supercooling maintains the integrity of cells and tissues within foods that are prone to damage in normal cooling processes where ice crystals do form (Usta et al., 2013).

The ability of the supercooling process to reduce microbial growth and preserve quality has been observed in numerous trials of various food types, including fruit, vegetables, meat and fish (Stonehouse & Evans, 2015), but no specific data on the effect of supercooling on fresh raspberry quality is publically available. Fresh raspberries in Australia are an expensive, seasonal, high demand, but also highly perishable fruit, and there are currently no effective methods of extending the shelf-life of fresh (i.e. not frozen) raspberries over more than a few days.

Spoilage by mould is the major problem that limits the shelf-life of fresh raspberries. The fragile nature of the raspberry structure makes it particularly difficult to reduce numbers of spoilage microorganisms on the fruit by washing or sanitising without damage to the epidermal layer. Beyond microbial spoilage, the other factors most likely to limit the appeal of fresh raspberries are changes in texture and colour. We tested the effectiveness of supercooling for preserving the microbial and non-microbial quality of raspberries over a storage period of six weeks, comparing supercooled raspberry quality with quality of refrigerated and frozen raspberries.

METHODS AND MATERIALS

Storage trial set-up

Twenty punnets of fresh raspberries were purchased from a single retailer; best quality fresh raspberries were selected.

Each punnet of raspberries was opened and separated into two parts. Raspberries were gently sorted using gloved hands, with half of the raspberries placed into the lid of the punnet and half left in the base of the punnet. Any raspberries showing gross evidence of spoilage at this time were discarded (less than 1% of raspberries). Raspberries were covered with cling wrap; both punnet lids and bases contained air holes to allow fruit respiration.

Half-punnets of raspberries were divided between the three storage conditions of supercooled fridge (-4°C), standard fridge (4°C) and freezer (-18°C).

Assessment of non-microbial quality of raspberries

Assessment of the non-microbial quality of raspberries was undertaken at week 0, 3 and 6. At each time point two half-punnets, A and B, were randomly selected from each storage condition.

The overall appeal of individual raspberries was scaled subjectively from 1 - 5, with 1 being the most appealing to the eye and 5 being the least. The number of badly mould-affected raspberries in each sample was tallied and noted, and these raspberries were not included in further assessments of non-microbial quality.

The colour of individual raspberries was assessed using the “RGB ColourMeter” smartphone application (app). The results for the L, a and b colour space as well as the definitive name for that colour (as attributed by the app) were recorded.

Texture assessment of individual raspberries was undertaken using an in-house measuring device developed by Emma Winley, of Quantal Bioscience (and affectionately christened the Raspberry Squishy Metre (RSM) (Photo 1)). By adding weights to the RSM, the amount of weight required to close the cavity of each raspberry was determined, thereby determining the relative firmness of each raspberry. Raspberries were placed on an absorbent wipe and weighed. Then 0.60 g weights were placed on top of each raspberry until the parallel receptacles were in contact. The absorbent wipe was then weighed and the weight variation was noted as the exudate produced. Exudate is an indicator of rupturing of the cell walls which can also suggest changes in texture.

Assessment of microbial quality of raspberries

Assessment of the microbial quality of raspberries was undertaken at week 0 and then each week, up to six weeks for samples stored under supercool and normal refrigeration conditions. No samples were taken for microbial quality assessment of frozen samples, as no microbial growth would be expected under these conditions.

A half-punnet of raspberries was randomly selected from each storage condition. Strawberries showing gross microbial spoilage (i.e. mould growth) were removed, then approximately 25 g of raspberries (4-5 raspberries) were weighed into a stomacher bag, and diluted 10fold in 0.1% (w/w) Peptone Diluent. The mixture of raspberries and diluent were mixed for 30s in a stomacher or hand blended for 2 min. The raspberry samples were further serially diluted up to 1000fold, depending on observed microbial counts from the preceding

weekly samples. Appropriate dilutions were spread plated (0.1 mL) on Dichloran Rose Bengal Agar (DRBC) and Plate Count Agar (PCA), in duplicate. All plates were incubated for seven days at a constant temperature of 27°C. Following incubation, numbers of mould and yeast colonies on DRBC, and numbers of mould and bacteria colonies on PCA were counted. The average number of mould, yeast and bacterial colony forming units/g (CFU/g) of raspberries were calculated.

RESULTS

Non-microbial quality of raspberries

The results of non-microbial quality assessment of raspberries are shown in Figures 1 – 3 and Table 1. In terms of overall visual appeal, raspberries stored under standard refrigeration conditions had the least appeal throughout the storage period (Figure 1). Until three weeks of storage raspberries stored under supercooled conditions were the most visually appealing, but after six weeks of storage raspberries stored under normal refrigeration conditions had the most appeal.

1 Average subjective overall appeal of raspberries over six week’s storage under standard refrigeration and supercooled conditions (1 = most appealing to 5 = least appealing)

Figure 2 Average firmness of raspberries over six week’s storage under standard refrigeration and supercooled conditions

Photo 1 In-house measuring device for measurement of raspberry firmness

Figure

Figure 3 Average exudate from raspberries over six week’s storage under standard refrigeration and supercooled conditions

As expected, raspberries stored frozen (and then defrosted prior to assessment) were much softer than raspberries stored supercooled or under standard refrigeration (Figure 2). After three weeks of storage supercooled raspberries were marginally firmer than raspberries stored refrigerated, but this result was reversed after six weeks of storage. Firmness results were reflected in the results for assessment of exudate (Figure 3), with six-week supercooled-stored raspberries producing more exudate than raspberries stored refrigerated. However, after six weeks storage it was noted that supercooled raspberries appeared somewhat “frozen”, and the decreased firmness and increased exudate noted likely reflect this observation. The results of L.A.B. colour assessment are shown in Table 1. After three weeks frozen storage the majority of raspberries ranged in colour from light coral/crimson/fire brick and rosy brown, compared with maroon for all raspberries stored supercooled and refrigerated. After six weeks of storage the majority of raspberries under all storage conditions were scored as either maroon or brown.

or frozen conditions

Microbial quality of raspberries

The mould, yeast and bacterial counts are shown in Figures 4– 6, respectively for samples stored supercooled or under normal refrigeration conditions.

As the storage trial progressed, a larger number of raspberries showed gross spoilage under standard refrigeration conditions, and were therefore excluded from plate count assessments per se. Excluding these grossly spoiled raspberries, in general the results indicated no significant difference in microbial quality for raspberries stored under either condition. However, some significant variability in results across time points occurred, particularly in the case of week two counts of bacteria under supercooled conditions (Figure 6) and week four counts of moulds on PCA under normal refrigeration conditions (Figure 4).

4 Average counts of moulds on raspberries (non-grossly spoiled) over six week’s storage under standard refrigeration and supercooled conditions (counts from Plate Count Agar (PCA, non-selective) and Dichloran Rose Bengal Agar (DRBC, selective))

Figure 5 Average counts of yeast on raspberries (non-grossly spoiled) over six week’s storage under standard refrigeration and supercooled conditions (counts from Dichloran Rose Bengal Agar (DRBC, selective))

Figure

Table 1: Results of L.A.B. colour assessment of raspberries after 3 and 6 weeks storage under refrigerated, supercooled

Figure 6 Average counts of bacteria on raspberries (non-grossly spoiled) over six week’s storage under standard refrigeration and supercooled conditions (counts from Plate Count Agar (PCA, non-selective))

DISCUSSION

This research aimed to compare the effect of supercooled storage with standard refrigeration and frozen storage on the quality of fresh raspberries. Overall, after six weeks storage, supercooled raspberries were of greater visual appeal than raspberries stored under either of the other conditions. The improved appeals of supercool-stored raspberries was largely due to the decreased observation of grossly spoiled fruit, since the lower temperature of supercooling slows the rate of growth of microbes compared with standard refrigeration temperatures, moulds in particular.

Leaving aside grossly spoiled fruit, there was substantial variability in the mould count data among storage conditions and across time points, reflecting the difficulties of sampling these types of microbes where small numbers of colonies can produce large numbers of spores, or large numbers of colonies can produce few spores when growing on the fruit. Yeast and bacterial counts were similar for supercooled and refrigerated fruit across the storage period.

After three and six weeks of storage, the firmness and colour of raspberries stored supercooled and in the fridge were more similar than raspberries stored in the freezer. Raspberries stored supercooled and refrigerated were firmer than those stored frozen, but darker red in colour. After six weeks of storage, refrigerated raspberries were firmer than raspberries stored supercooled, but this firmness was offset in overall quality terms by the greater proportion of grossly spoiled raspberries present after six weeks under refrigerated conditions, compared with supercooled.

CONCLUSION

In conclusion, it appears unlikely that supercooling can achieve a six-week shelf-life for fresh raspberries. However, the shelf-life of supercooled raspberries in this trial was more limited by changes in texture rather than microbial spoilage, which is the usual limitation for fresh raspberries. It is possible that a shelf-life of three to four weeks may be achievable for fresh raspberries stored supercooled. In order to test this, it is recommended that greater attention be given to temperature control throughout the supercooled storage period. Improvements in packaging may also be valuable in preserving fruit quality during very small freeze-thaw cycles that might occur under supercooled conditions, by decreasing the potential for moisture migration into and out of fruit packaging.

ACKNOWLEDGEMENTS

This work was undertaken jointly with students from Doonside Technology High School. We would like to acknowledge the valuable technical assistance of Naomi Yeatman, Brendan, Felivic Aserios, Abhinav Dagg, TJ Stokes, Eric Soriano, Osbual Xavier and Peter Cabanding in the initial set-up and monitoring of the trial (Photo 2).

REFERENCES

Schülli, T.U., Daudin, R., Renaud, G., Vaysset, A., Geaymond, O. & Pasturel, A. (2010). Substrateenhanced supercooling in AuSi eutectic droplets. Nature. 464(7292): 1174–77.

Stonehouse, G.G., & Evans, J.A. (2015). The use of supercooling for fresh foods: a review. Journal of Food Engineering. 148: 74–79.

Usta, O.B., Kim, Y., Ozer, S., Bruinsma, B.G., Lee, J., Demir, E., Berendsen, T.A., et al. (2013). Supercooling as a viable non-freezing cell preservation method of rat hepatocytes. PLOS ONE. 8(7): e69334.

Photo 2 Students from Doonside Technology High School

Development of a Quality Control test method for PrIME Albumin Separation Membrane

The Protein Paramedic

Our work with PrIME this year involved developing and refining a testing method for the membranes that are used for PrIME Separation. Albumin, a plasma based protein, can be used therapeutically in a variety of different medical situations such as in burn therapy and in renal dialysis. Our aim was to establish a test method by optimising testing conditions to ensure the method has the sensitivity and accuracy to detect consistency or variations of membrane pore size. This means we will have a way to test and ensure the membranes are of high quality for PrIME’s plasma fractionation.

The Future Project has really opened my eyes to how Science is used and applied in real-world scenarios; learning about the day to day functioning of PrIME Biologics was a unique experience that I would not have otherwise had. Working in a laboratory environment has really taught me the importance of being mindful and concentrating on whatever you are doing in order to pull it off successfully. The Future Project has also highlighted the need for good communication and teamwork.

The Future Project has been an enlightening experience into the world of hands-on scientific applications. It isn’t often that an average Year 11 student obtains an opportunity to witness and partake in research on the industrial processes of biopharmaceutics with a rising protein extraction firm such as PrIME Biologics. With the presence of a fully equipped laboratory on site and an active, interesting and inspiring environment created by experienced researchers and fellow Senior Interns, it has been a memorable undertaking. The skills that I developed on time management, communication, concentration and scientific conventions will be employed for years to come.

Sami

Tehilan

T. Tampoe1, S. Al-Hilfi1, E. Lin2, K.Wang2, H. Nair2 THE FUTURE PROJECT1 AND PRIME BIOLOGICS2

ABSTRACT

Albumin has a molecular mass of approximately 66 kilodaltons (kDa) based on its protein composition and an approximate isoelectric point (pI) of 4.7. Human serum albumin (HSA) is the most abundant protein in human blood plasma and constitutes approximately half of the serum protein. Preparative Isolation by Membrane Electrophoresis (PrIME) Separation is a form of plasma fractionation that separates molecules based on their particle size and charge through membrane electrophoresis. This study is an investigation into the establishment of a test method for the Albumin Separation Membrane manufactured by PrIME Biologics. An establishment of an optimised and robust test method from testing the membrane pore size enables regulated Quality Control (QC) for PrIME under the standards of Good Manufacturing Practice (GMP). A test method was developed for membrane QC and the results have demonstrated reproducibility of the method by testing several batches of PrIME Membranes.

INTRODUCTION

Plasma fractionation is the process of isolating, purifying and concentrating plasma proteins from blood plasma (CSL Behring, 2016). The core purpose of plasma fractionation is to isolate proteins that are present in plasma that are in global demand as biopharmaceutical products. Albumin is one of the most abundant proteins in human blood plasma and it is vital for the maintenance of oncotic pressure to stabilise extracellular fluid volume and primarily functions as a carrier protein for steroids, fatty acids and thyroid hormones (Baker, 2002). As a therapeutic product, albumin has been clinically indicated to assist in the treatment of shock, sepsis, trauma, acute respiratory distress syndrome, burns and liver cirrhosis (Garcovich et al, 2009). Its scarcity and high cost limit access especially in developing countries, hence it is rarely administered. PrIME Biologics extracts albumin from blood plasma through its revolutionary membrane technology, which is a disposable, modular manufacturing process that works in an electrical environment and is fully scalable. PrIME technology can deliver a highly purified albumin product that is cheaper, enables more accessibility for developing countries and hence will improve the quality of wellbeing in many countries. Currently, PrIME is manufacturing an Albumin Separation Membrane for this process. Human Serum Albumin (HSA) has an isoelectric point (pI) of approximately 4.7, which is utilised as a key factor in the size- and charge-based separation by PrIME technology (Corthals et al, 1996). The aim was to establish a reproducible QC test method on the PrIME Albumin Separation Membrane with optimal running conditions that ensure consistent results.

METHOD

Tris-borate (TB) buffer preparation

325 grams (g) of Trisaminomethane and 63.75 g of boric acid were weighed and dissolved in ultrapure water in a beaker to produce a total volume of 5 litres (L) of 540 millimolar (mM) TB stock buffer. It was then mixed with a magnetic stirrer until no visible particles were apparent and stored at room temperature for up to three months. The 540 mM

stock buffer was then diluted with ultrapure water at a 1:10 dilution factor to create a working 54 mM TB buffer.

Bovine serum albumin (BSA) solution preparation

Lyophilised BSA was dissolved in the 54 mM TB buffer to produce a working BSA solution for Stream 1 (S1). The streams are lines through which the BSA solution and 54 mM TB buffer are drawn and passed through a membrane cartridge within a laboratory scale protein separation unit (BF400 instrument). These BSA solutions were prepared in 1 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 8 mg/mL, 10 mg/mL, 15 mg/ mL and 25 mg/mL concentrations.

Cartridge assembly

A membrane cartridge was prepared using two Restriction polyacrylamide membranes and one Albumin Separation Membrane. One Restriction membrane was placed between the outer housing and a plastic grid, followed by the Separation membrane, another plastic grid, the other Restriction membrane, and the inner housing. The channels within the plastic grid were oriented to face the Separation membrane and the inner housing was then locked into the outer housing. The Restriction membranes work to prevent the loss of protein into the buffer while permitting buffer flow.

Figure 1 PrIME Separation Principle. A schematic drawing of separation cartridge facilitating the separation flow from S1 to S2, or vice versa. The separation of albumin is from S1 to S2. Albumin Separation by PrIME

Albumin Separation by PrIME

The albumin was separated from the BSA solution using a BF400 instrument. The BF400 instrument performs tangential flow electrophoresis to separate macromolecules by applying a charge to transfer proteins (Figure 1). The albumin in the BSA solution in S1 is transferred through the Separation membrane into the 54 mM TB buffer in Stream 2 (S2).

The BF400 instrument was cleaned following a documented cleaning procedure. After cleaning, 54 mM TB buffer was poured into the buffer reservoir until the cooling unit was completely submerged. The prepared membrane cartridge was then inserted and installed into the BF400. A leakage test was performed to determine the integrity of the membrane by running the BF400 for 10 min with 20 mL of the 54 mM TB buffer in both S1 and S2 containers.

The temperature and the end volume of buffer from S1 and S2 were recorded and the process continued as the difference between the final and original volumes of buffer in each container was equal or less than 1 mL. Once the leakage test was passed, 16 mL of BSA solution and working buffer were poured into two clean stream containers. 1000 microliters (μL) from each sample were then aliquoted into two Eppendorf tubes labelled S1T0 and S2T0 respectively, making 15 mL of BSA solution and working buffer. The BF400 instrument was set to 250 volts (V) and the samples were run for 30 min, with BSA solution in S1 and working buffer in S2. The temperature was measured at T0 and T30 and the current was measured at T1 and T29. After the run, the volumes of samples in each container were measured. 1000 μL of each sample after the run was aliquoted into two Eppendorf tubes labelled S1T30 and S2T30. The runs were repeated with different running conditions such as changes in concentration (1 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/ mL, 8 mg/mL, 10 mg/mL, 15 mg/mL and 25 mg/mL) and voltage (100 V, 150 V and 200 V).

Bradford assay

980 μL of working buffer was pipetted into four Eppendorf tubes. 20 μL of each sample from the albumin separation step was diluted into each Eppendorf tube separately and then vortexed, making a 1:50 dilution factor. Other dilution factors of 1:5, 1:10, 1:15, 1:20, 1:30 and 1:40 were also tested with the amount of working buffer and samples adjusted accordingly.

10 μL of each diluted sample was pipetted into a 96-well plate in triplicates. 250 μL of Bradford reagent was pipetted into each well containing the samples. The plate was incubated in the dark for 5 min, then read in the Bio-rad iMark Microplate Absorbance Reader. The absorbance of the wells was measured and the results were exported and collated using an Excel spreadsheet.

RESULTS AND DISCUSSION

Figure 2 and Figure 3 display the 11 separation runs conducted by three different operators, running five different batches using 10 mg/mL BSA at 250 V for 30 min in accordance with the chosen running conditions. The results were generated using 1:50 dilution factor for the samples. The %Protein S1 refers to the percentage of total albumin retained in S1 after a run. The %Protein S2 refers to the percentage of total albumin in S2 after a run, which determines the transfer rate. The %Mass Balance refers to the percentage total albumin present in S1T30 and S2T30, relative to S1T0.

Figure 2 % Protein S2 (Transfer Rate) chart. The range of the preliminary specification is 12% - 32% and all 11 runs are within the range. The error bars illustrate the standard deviation of the intra-batch results from three cartridges.

Figure 3 % Mass Balance chart. The range of the preliminary specification is 87% - 100% and all 11 runs are within the range. The error bars illustrate the standard deviation of the intra-batch results from three cartridges.

The parameters that require specifications for QC testing are the transfer rate and mass balance. The allowable limit for the transfer rate is 10%-90% and the allowable limit for mass balance is in the vicinity of 100%. This is necessary in order to establish preliminary specifications of mean +/- 2 Standard Deviation (SD).

The specific running conditions of 10 mg/mL BSA at 250 V for 30 min were chosen as the results from those conditions were the most consistent and fell within the allowable limit for transfer rate and mass balance. The preliminary specifications of the transfer rate and mass balance were set using the results from five different batches conducted by three different operators. The specifications were statistically calculated using the mean +/- 2SD. From the calculations, the range of %Protein S2 is 12-32 and the range of %Mass Balance is 87-100.

The results were deemed to pass if they fell within the preliminary specifications of %Protein S2 and %Mass Balance. Out of 33 cartridges in 11 runs, 29 passed, while two of the cartridges did not fall within the preliminary specifications and another two results were declared invalid due to technical errors and were discarded. Figure 2 and 3 illustrate that the batch results fell within the preliminary specifications.

Excluding the discarded results, all other results displayed adequate consistency between each cartridge and were within the allowable limits of %Protein S2 and %Mass Balance. As a result, the running conditions of 10 mg/mL BSA at 250 V for 30 min and 1:50 dilution factor used in Bradford Assay was most suitable and could be used as the test method for the 150 kDa QC test method. This experiment was also designed to validate the test method by performing intra-batch and inter-batch testing from different operators. The results using these running conditions proved that the intra-batch and inter-batch results were consistent (Figure 2 and Figure 3). Thus the running conditions were validated and could be applied to a test method.

As the specifications were based on the statistically calculated ranges from limited data, they are now preliminary specifications. More data is required to consolidate the ranges in order to consider them as approved working specifications, or product acceptance criteria. The development of working specifications will be handed to the QC Department of PrIME Biologics and more results will be gathered to ensure the accuracy, precision and reproducibility of the specifications.

CONCLUSION

Consistent results within the preliminary specifications for protein transfer rate and mass balance ensured that the testing conditions were valid and could be used as the QC test method for the 150 kDa Membrane. This test method was robust as demonstrated by the intra- and inter- batch testing from different operators. The accuracy, precision and reproducibility illustrated from the results concluded that the running conditions of the test method were most suitable for QC of the 150 kDa membranes. Thus, a test method was successfully established for the 150 kDa PrIME Albumin Separation Membrane.

ACKNOWLEDGEMENTS

We would like to thank Eric Han Lin for his guidance and mentorship throughout the year. Special thanks to Dr Vera Munro-Smith, Dr Meryem Jeffries and Dr Kailing Wang for their ongoing support and to PrIME Biologics for making this paper possible. Additionally, thanks go to Brad Papworth, Roger Kennett and the The Future Project staff for making this journey possible. Finally, we would like to thank The King’s School for providing the opportunity, laboratory space and facilities.

REFERENCES

Baker, M. (2002). Albumin, steroid hormones and the origin of vertebrates. Journal of Endocrinology, 175(1): 121-127.

Garcovich, M., Zocco, M. A. and Gasbarrini, A. (2009). Clinical use of albumin in hepatology. Blood Transfusion, 7(4): 268–277.

Corthals, G.L., Margolis, J., Williams, K.L., Gooley, A.A. (1996). The role of pH and membrane porosity in preparative electrophoresis. Electrophoresis, 17(4): 771-775

CSL Behring (2016). Protein Purification. Retrieved from http://www.cslbehring.com/quality-safety/ integrated-safety-system/protein-purification.htm

Role of anti-oxidant proteins during stress

INTRODUCTION

Sangui Bio studies inflammatory signalling in blood to look for diagnostic markers of disease and indicators of treatment effect. The research is focused on a set of proteins called cytokines and growth factors that are potent signalling molecules in the body. They control the regulation of inflammation, signalling to immune system cells and initiating tissue repair after injury. Sangui Bio has patent applications covering the analysis and potential uses of the proteins in this study.

A new area of research at Sangui Bio is the role of anti-oxidant proteins during stress. Most signalling proteins were initially discovered in experiments designed to explore cellular communication systems. Advances in protein analysis have revealed that many proteins have multiple functions beyond their initially described roles (Butler & Overall, 2009). For a number of inflammatory signalling molecules, a secondary role is antioxidant function, where they have been shown to protect cells against oxidative stress. Although the protective properties of these proteins during oxidative stress has been studied inside cells, there is a gap in the literature on their role in blood.

The investigation in this study was focused on what happens to certain proteins within the blood when they are exposed to Cigarette Smoke or Oxygen.

The experiments aimed to observe what happened in blood during exposure to cigarette smoke or elevated oxygen, by measuring the levels of certain proteins. The blood plasma level of inflammatory markers is a common test for inflammation across a wide range of health conditions, with millions of tests in Australia each year. The goal of this research is to enable tests that: provide an improved understanding of inflammation; may assist in diagnosis; provide an indication that treatment is required; and allow post-treatment monitoring.

CIGARETTE SMOKE

Over the last 50 years the detrimental impacts of cigarette smoking have been researched, documented, and are now widely known. There are over 4000 chemicals in cigarette smoke that interact with tissues in the body and directly cause or contribute to a multitude of diseases. Over 60 of the chemicals in cigarette smoke are known to cause cancer and smoking is a leading cause of premature death. Cigarette smoke damages the membranes of cells in the blood, which compromises blood function.

In addition to toxins and carcinogens, cigarette smoke contains stable and unstable free radicals and reactive oxygen species (ROS) in the particulate and the gas phase with the potential for biological oxidative damage. These ROS readily react with surrounding biological tissues,

damaging cells, proteins, and DNA. Because cigarette smoke is inhaled into the lungs, which have evolved to provide gas transfer in and out of blood, the cells within it are the primary tissue that is damaged by ROS as a result of smoking (Valavanidis, Vlachogianni & Fiotakis, 2009). Over time, chronic oxidative stress can interfere with the body's protective functions, which may contribute to the progression of diseases such as cancer and neurodegeneration.

OXYGEN

Animals and humans usually obtain a sufficient level of blood oxygen (O2) by breathing air, which is 21% O2. In certain conditions where low O2 saturation in the blood is common, such as pneumonia, chronic obstructive pulmonary disease and in some newborn babies (National Heart, Lung, and Blood Institute, 2012), pure O2 is also used as a therapeutic. Although O2 therapy can save lives, there is a balance between the biological benefit and O2 toxicity effects. The process of oxidative stress is associated with increased levels of O2 and results from the production of reactive O2 species (ROS) when high levels of O2 are administered. During aerobic metabolism, ROS are produced as naturally occurring by-products of cells and have important roles in cell signalling and homeostasis (D'Autréaux & Toledano, 2007). However, in hyperoxic environments, excess ROS can overwhelm the anti-oxidant capacity and then readily react with surrounding biological tissues, damaging cells, proteins and DNA. Blood and the cells within it are the primary tissue that is damaged by ROS during O2 therapy (Mach, Thimmesch, Pierce & Pierce, 2011).

METHODS

Experimental setup

For each experiment, four different individuals donated venous blood samples, which were collected with EDTA anticoagulant. The samples were donated following collection of informed consent under ethics approval (Northern Sydney Coast Human Research Ethics Committee of NSLHD and CCLHD; approval number: 1201-046M). Whole blood samples were aliquoted into 2 mL quantities and were placed into four six-well plates according to the sequence shown in Figure 1.

Figure 1

R. Chua1, A. Guo1, B. Hines1, A. Quattrocchi1 and B. Herbert2 E. Krasten2

THE FUTURE PROJECT1 and SANGUI BIO2

Smoke from individual cigarettes was obtained and contained in a syringe as shown in Figure 2. As the plunger was engaged, smoke was drawn into the syringe and the volume could be determined. Multiple doses of smoke could be obtained from each cigarette using this method. Smoke was introduced into a sealed plastic box as shown in Figure 3. Each six-well plate containing blood samples was exposed to cigarette smoke and a control sample was held outside the treatment box for the maximum time period. The blood samples were exposed to smoke from half a cigarette for 30 min. The box was then opened and aliquots of blood removed from each sample. The box was resealed and the blood samples were exposed to smoke from half a cigarette for an additional 30 min. This procedure was repeated once more, producing blood samples exposed for 30, 60 and 90 min.

REFERENCES

Butler, G.S. & Overall, C.M. (2009). Proteomic identification of multitasking proteins in unexpected locations complicates drug targeting. Nature Reviews Drug Discovery. 8: 935-948.

D'Autréaux, B. & Toledano, M.B. (2007). ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nature Reviews Molecular Cell Biology. 8: 813-824.

Oxygen treatment

The blood samples in six-well plates were contained in a sealed plastic box as shown in Figure 4. Oxygen was introduced through tubing and sealed hose connectors added to the box. Positive pressure was controlled via a tap on the exit tube. Each six-well plate (as prepared in Cigarette Smoke Treatment) was exposed to pure O2 for a different interval of time consisting of one or more of the following: 0 min (control), 15 min, 30 min, and 45 min. Whole blood samples were exposed to O2 in a sealed chamber which was connected to a cylinder of O2 as seen in Figure 5.

Each six-well plate was oxygenated in five min intervals. This consisted of a period of flushing the chamber of O2 with the nozzle of the chamber opened, followed by a period of low amounts of flushing the chamber with O2 with the nozzle of the chamber closed. This sequence was continued until the end of each time interval, removing the plates after their allocated time. Immediately after each plate removal, the dissolved oxygen (DO) mg/L was measured in each biological replicate with a dissolved O2 probe (Vernier DO Probe extrapolated with Vernier LabQuest 2 data synthesiser).

RESULTS

In 2015 Sangui Bio filed patent applications covering inventions that are in part related to these projects. There was an expectation that by October 2016 the work would no longer need to be kept confidential. However, the datasets obtained in these projects have provided additional data in support of the Sangui Bio patent applications and are subject to confidentiality provisions that prevent publication until mid2017. It is the intention of Sangui Bio and The Future Project to publish these projects in 2017.

Mach, W.J., Thimmesch, A.R., Pierce, J.T. & Pierce, J.D. (2011). Consequences of hyperoxia and the toxicity of oxygen in the lung. Nursing Research and Practice. 2011.

National Heart, Lung, and Blood Institute. (2012). What is oxygen therapy? Retrieved from http://www. nhlbi.nih.gov/health/health-topics/topics/oxt.

Valavanidis, A., Vlachogianni, T. & Fiotakis, K. (2009). Tobacco smoke: involvement of reactive oxygen species and stable free radicals in mechanisms of oxidative damage, carcinogenesis and synergistic effects with other respirable particles. International Journal of Environmental Research and Public Health. 6: 445-462.

Figure 2

Figure 3

Cigarette smoke treatment

Junior Interns

Our Year 10 Science rotation allowed us three weeks to work with various scientists from The Future Project, in order to develop further practical skills in Biology. Our introduction to research began with a discussion with Dr Ben Herbert from Sangui Bio on the role of proteins in blood and we then examined the possibilities of studying the effect of different environmental conditions on blood proteins.

This was followed by some laboratory work with Dr Michelle Bull and Dr Belinda Chapman from Quantal Bioscience who guided us through an investigation into the possible growth of microbes in sports drinks. For example, Lodderomyces elongisporus is a unicellular yeast microbe that can grow in sports drinks after being triggered by certain environmental conditions. The aim of our research task was to investigate the impact of these conditions, such as temperature or pH, on the form or structure of any microbes which grew.

This research was also extended to measure the concentration of red food colouring (allura) in a sports drink. We were given instructions on how to use a spectrophotometer, which is an instrument used to measure the intensity of light in a sample of a particular wavelength or colour. This allowed us to establish a standard curve for allura; that is, a graph of samples of known concentration against the intensity of their red colour. From this we were able to run a sample of sports drink and use the standard curve to calculate the exact concentration of allura in the sports drink.

Our intern experience was a great opportunity to experience real research and conduct experiments not possible in a normal school science laboratory. It challenged us to think at a higher level and to make sense of how scientific research helps society.

The Junior Intern program has helped us experience and learn many new practical research skills. We would like to thank Dr Herbert, Dr Bull and Dr Chapman for their time and expertise in putting these projects together.

Shaeel Hassan

GROUP MEMBERS

Pravin Chanmugam, Hanson Chen, Jason Chhoeu, Jack Chu, Shanith de Mel, Anthony Dingwall, James Dugdale, Nathan Dugdale, Christopher Gailey, Zane Gale, Oscar Han, Shaeel Hassan, Mohan Huang, Darcy Keogh, Charlie Lowe, Aaron Manton, Jacques Millichamp, Alex Noh, Steven Pham, Samuel Rider, Christian Saad, Rohan Shankar, Alexander Siu, Charlie Webster, Leo Zhang

REFLECTIONS

I thoroughly enjoyed my experience as a Future Project Junior Intern. I especially enjoyed working hands-on with real-life scientists. I can say with confidence that this experience has given me a taste of real-world science, and an insight into the importance of science in our rapidlychanging world.

Samuel Rider

I found The Future Project Junior Intern program very fascinating this year as our research delved into areas of real-life science we never really considered, such as sports drinks and blood cells; all of which are increasingly relevant to society.

Shaeel Hassan

I have enjoyed being a Junior Intern in The Future Project as it has allowed me to work with real scientists and do experiments I would never have done in normal science class.

Charlie Webster

I personally saw this experience as something different to the traditional science we normally experience in class. I enjoyed the opportunity of collaborating with those who may have greater knowledge than myself and working together with them to solve issues which may be affecting our society and the environment.

Hanson Chen

The Future Project was a great experience that really showcased the research that is being done out in the real-world, and some of the dilemmas we, as a society, must face moving into the future.

Aaron Manton

The projects provided me with a different perspective into the sciences and has helped me understand and develop my own knowledge of the subject.

Zane Gale

I found the Junior Intern experience great, enriching our own knowledge showing the application of science in the real-world, especially in our changing modern society.

Steven Pham

Communicators: Public Presenters

“Few people base their decision making on just being presented with good science. The communicator’s message must have meaning, be useful and acknowledge the needs, aspirations and concerns of each intended audience.”

Jesse Shore President of Australian Science Communicators (2010-2012)

Effective science communication has never been more important to our society. Recent advancements in science in fields such as genetic engineering, 3D printing and quantum computing (to name a few), have the potential to radically reshape the world in which we live. It is in all our interest to ensure that there is clarity and purposeful discussion about the appropriate use and implications of these emerging technologies.

It is the goal of The Future Project’s Communication Strand to develop the skills in our students to allow them to effectively communicate science to a lay audience. This involves the transmission of scientific information clearly, concisely and accurately. It’s about engaging audiences by explaining why this information is important and relevant to their lives. At its most aspirational, science communication is also about the wonder of science: the jaw-dropping, eye-popping excitement that comes with getting a glimpse into a natural phenomenon or human innovation that seems to open up infinite possibility and hope for the future.

This year, the Public Presenters of The Future Project Communication Strand have designed and created an exhibition housed in the Science Centre of The King’s School entitled “Soar: The Science of Flight”. The goal of this exhibition was to communicate past and present understanding of the science of flight, both in the natural world and with human-powered aircraft. The exhibition included student work such as posters, videos, animations as well as items on display. The exhibition opened in August of 2016 and remained in place over many months for the interest of The King’s School community and members of the public.

In this exhibition, a portrait gallery running along one side of the atrium of the Science Centre showed the four main types of flying creatures: insects, birds, bats and pterosaurs (now extinct). The gallery opposite had posters describing the working of the main kinds of aircraft currently in use: fixed-wing, rotary-wing, ornithopters and lighter-thanair aircraft, as well as an “unusual aircraft” category. In the central atrium, additional displays explained the important scientific principles behind flight such as the “Bernoulli Effect”, and the history of aircraft.

Also on display were a number of items on loan from the Temora Aviation Museum, such as burner cans, propellers and ailerons, with interpretive signage explaining the role of each of these in powered flight. Hanging from the ceiling of the Science Centre was the centrepiece of the exhibition, an “Airborne Edge X” microlight aircraft with an impressive 11 metre wingspan. The students of The Future Project worked hard to ensure that all of these exhibits were as informative and engaging as possible.

One additional part of the exhibition worth a special mention was the work of the Animation Team of the Public Presenters. A large video screen showed two 3D fixed-wing aircraft models on which the boys worked. The models rotated and zoomed in to focus on the crosssection of the teardrop-shaped wing or “aileron”. This shape is crucial to most forms of powered flight as it helps to generate the all-important “lift force” that keeps aircraft in the air.

Over the course of the year, the students in The Future Project’s Intern Strand have been collaborating with the research partners of The Future Project to do authentic and innovative research in the Science Centre’s purpose-built biochemistry laboratory. This research has been drawn from a diverse range of areas, such as a search for better methods of food preservation, designing autonomous rovers to patrol a grassed area, or growing algae in farming tubes to harvest them for omega-3 fatty acids. In the second half of this year, the Public Presenters were commissioned to produce a series of short documentaries showcasing this research.

To help explain the significance of this research to a lay audience, the students filmed the interns at work in the Science Centre, and then interviewed them about the details of their research. This footage was then edited by the students along with other elements such as music, banners and titles. It is hoped that these films can be used to raise awareness and understanding of the science behind this research. 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 Public Presenters of The Future Project Communication Strand. All of the students involved have enjoyed the opportunity to learn more about different areas of science while honing their communication skills in print and digital media. It is hoped that the work of these students has produced has provoked interest, understanding and excitement.

Matt Purser

Overall, the main thing about the experience which I enjoyed the most was when it actually came to teaching the visiting students. I believe communication of science is extremely important as it is an essential part of understanding science.

Zain Khan

I found that The Future Project allowed me to gain the life skill of talking to people and making things easier for them to understand.

Joshua

White

Being a School Presenter challenged me with talking to younger children and it really pushed me regarding communicating science. Science is a universal language and no matter your age, it is always good to inspire young minds with the wonders of science.

Keshav

Mohan

It is wonderful giving younger students a better insight into certain concepts and ideas, and as a presenter, the communication part was quite important. I think our hands-on activities they managed to take home new understandings about the science of flight.

Matthew Chamoun

I believe that The Future Project is a great program. I enjoyed every moment and I'm glad I signed up. Science communication is important for today’s society so general citizens are able to understand the current technology and discoveries in the field of science.

Ben Billyard

Communicators: School Presenters

This year the School Presenters continued with the theme of flight in their workshops to visiting primary school students. This theme also supported the new exhibition, Soar, developed by the Public Presenters. Participants included Year 5 students from Tudor House and The King’s School Preparatory School. Students rotated through four workshops based on the science behind lift, thrust and drag. By all accounts, the chance for School Presenters and primary students to engage in scientific inquiry was well received by all.

THE SHAPE OF A WING MATTERS

Why do airplanes have wings? Is there something special about their shape? The science behind how wings provide lift for the plane is a little harder to demonstrate. In our workshop, we had students make the shape of the wing by folding a piece of cardboard over to make a curved shape at the front. Then came the truly hard part. Punching a hole through it and inserting a straw, through which to thread a piece of string. This allowed the wing shape to slide up and down. By running forward or spinning around quickly, students could get the air to move quickly over the top and bottom of the wing. Why was this important? Because it is the difference in air speed over the top and bottom that generates an important force: lift. Why does this occur? Because the air has to travel faster over the curved top surface than over the bottom surface. This creates a difference in air pressure: low pressure on the top, high pressure on the bottom. Naturally, air always wants to move from areas of high pressure to areas of low pressure, and for the wing, this causes lift.

EXPLOSIVE THRUST

There is no lift without thrust; that is, without something pushing the aircraft forwards. Think about it: you are sitting on the runway ready for take off, then all of a sudden the engines roar, sending huge amounts of air backwards and the airplane accelerates forward down the runway and you’re up in the air. In this activity, students made a rocket by partly filling a plastic soft drink bottle with water and then screwing on a special cap with tail fins. Then they got to pump air into the bottle. As the air goes in it gets compressed under pressure, with the pressure building up until it eventually causes the water to explode out of the bottle, creating enough thrust for the rocket to fly across the field in front of the Science Centre. While not exactly how engines work, it did show how thrust can make an aircraft fly through the air.

UP, UP AND AWAY

How can we demonstrate the science behind hot air balloons? Take a toaster and lightweight dry cleaning bag, and you have a ready-made simulation of how hot air rises. Although it was not quite as simple as that. It took us a number of prototypes and burnt plastic bags to work out which one could capture enough hot air, be light enough, and not melt. First we had the students use the outside material of a teabag to make a long cylinder. Then we lit it and it burnt down to a very small amount of ash. The ash was then carried up on the hot air produced. While this was fun, it was not an easy way for students to see what was happening. Hence, our second activity was a demonstration of how hot air from the toaster fills the plastic bag, causing it to inflate and rise up. Then, up, up and away it travelled until the air cooled, causing the bag to float back down again. Do not try these activities at home unless you have adult supervision!

IT’S A DRAG

What better way to learn about the science behind how parachutes work than through a design competition? Our group had students work in pairs to design their own parachute from a piece of plastic, four pieces of string, and some Play Doh that acted as a weight. Each pair’s design was a little different depending on the size of the plastic, length of string, or amount of weight added. Then it was time for testing. Which design fell faster and which design fell slower? It all came down to the surface area of the parachute compared to the amount of weight pulling down on it. The larger surface area worked well but if there was not enough weight pulling down when it was dropped, then the parachute had trouble opening. This activity was a little bit of science and a lot of fun.

While these activities were simple, they were also very effective. They allowed the visiting students to get a hands-on experience and see the science for themselves. It also gave the School Presenters a chance to guide them, as they developed an understanding of each important concept behind the science of flight.

Ben Billyard, Matthew Chamoun, Navneet Gantasala, Zain Khan, Marcus Lim, Keshav Mohan, Darcy Penman, Aleks Sasic, Tej Shah, Benjamin Stewart, & Joshua White

Under the microscope

ASSOCIATE PROFESSOR

BEN HERBERT NZCS (Chemistry) CPIT, PhD Macq

What do you get if you cross a New Zealander with a passion for scientific research and innovation? Ben Herbert. Born in Christchurch, he started full-time work and part-time study in 1986, working for the chemistry division of the Department of Scientific and Industrial Research (NZ equivalent of CSIRO). He studied chemistry at Christchurch Polytechnic Institute of Technology and graduated with a Certificate in Science after four years. At that time, most of his work had been in protein chemistry and he was lucky enough to get involved in research quite early in his career. It was this early time, simultaneously studying and working, that would put Ben on a path of innovation and the rest of his career.

After a five year stint at the Wool Research Organisation of New Zealand investigating wool protein chemistry, Ben moved with his family to Sydney in 1995 to complete a PhD at Macquarie University. Here, he was part of the team that established the Australian Proteome Analysis Facility, helping to pioneer the way in protein analysis. From there his career took off in the biotech space, co-founding two ASX listed companies. He later returned to Macquarie University as the Vice Chancellor’s Innovation Fellow. Career highlights include: 66 peer-reviewed scientific papers and book chapters; nine PhD students supervised; eight patents in the US, Europe and Australia and several more pending; numerous products developed and commercialised; and co-founder of three biotech companies. He is now in the medical school at the University of Sydney, based at Royal North Shore. It was research into stem cells that led him to spin off his latest company in a different direction, Sangui Bio.

Ben is passionate about science saying, “Science and technology have produced the modern world we see around us. A world where children born now will live for 100 years and where we enjoy rapid, safe travel and communication networks that have enabled global collaboration in real-time. The challenges thrown up by our dominance of the planet cut across medical science, the provision of food, water and sustainable energy”. It is for these reasons that he sees a pressing need for innovative scientific solutions.

What is profound is Ben’s desire to provide young people with the opportunity to be part of the process of research, innovation and commercialisation. In The Future Project, he says, “Students are involved in formulating the research question, experimental design, lab work, analysis and writing. This is well beyond usual school science experiments.

“Science teaches a powerful form of open-mindedness and critical thinking, which is the spark of innovation. This century will see an

increasing demand for scientists and engineers and we must do a better job of ensuring that girls make the transition into these careers. Outside the mainstream science and engineering vocations, businesses of all types value science and engineering graduates for their critical thinking and it is becoming increasingly common for university students to graduate with a double degree in another area.”

Ben sees The Future Project as an opportunity for researchers in terms of science communication as they need to be able to explain their work and engage students in meaningful experiments. It also provides researchers with an alternative way of thinking about their work, leading to new ideas.

Programs such as The Future Project enable students to engage in formal and informal learning simultaneously, which is very beneficial. It adds the important real-world context around their formal learning and encourages creativity. Ben has been involved as a collaborator since the early piloting phase, having seen the building emerge and come to life with students, teachers and many more collaborators. He has been a champion of The Future Project and without his early involvement it may well have failed.

“It has been very exciting. I get a lot of pleasure in seeing students engaged in science and coming in with questions and ideas and being part of discovering something new.”

If there were any advice he would give to students thinking about a future in science or engineering, what would he say?

“Passion is more important than brilliance. Do what you’re passionate about and don’t be dissuaded or embarrassed into conformity. Don’t specialise too early – there’s reward in diversity of knowledge.”

RIYA GAIKAIWARI

YEAR 11, BAULKHAM HILLS HIGH SCHOOL

Riya was one of four students selected from Baulkham Hills High School to join The Future Project this year. She worked as a Senior Intern on Quantal Bioscience research projects along with Naida Rasheed, Joseph Rylance, Bradley Gardner, and Zoe Marshall under the mentorship of Dr Belinda Chapman and Dr Michelle Bull. So what was the spark that prompted her to apply and what did she gain from the experience?

Riya says that her interest in science at school stems from the relevance it bears in daily life. “It is a subject that is always innovative and interactive, and presents challenges in learning, unlike other subjects. Science at school is well-rounded, and allows any student to gain an insight into each of the branches of science, which are all interconnected.”

This year Riya has been studying Chemistry, Legal Studies, Modern History, Extension 1 Mathematics and Extension 1 English as part of her Preliminary HSC course. But while she has a passion for studying science at school, she also saw an opportunity to enrich her understanding and experience through The Future Project. She elected to apply to be part of the program because it provided first-hand involvement in meaningful research.

“While school gives exposure to the core subject of science, most students have minimal exposure to real-world science, especially being able to participate in the entire research process.”

Riya’s involvement in The Future Project gave her a chance to see applications of science, to learn new skills outside of the classroom, especially with regards to conducting rigorous research, analysing results, and reporting the findings of her team’s research.

Her team had two main research projects. One was to investigate the effect of supercooling on the preservation of raspberries and the other to develop a method to test the tolerance of capsules filled with Dietzia, a probiotic being used in a Quantal Bioscience clinical trial.

Why are these two studies important? The first study into the supercooling of foods was based on the premise that it would be a good way to maintain the integrity of delicate foods such as raspberries, especially during periods of transportation, rather than using conventional refrigeration or freezing. Their experiment was based on similar investigations into the supercooling of fruit but sought to add further insight into modelling its effects. For the food industry, this is valuable in extending the life of food and maintaining important taste and texture qualities. The second study aimed to improve delivery of

Dietzia as a treatment for Crohn’s disease, a long term inflammatory disease of the intestines. Earlier clinical trials relied on giving each patient a small liquid dose by mouth each day; however, this posed a number of problems in terms of consistency and reliability between patients. The Quantal Bioscience researchers hoped to simplify the process by giving patients a capsule containing the Dietzia, but this required further investigation into which capsule would allow Dietzia to get to where it was needed in the gut. Given the global population of people suffering from Crohn’s disease exceeds 25 million, this research is critical to aiding the health of many people.

Writing up these investigations was not easy. But even this process sheds insights into the process of research for Senior Interns. Riya says, “I believe each and every intern in our group has glimpsed the complexity of research and the work that takes to put together a comprehensive report.”

Although the research was challenging, Riya also found it very enjoyable.

“It was great meeting each and every member of my research group, and working, learning and problem-solving alongside them. Our researchers have been amazing to work with, and have been patient in fostering our learning, teaching us new methods both inside and outside the laboratory.”

A key thing she takes away from her involvement in the program is a greater understanding of the practical applications of science, especially in the field of microbiology that her team was involved in, as well as the impact of science, innovation, and research on our daily lives.

After school, Riya hopes to pursue a career in either Law, Medicine, or Medical Research. The skills learnt during The Future Project, as in many science or engineering degrees, are an asset she will take into one of these careers.

PrIME Biologics (PrIME) is a Singapore-based plasma fractionation company which aims to address the US $1billion Asian therapeutic plasma products market. PrIME has developed an integrated PrIME+ process, using their signature technology in combination with current existing processes.

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, of 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 with 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’ mission is summarised in its motto: ‘improving lives through sustainable innovation’.

Single-use membrane cartridges ensure the elimination of possible batch-to-batch contamination. This allows PrIME to process plasma which might otherwise not be able to be processed. Further, the PrIME technology provides an additional level of product safety by its ability to remove viruses and pathogens.

Earlier this year, PrIME Biologics achieved its first goal of attaining cGMP accreditation for its plasma fractionation plant in Singapore’s Science Park II. Now, PrIME is working towards scaling up its plasma fractionation processes for commercial manufacturing, to become the first plasma fractionator in South-East Asia. PrIME’s first products will be Albumin and IVIG from human plasma, with a following pipeline that includes Factor VIII, Factor IX, and much more.

Vitramed Mechatronics is part of Vitramed, a company focused on gastrointestinal health. Vitramed was founded in Sydney, Australia and serves Australia and South East Asia, with offices in Kuala Lumpur, Malaysia and Singapore.

The main objective of Vitramed Mechatronics is to design and develop medical devices using the latest sensor, electronics and microcontroller technology. More broadly, Vitramed Mechatronics is involved with robotics, image processing and the emerging idea of the Internet of Things (IoT). Vitramed Mechatronics is also responsible for the support and servicing of medical devices distributed by Vitramed Medical Devices.

Rapid advances in computing and small scale manufacturing techniques, such as 3D printing and desktop CNC machining, has made this sort of device development work possible in smaller scale environments. In the 1980s, the desktop publishing revolution had a similar impact in terms of being able to write, print and publish words.

In mechatronics terms, the tools required to design and prototype medical devices are available without needing to rely on traditionally more expensive methods of prototyping. This allows Vitramed Mechatronics to experiment with the latest technologies and to allow for constant revisions to hardware such as electronics boards or device enclosures. Vitramed Mechatronics, and Vitramed as a whole are proud to be a partner of The Future Project.

Sangui Bio was founded in 2015 by A/Prof Ben Herbert, Elisabeth Karsten and Alan Liddle. A/Prof Herbert leads the Translational Regenerative Medicine research group, which is based at the Kolling Institute; part of the University of Sydney’s Medical School. The group studies inflammatory signalling in disease and its effect on immune function. Their primary focus is on signalling proteins and associated factors that control the regulation of inflammation, signalling to immune system cells and initiating tissue repair after injury. They are working with internationally recognised experts in haematology, cancer, infectious disease and pregnancy. Over the last two years the group made a series of discoveries related to immune function, which are covered by patent applications. Sangui Bio Pty Ltd was established to drive the commercialisation process. The company is currently exploring commercial opportunities in diagnostics and therapeutics with international partners.

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:

• Aeronautical, Biomedical, Chemical, Biomolecular, Civil, Computer, Electrical, Environmental Fluids, Geotechnical, Mechanical, Mechatronics, Power, Software and Structural Engineering;

• Computer Science and Information Technology;

• Project Management;

• 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.

Quantal Bioscience is a small private research company specialising in applied microbiology and microbiomics. Quantal Bioscience was founded in 2015 by Dr Belinda Chapman and Dr Michelle Bull, previously of Vitramed Bioscience, itself one of the original collaborators in The Future Project. Together with Emma Winley, Andy Ryland and Dr Janelle Brown, Quantal Bioscience offer research consulting, science management and a range of specialist testing services across the Health and Medical, Environment and Agriculture, and Food Industry sectors.

In the Health and Medical area, Quantal Bioscience has a particular interest in gut health. Through Vitramed Bioscience, and now as Quantal Bioscience, a key focus area of research is in the development of a novel probiotic treatment for Crohn’s Disease. Crohn’s Disease is one of the major forms of Inflammatory Bowel Disease (IBD). Crohn’s Disease most commonly affects the large intestine, but may also be present in the small intestine, and indeed throughout the gastrointestinal (GI) tract. The prevalence of IBD is rising around the world, and currently affects more than 75,000 Australians. There is at present no cure for Crohn’s Disease, and treatments for Crohn’s Disease use a variety of drugs including various antibiotics and corticosteroids that can have undesirable side-effects.

Beyond the human gut environment Quantal Bioscience is also involved in characterising the equine gut in health and performance. The equine gut is a fascinating environment for study owing to the incredible diversity of microorganisms that call it home. Like humans, the horse has a single stomach. However, unlike humans, the proper functioning of the equine gut relies not only on bacteria, but also a diverse array of fungi and protozoa, reflecting the much higher fibre diet that horses have evolved with. Quantal Bioscience’s focus on microbiome profiling in the Environment and Agriculture sector extends from the equine gut to other complex natural and agricultural environments, including soils and composts.

In the Food Industry sector, Quantal Bioscience provides research consulting services to a number of small artisan and large multinational food producers. In this area, the team of scientists at Quantal Bioscience draw on their extensive experience working with the food industry, gained during their combined 30 plus years working with the CSIRO Division of Food Science. An area of current focus here is in bringing the new technologies of microbiome profiling to traditional areas of factory spoilage investigations and complex mixed culture fermentations.

Staff Changes

DR ANTE (TONY) JERKOVIC

Tony has extensively studied at Macquarie University, completing a Bachelor of Science majoring in Medical Sciences in 2004, a Masters of Science in Plant Biochemistry in 2007 and a PhD in the same field in 2011. He has worked with the Department of Chemistry and Biomolecular Sciences, and the Faculty of Medicine and Health Sciences at Macquarie University, as well as various roles in the private sector and as a Postdoctoral Research Fellow with the Australian School of Advanced Medicine.

DR OLIVIER LACZKA

Olivier obtained a degree in Biotechnology and Applied Microbiology in France, followed by a PhD in Biotechnology and Biochemistry in 2009 from the Autonomous University of Barcelona, Spain. He has worked in development and validation of diagnostic tools for medical, environmental and food industry applications, in both academic and industrial environments, and has expertise in molecular biology, microscopy, optical methods (chemical and electrochemical), and microbiology.

MS EMMA WINLEY

Emma is an applied research scientist with Quantal Bioscience. She is a horticultural scientist and applied microbiologist with experience in: postharvest physiology and pathology; storage and transport of perishable food; minimal processing of fruit and vegetables; food safety and hygiene; and food microbiology. Emma has previously worked for the NSW Department of Agriculture, the CSIRO, the NSW Food Authority, Western Sydney University and Applied Horticultural Research.

MS MERYEM JEFFERIES

In July, Meryem left The Future Project to pursue other goals. Her work with the interns was invaluable and she will be missed.

MR TOM RILEY

Farewell to a fantastic TFP innovator. Mr Tom Riley has been a vibrant and energetic contributor to the success of The Future Project. Over two years, Tom edited and produced the Journal of the Future Project, which is an incredibly complex and demanding job. This Journal rivals many professional productions and Tom was the key driver of its success. Tom also introduced the Year 10 Junior Interns Program and worked tirelessly with our collaborators to make this effective and meaningful for our students. Tom also managed the corporate relationships, being the “chief collaborator” and a significant interface between the many collaborators involved in the Project. His management of the safety, cooperation and curriculum set him apart as a truly inspirational educator. Tom has moved on to different pastures and we will miss his enthusiasm, energy and whirlwind work ethic.

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

Jenny Tan for her work as the Bursarial representative for The Future Project

Tina Moshkanbaryans for her work as Editor of The Journal of the Future Project

Karl Sebire for his brilliant graphic design of this Journal

Julie Saad for her endless help with Future Project matters

The entire science faculty for their generosity in time and willingness to be involved

Our collaborators who make The Future Project possible

Baulkham Hills High School, Cherrybrook Technology High School, Mamre Anglican College and Doonside High School for their willingness to participate in The Future Project

Everyone who assisted with Science Week

The Industrial Arts Department for their ongoing support of the Mechatronics Strand

Professor Mary O’Kane for her support of The Future Project and Science at The King’s School