A recent ‘person specification’ for a senior consultant to be recruited to a scientific consulting practice read like it was describing a super-being. Competencies spanned the ability to present at international conferences, development and use of models for systems analysis and synthesis, use of Excel, development of software and application of geographical information systems.

This was before the addition of sector specific skills and knowledge, such as the ability to demonstrate fundamental knowledge in biological science and basic workplace and academic competencies like writing and personal organisation. You’d be right if you concluded that science is a pretty competency-rich environment.

But, like most things in life, the fewer degrees of freedom we’re prepared to allow, the fewer the opportunities – in this case, the fewer folk there out there who can excel in the job. And when there are fewer folk who can excel, the laws of economics apply – those who have the skills and the knowledge can expect inflated salaries. If the employer can’t meet the salary expectations (because perhaps the business simply can’t afford it) there is then pressure to relax the ‘person specification’, reducing those competencies in an effort to achieve a compromise.

Reports from the House of Lords Select Committee on Science and Technology make interesting reading. Government laments the numbers taking up STEM (science, technology, English and maths) subjects. Talking specifically about competency in maths, the Lords comment that ‘a high level of numeracy is of increasing importance to employers’. And in the same report, they go on to note that the UK is simply not producing enough science, technology and engineering technicians and graduates. If that’s true, our degrees of freedom look set to reduce further.

So, given this situation, how does a science employer go about getting their fair share of available applicants when recruiting? Indeed, if skills and knowledge are not abundant, how does the employer succeed, despite the tight employment market? Is it all about salaries or does approach matter too?

Traditional recruitment involves a three-step process; ask a recruitment agency to source CVs, select a shortlist from the CVs offered and interview. But how does one know that these are the only candidates available? In a tight market, only those actually unhappy in their work or those actually out of work will place their CVs on a jobs board to be found by a searching agency. Surely there must be a better way?

Traditionally, head-hunters succeeded where perhaps recruitment agents failed. They sourced by detective work, finding names and calling them in effort to encourage otherwise happy employees to look at new opportunities. And they charged over 25% of salary for their efforts.

Traditional selection involves a manager sifting the CVs and making a shortlist, perhaps seeking key attributes of applicant personal characteristics. Candidates from the shortlist are interviewed, often informally, and in meetings lasting around an hour. Research shows that few managers are trained in selection and, without some science in the process, the chances of a manager making a valid prediction of how well someone will do in a role are very small.

But things are changing and, arguably, they need to.

A recent report in Recruiter, the magazine of the recruitment industry, cited results of a survey showing that the leading jobs boards each only enjoy around 7% of successful placements. There’s a raft of lesser boards that enjoy significantly less. Towering high above them all, though, is LinkedIn at a colossal 21% of successful placements. Today everyone wants to be seen. Everyone wants to profess skills and knowledge to customers, future employers and colleagues alike. Only the recluse is below the radar.

It doesn’t take a detective to find people today. It just takes sound analysis of the requirements and a robust search. Finding staff is no longer a black art. Of course, it’s a different sort of person that’s found and managers need to be ready to unseat someone who perhaps wasn’t actually looking at that point in time.

Over the past 15 years or so, the science of selection has galloped ahead. Gut-feel is out and predictively valid selection instruments are in. Psychometrics help measure those numeracy skills. They assess those desirable workplace competencies, such as verbal and abstract reasoning. Managers know that it’s those who are pro-active and conscientious that make the best scientists: personality profiles measure these characteristics too. Managers know too that future staff must fit in the firm and person-environment fit can be assessed as well.

When it comes to interviews, the best include work sample tests. If the ‘person specification’ calls for competency in developing models to describe phenomena, ask the candidate to use Excel to build a model. And give them just 15 minutes to do it. The combination of test and pressure will show who will excel with Excel.

Those old interviews too can be modernised. Using competencies to sift CVs into a shortlist quantifies the task – the same competencies can be used to develop the interview questions and developing scored model answers makes decisions simpler.

The combination of psychometrics and improved testing and interviewing has raised the predictive validity from an R value of around 0.2 to around 0.6. For those statistically minded, R2, the percentage chance of successful recruitment from these selection instruments alone rises from 4% to 36%. While selection still isn’t perfect, it’s a lot better than the traditional ways.

In a tight market, where there are just too few folk to go round and where the competencies available fall short of that required, two huge changes are upon the science industry. The first is a revolution in the search activity: people can be found. And the second is an evolution in selection instruments: selection is now a science.

Technology has a habit of changing the goal-posts. There was a time when it was a huge skill to draw using tools like Harvard Graphics. Today, everyone can drive PowerPoint and the skill is in developing the message in the slide. With traditional recruitment, it was an art to head-hunt and interview. Today it’s a science and the skill is in describing the job right, developing valid queries, attracting candidates, building appropriate psychometrics and tests and making sure the selected candidate actually joins.

The old three-step process is now six: specify, search, recruit, select, appoint and induct. And the job’s not over until all six are complete.

Moving from art to science hasn’t changed the fundamentals though. Absence of future candidates entering education to eventually be in the labour market seeking scientific careers can’t be changed by changes to process. We’re still maybe after super-beings and there will still be a skills and knowledge shortage. But at least employers with vacancies can get a chance to illustrate what life would be like working for them. They’ll be able to persuade highly competent staff to move for increase in job-satisfaction and responsibility, even if the balance sheet won’t allow inflated salaries. If we can’t shake out more degrees of freedom, at least excellent employers can trade easier in the labour market.

Improvement in selection methods is in itself useful. Optimising predictive validity is a good thing. But there’s a spin-off. It’s not all one-sided when a candidate visits an employer. The candidate is evaluating the firm.

Splitting the recruitment phase from the selection phase serves two purposes. Firstly it allows the employer to focus on attracting the candidate at an initial meeting. An informal discussion can take place at which the bias is towards attracting rather than selecting. Secondly it allows a scientifically valid selection to take place at a second meeting.

Research shows that candidates who were put through a robust and valid selection process are more likely to accept a job offer, when compared with those who went through a traditional single interview.

So, while we can’t change the state of skills and knowledge in the UK in the short term, we can change the way employers approach the recruitment task. We can ensure that our firms are survivors and that we get to make choice from an adequate candidate pool using valid selection methods.

Most modern high energy batteries, not only Li-ion, contain highly reactive and potentially explosive chemicals as exemplified by the recent Boeing Dreamliner incident. Testing techniques exist that can predict these problems yet are rarely used and not widely understood. The most important of the tests aim to define the safe working limits: safe temperature, maximum discharge current and maximum safe voltage. Yet some of these parameters are often missing on battery data sheets and the even when they are quoted, there is little supporting evidence.

These important working parameters are often mixed up with the whole range of so-called “abuse” tests that are specified for Li-ion batteries. In reality these particular “abuse” tests are special, both because they are much more fundamental to the use of batteries and also because the data can only be obtained on special equipment, called an adiabatic calorimeter.

The United States’ FAA has listed 132 previous aircraft incidents between 1991 and 2012 that involved “smoke, fire, extreme heat or explosion” in which battery powered devices were implicated and 62 of these incidents involved Li-Ion batteries. The result of the Boeing incident was mostly confined to the battery enclosure and though expensive for the companies involved, it did not lead to any injuries. This will not always be the case and especially when larger numbers of more powerful cells are used, for example in EVs, the situation can be far more serious. Commercial pressures such as the need to drive EVs more quickly, over greater distances and charge the batteries in minutes rather than hours will increase the importance of battery testing.

The importance of testing in an adiabatic calorimeter lies in the fact that it represents a reasonable but worst case situation. When the same test is done, for example to examine limits of overcharging, without such device, the hazard is understated and might even imply the absence of any risk. The other important aspect of custom designed Battery Testing Calorimeters (BTC) is that large batteries (more 50cm x 50cm) and even packs can be tested as they would normally be used and the intensity of the fire or explosion, the amount of toxic and hot gases generated and the speed at which the disaster develops are there to see.

Essentially, these calorimeters allow the heat generated by the mal-function of a battery to be retained within the battery so that its temperature rises in proportion to the heat liberated and thus enables the consequences of malfunction to be realistically and unambiguously measured. In extreme cases, the temperature can continue to rise more and more quickly (often called a thermal runaway) leading to the generation of vast amounts of toxic chemical gases and the battery can physically disintegrate and possibly catch fire too. This is therefore a realistic simulation of an accident, using real battery samples, but performed in a laboratory and possibly photographed.

The most basic adiabatic calorimetry test determines the battery temperature at which problems of thermal runaway start and hence defines the maximum safe temperature. This involves use of a heat-wait-search (HWS) procedure that starts by heating the sample in small steps (see figure 1) and at the end of each step, the system “waits” to see if the battery is generating heat that can be measured by a temperature rise (so called “search” step).

In this experiment a pouch type Li-ion battery was being tested and the “search” procedure starts at around 35^oC and since no heat generation is detected (temperature remains constant) the battery is heated again. This stepwise heating followed by a wait period before search, is repeated until self-heating within the battery can be detected (at around 120^oC); this is essentially the maximum safe temperature.

Doing the same “abuse” test without an adiabatic calorimeter would have three main drawbacks:

-          The correct maximum safe temperature would not be determined. More likely a temperature higher than the safe figure would be obtained (ie the battery would be perceived to be less hazardous).

-          The consequences of the thermal runaway would be understated in terms of severity and speed of incident. For example how hot the battery gets, the amount of fumes generated, the damage to the battery itself and of course how long it takes to produce these conditions would be less severe in a non-calorimetry test.

-          A custom-built calorimeter provides a safe environment for operators to carry out the tests. The absence of such provisions can present a serious hazard to the operators.

While the thermal stability of normal (undamaged) batteries is of huge interest it is also important to determine how the results change under abnormal conditions or when a battery is charged and discharged at too fast a rate. There are huge commercial pressures to speed up charging/discharging; in the context of EVs, charging is the equivalent of filling a tank with fuel and the discharge rate determines the car speed. If the battery is connected to a cycler while placed in the BTC changes in the battery temperature during charging/discharging cycles can be measured. The cycler can be programmed to repeat this operation for many days and thereby also provide information about the longer term stability of the battery.

A Li-ion polymer battery, used to fly model airplanes, was subjected to higher charging and discharging currents than recommended, while inside the BTC. The temperature rose during discharging and fell during charging, but overall there is was a continued rise in temperature and after only a few cycles the battery went into thermal runaway – the temperature being around 110^oC when it did so.

Clearly, at this discharge current, the battery would most definitely need to be cooled to prevent runaway, though the cooling duty is not known from this test.

The overcharing (voltage) test is similar to the over-discharge (current) discussed above except that now the current is at a normal (and safe) value but the battery is charged to higher voltages until it starts to thermally degrade.

Battery technology to tackle current demands, let alone higher energy applications in the future, already has the potential to cause violent fire and explosion incidents. Fully developed testing methods, widely used in the chemical industry for more than 30 years, exist which can reveal the limits of use and demonstrate the hazard. However, at present, far too little approriate testing is being done and as a consequence, accidents will continue to occur. The expensive problems with the Dreamliner could almost certainly have been avoided with a better understanding of the risk through use of adiabatic calorimetery testing with a device such as the BTC.

Author: Jasbir Singh, Founder and Managing Director, HEL Ltd

Contact:
www.helgroup.com

When we set out to move a new tumour-busting chemical entity from its academic origins within the University of Bradford, we knew it would not be easy. Drug development is time consuming, costly, and given that most drugs fail somewhere in development, funding a new drug development programme is inherently risky.

But ICT2588 was designed to kill tumours without causing any nasty side effects and the in vivo data backs this up. The science is great, supported by a well thought out business plan and a credible management team – essential to overcome the risk-averse world of biotech financing. Despite the inevitable difficulties, we were confident that potential investors would be interested. In addition to the tumour-reducing element, other patient benefits include improved life expectancy, fewer side effects, and reduced frequency of treatment, resulting in potential cost savings for medical professionals and less disruption for patients. We just had to ‘de-risk’ the project as far as possible to increase its appeal to potential partners.

In science-based commercial opportunities, ‘de-risking’ is a practice regularly used by companies in order to make an offering attractive to those investors who prefer to avoid anything that is too early stage or doesn’t have a “hot” target. Potential investors who like an idea presented to them will often invite the developer to contact them again when they have more data – at least Phase I clinical safety. This approach has led to the so-called ‘Valley of Death’ for many promising drug developments. The sums required to cross the valley are relatively modest in terms of overall drug development. But new start-ups are immediately limited to a small group of funders who are prepared to invest pre-clinically.  Having a credible management team with the right networks is critical to getting the attention of this small group.  Many promising projects fail at this stage, and the ones that attract funding are those with both appropriate connections and great science.

Developers fortunate enough to secure a meeting with one of pre-clinical funders usually find that the more fashionable and ‘hot’ the drug target is, the greater the interest. In cancer drug development, for example, cytoxic agents are seen as somewhat old-fashioned even though they still represent some of the best cancer therapies available. A “hot” drug target generally equates to something that is popular, with consequent clustering behaviour around a small number of such targets. The latter appears to give both drug developers and investors comfort in that they can see others have already invested in something similar which re-enforces their belief that the development is viable. The downside to this, which seems to be largely ignored, is that you then end up with an unrealistically large number of projects clustered around the same target. This can be further exacerbated by similar clustering around accepted sets of chemistries leading to a densely populated patent space with little room for novelty or breadth of claims.

Current projects in oncology provide a fine example of the fundamentally risk-averse nature of the investment and drug development industries, with the subsequent consequences of ‘clustering’.  Recent analyses of nearly 1,000 cancer projects in the US (PhRMA 2012) show that a mere eight drug targets account for around 40% of all projects in pre-clinical or clinical stages of development¹.

Furthermore, all eight of these targets are kinases. Each of these eight targets was identified as having at least 24 individual clinical programmes, VEGF/VEGFR topping the table with a whopping 70 molecules in the clinic. Add in the known pre-clinical projects and VEGF/VEGFR alone accounts for nearly 150 projects out of the 1000 in total. This presents an issue for developers, as later entrants in the kinase race have to rely on failure of earlier entrants to have any hope of success. Being the 15^th new VEGF inhibitor to market is hardly a reason to celebrate, so the 150^th could find the task almost impossible. The consequence of needing mass failure for your own success is that your target no longer looks so hot.

Trying to sell the technology behind ICT2588 was therefore harder than it could have been because it was unfashionable as a cancer cytoxic; It was too early stage (no clinical data) and it wasn’t in the “good” target cluster. Even worse it had some ‘bad’ target associations – ‘bad’ being basically anything that has failed spectacularly or repeatedly in clinical trials.

Just saying the words “ICT2588 is an MMP targeted cytotoxic pro-drug with vascular disrupting properties” immediately induced a quasi-Pavlovian response in otherwise rational technology assessors. Many potential investors heard the words ‘cytotoxic’, ‘vascular disrupter’, ‘MMP’ and instantly seemed to implement the ‘three strikes and you’re out’ rule.

Strike 1 – Cytotoxics, as mentioned above are generally regarded as old fashioned. To a large extent so are pro-drugs, possibly through association with the much overhyped magic bullet so beloved of the popular press.

Strike 2 – Data from in vivo studies with ICT2588² shows that the cytotoxic effect manifests itself in vascular disruption within tumours. In recent years there has been a succession of vascular disrupting agents (VDAs) that have had serious problems in clinical studies due to cardiotoxic effects.

Strike 3 – Targeting MMPs (matrix metalloproteinases) has a long history of failure. Roche, Bayer Pfizer and BMS all had MMP targeting compounds that failed in Phase III clinical trials. Most significantly in this country, British Biotech progressed 5 different MMP targeting compounds as far as Phase I to III clinical trials. The fiasco that killed off British Biotech over its Phase III results with Marimastat lingers in the minds of many to this day.

So how do you convince an investor (Incanthera now has several) that a pro-drug targeting MMP and acting as a VDA is a good idea? The imperative is to focus on the outcome, stunningly good anti-tumour activity with no systemic toxicity, then deal with the objections one by one.

Scientifically, the matrix metalloproteinases are an interesting family of enzymes that are generally inactive in the healthy adult body. They are involved primarily, but not exclusively, in tissue remodelling and are naturally expressed during wound healing, for example, where they are involved in revascularisation. MMP-associated diseases include arthritis and cancer. In the latter, developing tumours express MMPs in order to remodel their local environment, permitting tumour growth. Growing tumours also require the development of new vascular tissue to supply oxygen and nutrients to the expanding tumour mass. This neo-vasculature also expresses MMPs. Previous MMP targeting drugs have all focussed on inhibition of MMP activity as a way of controlling tumour progression.

ICT2588 is different in that it does not inhibit MMPs but instead becomes the substrate for the natural activity of specific membrane bound MMPs (MT-MMPs). Enzymatic cleavage of ICT2588 by these MMP’s at the site of the cancer, releases the cytotoxic moiety. As can be seen in Fig 1, the central linker in ICT2588 is a nine amino acid peptide attached at the NH₂-terminus to FITC and COOH-terminus to azademethyl colchicine, a colchicine derivative. This peptide sequence has been designed to be specifically cleaved by MT1-MMP and is key to the localised targeting of ICT2588.

The N-terminal FITC molecule acts as an end cap, stabilising the molecule by blocking non-specific exopeptidase activity. This is crucial to the targeting of the molecule as non-specific breakdown would result in systemic distribution of the active moiety, probably leading to unwanted cardiotoxicity. The choice of FITC as an encap was almost entirely serendipitous and was originally used as FITC is designed to conjugate to peptides and is readily available in the lab. Subsequently scores of other end caps have been tried but none have proven as effective as FITC.

Colchicine, as a plant extract, has been used medicinally for thousands of years and is currently, in purified form, used to treat gout. Like many VDAs that have been developed, colchicines bind to the β-subunit of tubulin dimers, causing disaggregation of microtubules. In dividing cells this results in mitotic arrest and subsequent cell death. In vascular epithelium, microtubules play a key role in maintaining the cellular architecture and loss of microtubular structure causes cellular collapse and loss of epithelial integrity.

Xenograft models have been used extensively to show that ICT2588 not only has excellent anti-tumour properties but pharmacokinetically is also very stable systemically with approximately 99% of all free azademethyl colchicine being found only in tumour tissues. Ex vivo work with a number of human tissues including liver and heart again confirmed the stability of ICT2588².

So far, our approach has worked. Since launching in 2010, Incanthera, which is backed by the University of Bradford where its academic founders benefit from the support of Yorkshire Cancer Research and Cancer Research UK, has secured investment from SPARK Impact, managers of the North West Fund for Biomedical. Incanthera has also raised funding from private individuals who share a concern for improving the chances of surviving cancer, and are now in the final stages of securing further investment to progress to Phase 1 clinical trials. Finally, Incanthera recently won Bionow’s Start-Up Company of the Year award.

So ICT2588 may not be fashionable or have a hot target, but maybe it shows that true innovation in drug development means not following the crowd. Certainly ICT2588 shows that MMPs are not dead as a target and that the unwanted toxicity of VDAs can be tamed. As Aristotle said “the whole is greater than the sum of the parts”.

Authors: Dr Kevin Adams, Programme Manager and Dr Simon Ward, CEO of Incanthera (simon.ward@incanthera.com)

references:

1. Booth, B (2012): Cancer Drug Targets: The March of the Lemmings

http://lifescivc.com/2012/06/cancer-drug-targets-the-march-of-the-lemmings/

1. Atkinson, JM, Falconer, RA, Edwards, DR, Pennington, CJ, Siller, CS, Shnyder, SD, Bibby, MC, Patterson, LH,  Loadman, PM and Gill, JH (2010): Development of a Novel Tumor-Targeted Vascular Disrupting Agent Activated by Membrane-Type Matrix Metalloproteinases. Cancer Res; 70(17); 6902–12

When Alice Sheppard looks back to her first few days of classifying galaxies, she remembers initial confusion and apprehension but also experiencing an incredible thrill. Back then, little did she know the impact citizen science would have on her life.

Alice was one of the first recruits to Galaxy Zoo – an online astronomy project that enlists members of the public to assist in the morphological classification of large numbers of galaxies.
In 2007, the Sloan Digital Sky Survey telescope had imaged and released about two million objects – nearly half of which were galaxies. Oxford University PhD student Kevin Schawinski was looking for rare blue, elliptical galaxies in the images but was overwhelmed by the sheer amount of data he would have to look through. “I classified 50,000 galaxies myself in a week, it was mind-numbing.” He told the BBC at the project’s launch. Kevin’s research was a classic example of a “data deluge” where modern research produces vast amounts of information but the teams involved don’t have enough time or resources to analyse it all.

Galaxy Zoo - The general public are shown images of galaxies from the Sloan Digital Sky Survey and asked to classify their morphology

With one million images to classify all by himself, Kevin was drowning in data and desperately in need of a pint. Galaxy Zoo was born after he and his team headed to The Royal Oak pub in Oxford for a few drinks with Chris Lintott (astrophysics researcher at Oxford and presenter of The Sky at Night). There, the academics realised that if they could attract members of the public to help them cope with the abundance of information, the project would be completed far more quickly and efficiently.  Web designers Phil Murray of Fingerprint Digital Media and Dan  Andreescu were enlisted bring the site to life and shortly afterwards, Galaxy Zoo was launched into cyberspace, instructing first-time visitors to take a short tutorial before sorting galaxy images into two main categories: spiral and elliptical. The site was inundated with visitors almost immediately; receiving 70,000 classifications per hour after the first day, leading to one blown circuit breaker and an absolutely astounded team of researchers who estimate there are now more than 250,000 active armchair astronomers from all ages and backgrounds. These individuals with no astrophysical background have created a more detailed map of the universe and their work has given rise to over 30 scientific papers, exciting discoveries and numerous online friendships. Galaxy Zoo has evolved to become the Zooniverse, a web portal which hosts more than a dozen citizen science projects covering a range of scientific disciplines for those who want to try their hand at real research.

Citizen science is simply science undertaken by members of the public that aren’t professional scientists; individuals who voluntarily contribute their time, efforts or resources to research without needing a science background. The presence of massive online scientific datasets and the availability of high-speed internet access to ordinary people are providing new opportunities for citizen scientists from all over the world to contribute to growing our knowledge. There is now a myriad of assignments that ‘citizens’ can get involved with. These could take many forms; some use smartphones and computers to classify images such as Cancer Research’s CellSlider where real images of tumour samples are analysed by users in the form of a simple game of snap. Video-game enthusiasts can choose to contribute to biochemistry with projects such as Fold-It where the objective of the game is to fold the structure of selected proteins using various gaming tools. Or, for a commitment-free contribution, volunteers can even lend their computers’ excess power to solving biological puzzles such as folding@home, or pioneering citizen science project SETI@Home, which harnesses the idle computing time of millions of participants in the search for extraterrestrial life. Some projects even require their participants to put down their modern gadgets and head into the big outdoors to undertake ecological research with do-it-yourself sampling kits.

While it has certainly experienced a renaissance in recent years, citizen science is not exactly a new concept. Amateur scientists have throughout history contributed to academic research1. Even Charles Darwin had correspondents that would write to him about the naturalist work they were undertaking.
“Citizen science is in many senses an old idea – scientists have been asking volunteers to help with their work for centuries, whether it’s amateur astronomers monitoring the skies for supernovae or birdwatchers providing data to ornithologists. What’s changed recently is that in many fields the traditional pattern – amateur data collection and professional analysis – has been reversed in order to cope with the sheer volume, velocity and variety of modern datasets,” explains Chris Lintott, Citizen Science Project Lead.

However, more traditional forms of amateur science do exist with many projects still requiring citizens to go out and collect data. Dr David Jones from Imperial College London and the Natural History Museum runs the Soil and Earthworm Survey at the Open Air Laboratories Project (OPAL), a citizen science initiative led by Imperial College which, unlike Galaxy Zoo, requires people to leave their houses to rediscover the outdoors, motivating them to record local wildlife and habitat data for the first time. OPAL, made possible by a Big Lottery Fund grant, has led to scientists and the public working together to gather a wealth of new data about wildlife, their distribution across the UK and the condition of their habitats. Their networks of community scientists play vital roles in spreading the organisation’s philosophy to all sectors of society. The teams engage with schools, local groups and natural history societies as well as disadvantaged and hard to reach groups and organise local activities to allow people to contribute to ecological research as well as helping citizens to discover and value green areas on their doorsteps they may have not been aware of before. In January, The Community Environment report concluded that more than half a million people across the country have been inspired to discover their local environment through OPAL and the project has mapped more than 25,000 sites across England, including areas never sampled before by scientists.

OPAL's Soil and Earthworm survey

We get a whole host of advantages from citizen scientists, as they do a lot more fieldwork than scientists could do on their own,” says David. “These surveys are designed to ask researchers questions that we are interested in answering. And we get a lot of information from our standardised sampling approach.”

Helping professional scientists with data collection or analysis is obviously a substantial advantage, but what do the citizen scientists get out of it themselves? To find out, let’s go back to Alice, our intrepid galaxy classifier: “I think citizen science is important for two reasons,” she says. “Firstly, it massively increases the amount we can find out; secondly, science is something that involves everybody given the technical world we live in and therefore it’s only right that people get to take part.”
Alice found out about Galaxy Zoo by accident. “I bought myself BANG! A Complete History of the Universe by Brian May, Patrick Moore and Chris Lintott. They had a website where I could write in with questions, which I did. Chris e-mailed me back with great answers, so of course I started reading his website. A few months later, Galaxy Zoo was announced. It didn’t necessarily sound any more interesting than all the other amazing astronomy I’d started learning since reading BANG! And I didn’t think I’d be any good at it, but it was definitely worth having a go.”
Alice jumped into the Galaxy Zoo community and has never looked back. She now moderates the Galaxy Zoo forum, gives regular talks about citizen science, has a blog and runs skeptic events in London. She now juggles galaxy hunting and public engagement commitments with a Masters degree in Astrophysics.
“If I hadn’t discovered Galaxy Zoo, I think I’d still be looking for a home. I know that sounds over the top, but finding it really was like finding a home: scientifically curious people and a real mission. It’s set me on my career path and found my greatest interests.”

Dr Arfon Smith, technical lead at Zooniverse believes citizen science allows the public to become aware of how the scientific process works. “Citizen science improves public scientific literacy and increases the transparency about how science really happens. With projects like the ones we coordinate at Zooniverse, the way the public see how science happens is inherently opened up.”
In order to successfully design future citizen science projects, Arfon and his colleagues wanted to find out their participants’ motivation for taking part in Galaxy Zoo. The organisation added a survey to their website, encouraging participants to share why they wanted to be involved in the first place.
“We discovered that they wanted to contribute to real research projects and this eventually led to the Zooniverse: projects where people would be interested in doing better than machines.”
As Arfon points out, many citizen science projects, including Galaxy Zoo, exist because of the limitations of current technology – the fact that computer algorithms are currently not as good at recognising patterns as humans are. In several of these projects, researchers are using humans to teach machines, to bring them up to scratch.
“The point at which we need people is a shifting land camp. We can come up with algorithms but only if we’ve used people to make the decisions cognitively first. There will come a time when projects may be stopped because computers are as good as human pattern-recognition, but there will always be areas that will lack algorithms or will need human intervention first,” says Arfon. “Computers will slowly get better at classifying galaxies, but looking at an image and asking, “What’s that odd thing” remains uniquely human and we’ll always need that. Projects like these are a really good example of humans and machines working together.”
Chris echoes this outlook and says that he’ll stop running projects when computers can complete the task adequately, because they’ve learnt from participants’ pattern-recognition.
“In some ways the ideal is our supernova hunting project where new data provided by citizen scientists inspired the further development of machine learning to the point that the volunteers were no longer required. To me, that’s a successful outcome. As for how long there will remain things computers can’t do – I think that’s a bet on the race between the speed with which data sets are growing and the increasing percentage that computers can handle. I think that we might see that as datasets grow we’ll need humans to handle an ever smaller percentage but the total amount of work needed might stay the same.”

Cell Slider - Volunteers use images from Cancer Research UK to help to classify archived cancer samples

A symbiosis between humans and machines is certainly something to think about, but citizen science also raises questions about what it means to be a scientist these days. For all its advances, crowd-sourced science is a concept that has not been without criticism; David Weinberger from the Berkman Centre for the Internet and Society at Harvard has notably said: “These people are not doing the work of scientists…They are doing the work of scientific instruments.”

Arfon contests views similar to Weinberger’s: “I would disagree that being a scientist is solely about interpretation. A lot of the scientific process is about doing the hard work, which is what our volunteers are doing. It’s a really easy criticism to say these people aren’t really scientists, but I’m not entirely comfortable with any definition of a ‘scientist’ that I’ve come across yet.”
Likewise, Alice is not hugely interested in getting bogged down with definitions and wants to focus on the long-term outcomes of citizen science: “What I really hope for the long run is that people will take more ownership of science. Science is something lots of people love, but a lot more remember as a miserable time at school – and they feel convinced they’re not clever enough to understand any of it.”

Regardless of whether armchair astronomers and the like can be counted as ‘scientists’ amongst experts in the field, one thing is clear: these days you do not need to have a scientific background, have access to a laboratory or even leave your bedroom to contribute to real-world research. If executed effectively, citizen science can also be a great education and outreach tool, as well as providing space for a meaningful collaboration – something that conventional scientific research is built on. Chris says: “I think citizen science is a reversion to an old model in which it was possible for people from a wide range of backgrounds to feel like they were making a contribution to science – not just those of us with PhDs and university positions. I think the real magic of citizen science is that such authentic engagement is possible at every stage of learning about science, rather than just at the end.”

Reference: Silvertown, J. 2009. “A New Dawn for Citizen science,” Trends in Ecology and  Evolution, 24, 467

Imagine that you’re a field technician for a public water provider in a developed nation. Your company is committed to supplying clean, safe drinking water to millions of people, so it has implemented a sophisticated water sampling program to ensure water is free of contaminants. Across this water company’s territory, technicians just like you are collecting and testing samples from reservoirs, water treatment facilities and even customers’ homes.

Today you’re collecting samples from various points around a lake that supplies water to one of your company’s main processing facilities. After filling each sample bottle, you transcribe its barcode into your notebook. You check your watch and jot down the time. You remember to add a preservative that ensures the sample gives accurate readings after it has been transported to the lab. Finally, you pull out a GPS unit and note your exact location coordinates. When you get back to the lab, all of this information – recorded manually – will need to be re-entered into a database and combined with the test results.

As you pack up your notebook and GPS, it hits you: All of this data could more easily be collected – not to mention transmitted back to the lab – on a single device: the iPhone in your pocket.

Mobile devices are an increasingly critical component of modern life, and that trend holds true for laboratories as well. Regardless of industry, the incredible (and constantly evolving) features on these devices can enable technicians to easily capture new types of data more accurately and from more remote locations, but they also pose an interesting challenge. How do laboratories ensure that all the data collected via mobile devices is accurate, secure and organized? The answer is a laboratory information management system (LIMS). Just as a LIMS enables an automated, paperless environment inside the lab, it can integrate with mobile devices in the field to ensure that data collection, transmission and analysis are fully optimised.

The most straightforward benefit mobile devices bring to the lab is, quite simply, more data. The more data that labs have available, the more effective they can be, provided that the data is organized and accurate. That’s where the LIMS comes in. Going back to the example of the water sampling technician, if his camera, GPS, wristwatch and notepad were replaced with a company-issued smart phone, he would be able to record all information on a single device. This not only saves the company money (by purchasing and servicing a single device instead of several), it also improves the technician’s accuracy because he no longer needs to rekey data based on his notes from the field. It also walks him through all the necessary steps when collecting the sample – such as adding the preservative – to ensure consistency.

A LIMS enables users to transmit information directly from the field into the database, eliminating error-prone manual transcription. The ability to go completely paperless is no longer bound by the physical constraints of the laboratory itself. The LIMS ensures that location information, barcode reading, precise timing and any other data the technician collects are linked directly to the test results. In other words, combining mobile devices and a LIMS brings a sampling program’s entire chain of custody under one secure umbrella. This enables improved regulatory compliance, traceability and auditing, of course, but it also makes for better management.

One of the greatest advantages of mobile devices is that they’re a two-way street. Not only can users submit data to the LIMS, they can examine and look at data on a device without physically entering the lab. Modern LIMS offer connectivity with mobile devices that allow lab personnel to visualise results from high-level trends down to granular details.

For labs performing extensive chromatography runs, for example, data must constantly be monitored to ensure results match up with reference data. Scientists aren’t interested in general results alone, they want the ability to drill down to the level of individual components and peaks. A LIMS provides access to interactive data – not just a static image such as a JPEG or PDF – from a mobile device, allowing the lab to run far more efficiently. The same goes for automated alerts on sample runs – mobile devices linked to the LIMS allow users to make a decision from a remote location about whether an outlying result requires a retest or a full investigation, preventing unnecessary delays.  Because of these visualisation and connectivity advances, laboratory personnel are free to be far more creative as they design workflows. Data management is no longer a limiting factor in the laboratory; instead it is a driving force for innovation.

Another way mobile devices increase the amount of data available to lab managers as they create workflows is by enabling additional users to submit information to the LIMS. For example, a loading dock worker who has never heard of LIMS – let alone been trained on his organisation’s LIMS of choice – can use a mobile device to scan barcodes on shipments of samples or other materials, then upload that data directly to the LIMS. This works especially well for correcting issues with suppliers, since shipments can be checked for broken or missing items and compared to the shipping manifest at the loading dock without the need for a lab technician to get involved. A loading dock worker can record photographic evidence and store it in the LIMS until the problem has been resolved.

The benefits of mobile devices are undeniable, but new technologies also present new challenges for laboratories. Perhaps the most obvious of these is the “bring your own device” (BYOD) trend: Employees, from the C-Suite to individual technicians, now have preferred mobile devices. If a personal device has a certain capability that a user likes, why shouldn’t it be used in the workplace?

Data management is no longer a limiting factor in the laboratory; instead it is a driving force for innovation.We’ve passed the tipping point for mobile device adoption – at least in our personal lives – and it’s time for the revolution to move into the workplace.

As IT professionals know well, BYOD brings a host of issues, from information security and regulatory compliance to software compatibility. But these concerns have done little to stem the oncoming tide of hybrid personal/professional devices. Once a CEO becomes accustomed to his or her home tablet, there’s no turning back. Laboratories have so far typically avoided BYOD policies, especially in life sciences industries, due to sensitivities around intellectual property protection. In pharmaceuticals labs, for example, the risks involving counterfeit drugs are too great – and the regulations too rigorous – to allow technicians to use personal mobile devices to submit or analyse sample data.

Technologies which address these concerns are becoming more widely available, allowing companies to segregate corporate data from personal data on mobile devices. All company-related activity occurs in a “sandbox” over which the IT organisation has full control and which they can wipe remotely if the device is lost or stolen or if an employee leaves the organisation. This satisfies intellectual property and security concerns while allowing employees autonomy over their personal data. Enterprises are already using such technologies with company-issued devices, but it is clear that they have even greater applicability in BYOD scenarios.

Another challenge in integrating mobile devices with a LIMS is setting expectations. As anyone who has visited an app store can attest, devices are extremely well-suited to highly focused applications but not as strong when it comes to broad capabilities. Manual data entry, for example, is not an efficient use of a mobile device. That’s why some LIMS offer streamlined connectivity to mobile devices. The key is to design application workflows that are well-suited to the mobile form factor and that take advantage of these devices’ capabilities. With this approach, users quickly see the value in the right context.

While nearly all labs will integrate mobile devices with a LIMS in the future, some companies are still reticent. So which industries are early adopters? Process industries, such as water utilities and oil & gas, which require remote sampling across large plants or geographies, have been among the first to get on board. Using mobile devices to scan bar codes or radio frequency identification tags and submit samples to the LIMS gives management access to more data in near-real time. This timely information allows a company to be far more nimble, while oftentimes producing superior products.

Even regulation-saddled life sciences companies have begun using LIMS to integrate mobile technologies within the lab (albeit through company-supplied devices only) in both R&D and production environments. When samples are thawed in preclinical testing, for example, they lose some integrity, so pharmaceutical companies are now using mobile devices to scan barcodes each time a sample is removed from a freezer or replaced to be refrozen. While this could be done manually, as has been the common practice, readings wouldn’t reach the LIMS automatically and freeze-thaw events would often be missed, providing an incorrect evaluation of the sample’s condition.

Environmental monitoring in life sciences, which has sampling requirements outside of the laboratory, is another perfect fit for mobile devices. Mobile device/LIMS integration improves testing by providing precise timestamps and more timely submission of results. Issues can be detected early on, before an entire batch of product is contaminated.

The future importance of mobile devices in business is unquestioned. We’ve passed the tipping point for mobile device adoption – at least in our personal lives – and it’s time for the revolution to move into the workplace. For laboratory employees, this will be an exciting transformation, bringing new levels of efficiency and innovation. Integrating mobile devices with the latest LIMS technology is merely the first step. But given how it promises to speed sampling, improve data accuracy and provide faster access to critical information enterprise-wide, it’s an important first step that is worth taking right now.

One of the principle patterns of animal social behaviour is that males compete for females whereas females look after the young. In a number of insects, fishes and birds, however, these conventional sex roles are reversed: the males look after the eggs and young, whereas the females compete for males. In the latter species the females are often larger, more ornamented and more pugnacious than the males, whereas the males may have specific adaptations to look after the offspring. These sex role reversed species have caught the eyes of various naturalists including Charles Darwin since they appear to defy behavioural features that are often associated with “maleness” and “femaleness”.

Why sex role reversal evolved has puzzled sociobiologists and evolutionary biologists ever since Darwin, since it is not clear what ecological and life history variables have triggered the conventional sex roles to flip into sex role reversal. A large number of hypotheses have been proposed, for instance food availability may be limited for the female so that she is exhausted by producing the offspring, and thus she delegates the caring role to her mate so that she can replenish her resources. The empirical evidence, however, is scant for all of these hypotheses, since no environmental or life history variable has been found that would consistently distinguish the conventional species from the sex role reversed ones. Indeed, conventional and sex role reversed species often breed side by side in so contrasting habitats such as tropical marshes and Arctic tundra where they must share much of the ambient environment.

Recent theoretical models, however, emphasise the impacts of social environment on sex roles. Theory predicts that when there are many males in a population, the female should capitalise from the enhanced mating opportunities and abandon her family to seek out a new partner. For the male, however, the best is to stay with the offspring. The male behaviour has nothing to do with sacrificing his own Darwinian fitness for the sake of his partner: he would simply face stiff competition should he leave her and the offspring. Therefore, the best option for a male at male-biased sex ratio is to stay put and make most out of his existing family.

We recently tested these propositions using shorebirds (plovers, sandpipers and allies) as model organisms1. Whilst no shorebird has been studied to the same details as any of the traditional genetic or developmental model organisms (e.g. domestic mouse, zebra fish, fruit fly or Caenorhabdis elegans), shorebirds have an immense advantage over the latter model organisms having extremely variable sex roles that are well documented in nature. Shorebirds have diverse and well-studied courtship behaviours, and their body size dimorphism – an indicator of sexual selection – ranges from male-biased size dimorphism to female-biased size dimorphism. In addition, most shorebirds breed in open habitats that can be easily surveyed and therefore, assessing their social environment is less error prone than in other species that breed in dense habitats or in places that are difficult to access.

As theory suggested, we found strong relationships between adult sex ratio (an indicator variable of social environment) and sex roles. In populations where males outnumbered females, sex role reversal was common: the females competed for males whereas the males looked after the young. In female-biased populations the opposite pattern prevailed: the males competed for access to females, and the females looked after the young. These phylogenetically controlled results were robust to different phylogenetic hypotheses, and remained persistent in sensitivity analyses.

These exciting results have implications beyond birds, and suggest that adult sex ratios may influence courtship, mating and parenting behaviour in many organisms including humans. For instance, with increasing bias toward more men in the population, one may expect increased social pressure by unmated males since seeking out and competing for females often result in violence. Females may also respond to biased adult sex ratios by being more demanding in partnering relationships given that they have more man to choose from.

The study raises two intriguing questions. First: how does biased adult sex ratio emerge in the population? Adult sex ratios have not been studied in vast majority of organisms, and to answer this question one needs to investigate the genetic, developmental and physiological mechanisms that create male-biased sex ratios in some populations whereas in others female-biased sex ratios. Pilot studies from birds, fish and mammals suggest that closely related species can exhibit vastly different adult sex ratios, and these different sex ratios may have immense impact on social behaviour. Second: is the observed relationship between sex roles and sex ratios indirect and does it emerge via numerous incremental steps, or a direct (causal) relationship? To test the latter proposition one needs to manipulate adult sex ratios experimentally, and find out how the shift in sex ratio induces courtship behaviour, mating systems and parental behaviour.

Taken together, the recent work by Liker et al. has opened a Pandora’s Box in social evolution by showing that adult sex ratio predicts sex roles in birds. Further studies are urgently needed to establish the causes of adult sex ratio bias in natural populations, and to explore the full implications of sex ratio bias for social behaviour including pair-bonding, courtships and parenting.

Reference

1. Liker, A., R. P. Freckleton & T. Székely. 2013. The evolution of sex roles in birds is related to adult sex ratio. Nature Communications (in press).

Author: Tamás Székely Department of Biology and Biochemistry, University of Bath. Tamas is an evolutionary biologist interested in evolution of social behaviour. His team is investigating mating system and parental care in birds.

Contact:T.Szekely@bath.ac.uk

 Many people may be settling down over lunch to read their copy of Laboratory News. Those of you who have popped out for a sandwich, packet of crisps, yoghurt and a piece of fruit might find it surprising that a universal materials tester could have been involved in testing every part of your lunch, including the credit or debit card you used to pay for it!

Figure 1. Single and twin column materials testing machines

More about ‘lunch testing’ later, but first, let’s take a look at the basic principles of materials testing. Universal materials testing machines allow precisely controlled tensile or compression forces to be applied to the sample under test over a controlled period of time. Precision loadcells measure the material under test as a function of force against extension and time. Materials testers are available in a variety of sizes, and ar

e defined by the maximum force that they can apply (Figure 1). Bench mounted single column instruments can typically provide a maximum force from 1 kN (225lbf) to 5 kN (1124 lbf). For higher forces, twin column instruments are needed, going to floor-mounted versions capable of delivering a maximum force of hundreds of kN. Robust, high stiffness load frames are at the core of each materials testing system, with modern linear guide technology, pre-loaded ball screws and advanced software compensation ensuring high displacement precision. Depending on the machine, elongations of between 1 micron and 2.5 m (98.4 in) can be measured, meaning tests can be performed on anything from a human hair to large components as diverse as complete car seats, large cardboard cartons, mattresses and large diameter pipes. Although all materials testers basically apply a controlled compressive or elongation force, the secret to the versatility lies in the sample holders and jigs and fixtures. For example, support a sample at either end and apply a compression force to the middle and the system measures bend strength! Table 1 indicates just some of the tests that can be performed using a materials tester.

As materials testing is a well-established technique, it comes as no surprise that a vast range of internationally recognised standards (ASTM, ISO, FINAT etc) exist. Most modern machines will have a number of these standard operating methods stored in a software library so that they can automatically be called up when required. Although materials testing is well known as a laboratory technique, it can also be used throughout the design, development and manufacturing process, from the evaluation of raw material to the finished product. It can be used to test pretty well any material you can think of. One special branch of materials testing is found in the food industry, where the instrument can be used for texture analysis.

Texture analysis testing evaluates the mechanical and physical properties of raw ingredients, food structure and for pre- and post- Quality Control checks. Texture testing has applications across a wide range of food types, including bakery, cereals, confectionery and snacks, dairy, fruit and vegetables, gels, meat, poultry and fish, pasta and even pet food. Since texture is a characteristic relating to sensory perception, it is a property that can be measured easily by mechanical methods in units such as force, distance and time. In texture testing, standard tests such as compression, tension and flexure are used to measure hardness, crispiness, crunchiness and other properties listed in Table 2.

Figure 2. Compression testing of bread

Comparison of the results from texture analysis together with trained human sensory panels has shown that the measurements correlate well with the various sensory attributes associated with textural quality. For natural products, such as fruit, vegetables, meat and fish, texture can be related back to the way the product is grown, or reared, while for processed food, the texture can be used to optimise the process. Texture analysis can highlight quality improvement opportunities throughout the supply chain and the production process.  At the research and development stage, new or alternative ingredients can be compared with existing ingredients. In production, texture analysis is used for the measurement and control of process variations such as temperature, humidity and cooking time. As with ‘conventional’ materials testing, the key to the versatility of the texture analysis technique is the availability of huge number of grips and fixtures in a variety of sizes, gripping surfaces, styles and capacities.

Lunch testing

As mentioned above, a materials tester could have been used to test virtually everything associated with that lunch, which you may have finished by now!

Let’s start with the credit card used to pay for it. A special jig with an upper wedge grip and lower fixture containing rollers is available for peel strength testing of laminated layers of standard plastic identification cards. The jig allows 90˚ peel tests to be made at forces up to 200 N (44.96 lbf) for the measurement of peel strength between card layers in accordance with ISO/IEC 10373-1:2006 (E). The strip to be tested on the card is attached to an adhesive tape loop. This is passed through the roller assembly and held securely by the upper clamp so that the face of the card rests on the underside of the rollers. The grip therefore clamps the prepared card test layer so that a peeling angle of 90° is maintained during measurement. Up to four test strips per card may be prepared and tested.

Figure 3. Cutting resistance of cheese

Moving on to the sandwich, this probably arrived in plastic or cardboard packaging, and during design and manufacture, these packages could have been tested for properties such as tensile strength, puncture resistance, heat bond peel strength and tearing strength. The bread in the sandwich can be tested for firmness according to AACC (74-09) (Figure 2) and the spreadability of butter, margarine and other spreads can be tested. The cutting resistance for a filling such as cheese could be measured to ISO16305 (Figure 3).

For crisps, measurement of the crispiness and fracturability of crisps can be made using a penetration test (Figure 4), whilst burst strength, opening strength, seam strength, tear and peel are all parameters that can be measured on the crisp packaging.

For yoghurts, the consistency of yoghurt can be measured and a special jig is available for the measurement of the peel strength of yoghurt pot lids. When designing food container lids, a balance must be struck between a strong effective seal that prevents food contamination, and one that is easy to peel back. This jig consists of an angled platform that is positioned on a base plate and locked securely into position to suit the container. An upper fixture, connected by a non-elastic cord to the testing machine, grips the foil lid and the machine measures the peel forces in tension up to a maximum 500 N (112.5 lbf).

Finish off the lunch with a healthy fruit option, and texture analysis could also have played a role, since there are many factors that affect the texture of fruit and vegetables. The time of harvest and storage conditions have an effect on the rate of softening. Texture analysis can help determine the physical properties of fruit and vegetables, and how they change during ripening.

 There are plenty of other potential applications for materials testing and texture analysis even within the ‘lunch’ example we have given, so it is clear that this equipment offers extraordinary versatility. It is no exaggeration to describe it as a universal technique.

Figure 4. Fracture testing of crisps

Author: Carl Bramley, Export Sales Manager, Lloyd Instruments Ltd.  Bognor Regis, West Sussex,

Contact:

t: +44 1243 833 370

e: carl.bramley@ametek.co.uk

Single-cell microinjection is a powerful and versatile technique for introducing exogenous material into cells, or transferring cellular components between cells. It is normally performed using a fine glass capillary, under a specialised optical microscope setup incorporating a micromanipulator. Microinjection is frequently used in genetic engineering and transgenetics to insert genetic material into single cells. It is also used in basic genetics research, in the cloning of organisms, and in the study of cell biology and viruses. Common clinical applications include In Vitro Fertilisation (IVF) and Intracytoplasmic Sperm Injection (ICSI) to help achieve fertilisation for couples with severe male factor infertility. Other areas include ophthalmology procedures such as Retinal Pigment Epithelial (RPE) and Intra Occular (IO) injection

The technique of needle microinjection was first developed in the early 1900s. It has become an increasingly popular biological research method for studying living cell systems, being one of just a few viable ways to introduce non-genetic, large molecules into living cells. Microinjection is now routinely used to transpose cellular organelles, DNA and RNA, enzymes, structural proteins, metabolites, ions and antibodies, from test tubes into living cells. Other techniques such as vesicle fusion, scrape loading and electroporation have been introduced but using needles for cellular injection provides the most versatile option and the ability to be quantitative.

In the 1960s key biological studies focused on microinjection of organisms such as amoebae and mouse embryos. Following this, research expanded to investigate the use of microinjection to deliver organelles and molecules to other cells such as Paramecium, frog and mouse oocytes and eggs and mammalian cells of somatic origin, forming the basis of the future use of microinjection for the production of “transgenic” animals.

Early microinjection experiments with Xenopus oocytes studied gene expression relating to the molecular recognition pathways of oocyte maturation. Further exploitation of the technique identified receptors and ion channels of cloned genes from mammalian cells. In fact, oocytes as microinjection vehicles were instrumental in enabling expression cloning of numerous important neuronal receptors. Isolation or identification of desired clones became achievable by measuring newly expressed receptor activity, following injection of large pools of cDNAs from mammalian cells.

Today, microinjection is widely used for the study of different cell responses in a variety of systems. These include individual cell signalling responses in the regulation of metabolic pathways, second function of second messengers, the fate of injected proteins and cytoskeletal regulation, definition of apoptosis and survival signals and transformation of whole organisms such as nematodes.

Perhaps the most powerful aspect of microinjection is the ability to introduce several types of reagents into cells simultaneously, including DNA constructs, labelled dextran to mark the injected cells, antibodies, short interfering RNAs (siRNAs), and peptides. No other techniques currently available provide these capabilities.

Microinjection is a relatively complex and technically challenging method to perform, requiring specialist equipment and trained experimental technique. The work is done under a powerful microscope with a pipette holding the target cell in the field of view. The tip of a glass micropipette/injection needle (about 0.5 mm diameter), is injected through the membrane of the cell. The contents of the needle are delivered into the cytoplasm and the empty needle is gently removed.

Xenopus eggs and embryos have been widely used for microinjection studies in the fields of cell biology and developmental biology. Their use is cited in thousands of publications. Due to their extremely large size, microinjection into Xenopus cells is somewhat less demanding, requiring little practice before the researcher becomes proficient with the technique.

Due to the delicate nature of the technique, limiting the amount of vibration to the workstation is vital for effective, reproducible microinjection. It requires a suitable quiet location, away from sources of vibration (slamming doors, fridges, centrifuges etc.), fluctuating temperature, draughts and bright light. Select a sturdy table or bench with plenty of room around it, and ensure that the workstation components, including trailing wires and tubes, are not in contact with the floor or walls. If vibration is still detected when a micropipette is placed into the microscope field under high magnification, use of an anti-vibration table should be considered.

Microinjection applications are typically performed using an inverted microscope – the objective lens turret is located below a fixed stage, with the transmitted light source located above. The working distance of the condenser, measured from its bottom-most part to the stage, after the condenser has been adjusted correctly, is particularly important. To enable access and unobstructed movement of the micromanipulator headstages a “long” or “ultralong” working distance condenser should be used. Stage attachments, specimen holders etc. may need to be removed so they are not in the way. The microscope should have sufficiently good optics to be used for several hours without causing eye strain. A halogen light source with flexible fibre-optic arms is non-intrusive and provides a cold light that does not heat up the media in the culture dish. The addition of a camera and TV monitor should be considered and is essential if the system is to be used for teaching or demonstration purposes.

A micromanipulator is required to ensure precise positioning of the injection needle and to facilitate the delicate movements during the injection procedure. It is used to position and move the micropipette in proximity to the tissue to be manipulated or injected. The micromanipulator is securely mounted to the microscope using an appropriate adapter. Ultimately the success of any microinjection will depend significantly upon the stability of the micromanipulator mounting. For greatest precision, the micromanipulator should position the injector at approx a 45 degree angle to the injection dish.

A microinjector device provides the pressure needed to deliver the sample solution from the micropipette into cells. Depending upon the application being employed, different pressures may be required. Xenopus embryos are typically injected with volumes ranging from 5-50nl so this needs to be done very accurately and precisely. The microinjector can be a mechanically or electronically regulated air pressure device or a simple glass syringe where the plunger is screw controlled for precise adjustments of the pressure. There are various manufacturers of such equipment but one of the leading suppliers is Drummond Scientific.

The manual oocyte microinjection pipette has a non-rotating plunger that eliminates tip wobble, to allow more precise deposition and enables injection of 30nl or more with reproducible accuracy. It utilises capillary tips that must be heated until the glass becomes partially liquefied and then quickly stretched to form a very fine tip at the heated end. The tip of the pipette is about 0.5mm diameter and resembles an injection needle.

Using a manual microinjector clearly needs a very steady hand and considerable skill and patience. To facilitate complete control, automated microprocessor controlled instruments such as the Drummond Nanoject II Autoinjector have been specifically designed to perform ultra-delicate nanolitre injection procedures into cells, including oocytes and embryos.

The configuration of any microinjection system will depend upon the application for which it is used with the key differences being the needle size and pressures applied.

Needles are often prepared from borosilicate glass capillary tubing. To make an injection needle, the glass capillary tube is heated until molten in the middle and is stretched by weights to create a very thin, hour-glass shaped taper, about 30mm in length. The finely drawn out capillary is bent using fine forceps to identify the point where the needle resists bending. The forceps are used to very gently break the capillary at an angle at approximately the point of flexure. The ID at the tip of the needle should be around 10-30 microns. It is then completely filled with mineral oil to act as a non-compressible displacement medium. By using a hydraulic fluid instead of air in the syringe and tubing, an even greater level of control is attained.

For certain applications such as the injection of single cells into late-stage embryos, it may be desirable to inject volumes less than 2.3nl. In this case, it is necessary to use one of the gas-pressure regulated microinjectors that allow injection of extremely small volumes and permit continuous variation in injection volume. With these devices, it is necessary to calibrate the injection volume for different needles and for different pressure conditions, Suitable devices are supplied by Narishige Picospritzer, Harvard Apparatus, Sutter and others.

When using an automated microinjector, a foot pedal injection control is a valuable addition helping to keep the hands free and allow the user to pay constant attention to the cells.

Microinjection Applications
There are many documented applications for the technique but some of the most popular uses are listed here.

Zygote pronuclear DNA microinjection
The microinjection of DNA into the pronucleus of a newly-fertilized mammalian egg is now a
common and highly efficient method for creating transgenic offspring. Pronuclear microinjection was first described in the mouse, but now many different transgenic animals have been created in this way.

Embryonic stem cell transfer into blastocyst
Animals, usually mice, can be engineered with a specific gene function reduced or knocked out altogether by introducing genetically altered embryonic stem cells into the cavity of a blastocyst so that the stem cells contribute to the embryo. The resulting live animal is a chimera of both genotypes. Subsequent selective interbreeding of manipulated animals results in pure-bred gene“knock-outs” or “knock-downs” and can be used for subsequent gene function studies.

Somatic cell nuclear transfer
The enucleation of an oocyte followed by the transplantation of a somatic cell is a method of producing genetically identical copies (clones) of the animal from which the donor cell was taken.

Intracytoplasmic Sperm Injection
Intracytoplasmic sperm injection (ICSI) can be employed for veterinary in-vitro fertilization during rare species preservation or for any veterinary assisted conception. ICSI may also be used as a gene transfer technique when sperm are co-injected with exogenous DNA.

There is no doubt that microinjection is a valuable research procedure and the availability of affordable instrumentation makes it more accessible now than ever before. The development of tools and microanalytical techniques is likely to continue to make the process simpler and even more versatile. When combined with methods such as PCR the scope of possible microinjection experiments will further expand.

Author: Susan Mayfield is Product Manager for Liquid Handling at Alpha Laboratories
t: 02380 483000  w:www.alphalabs.co.uk

200 times stronger than steel and just one atom thick, graphene is the strongest and thinnest material ever measured, and also the world’s most conductive material. The National Graphene Institute will include two separate cleanrooms, laser, optical, metrology and chemical laboratories, seminar room and offices and ancillary accommodation.

The building is a compact 4 storey cube that occupies the full site foot print. The main cleanroom is located on the lower ground floor to achieve best vibration performance. Offices and labs are intermixed on all floors with most of the labs and all the offices having views and daylight. A top lit double height breakout space will link two floors providing welcome respite at the heart of the intense working environment and a roof garden will also form part of the top floor seminar room and social and public area.

The building is enclosed by an economic inner skin comprising a proprietary composite cladding panel system that provides weather tightness and thermal insulation and accommodates flush windows and other openings as required. Fixed to the outside of this inner skin is a separate perforated stainless steel ‘veil’ which wraps around the volumes of the different elements of the building continuously to provide a unifying texture and coherent, fluid shape.

The NGI will be designed in accordance with the current European Standards and associated national annexes and building regulations. These standards will consequently provide a design life of 50 years for the structure and generally define appropriate loadings for the specific use of the various spaces.

The most important overriding design feature requested by the users though was the assurance that the building will provide the requisite structural stability against vibration. This is clearly an important consideration for a research environment in which much of the activity will be carried out at the nano scale. The structural design therefore is heavily influenced by the need to achieve the necessary vibration criteria at the basement and first floor levels, where the cleanrooms are located, as well as the need to prevent EMI (electromagnetic interference) transmission. A structure with suitable mass to provide stiffness, damping, robustness and stability is required. The balance between vertical stiffness for vibration control, layout and economy has resulted in the adoption of a 6.6m by 6.6m structural grid.

The vibrations within the university cleanroom, at basement level, are required to be below VC-D, while the smaller first floor cleanroom, should not exceed VC-B. Achieving the VC-D vibration criteria for the main cleanroom is critical to the success of the project.

The results of the vibration survey indicate that traffic on the adjoining Booth Street East is the primary source of external vibration. The University is currently investigating options to have the road resurfaced prior to the NGI becoming operational and to put in place a maintenance regime that will ensure rigorous control of any future excavation works in the road.

Internally, the lifts, and the equipment within the Central Utilities Building (CUB) which houses the majority of the plant servicing the main building could transmit vibrations into the rest of the structure, so it has been designed as a separate block, physically isolated from the main building by a separation joint and a double line of structure. The slab thicknesses, column spacing and locations of shear walls also ensure that any locally generated vibrations, including footfalls, are not transmitted to the tools, which are themselves mounted on vibration dampers to further reduce any risks of vibration.

The Cleanrooms, which are the focus of the facility, are located on the lower ground and first floor levels of the facility, with the lower cleanroom maximising the available VC-D vibration criteria with a ground bearing slab over a stable layer just above bedrock at 4m below ground. The upper cleanroom is designed to provide flexible VC-B vibration criteria, which will accommodate future expansion. Both cleanrooms are immediately adjacent to the central utility building (CUB). They will be the most highly controlled spaces in terms of contamination, temperature and relative humidity.

The adjacency of the cleanrooms to the CUB allows for shorter distribution routes improving the efficiency of the services design and overall cost of the facility. Support accommodation associated with the cleanrooms such as gas stores/ hazardous waste and bulk gases are located in close proximity to the cleanroom, along with the cleanroom technician’s office.

The preferred option adopted for the lower cleanroom consists of a ‘bay and chase’ layout served via a central access/move-in corridor vertically, the clean room comprises a 1.2M raised access floor, with 3.0M internal cleanroom ceiling height, and a 3.0M plenum zone. There is a continuous viewing corridor along two sides of the cleanroom which allows visitors to view activities within without having to gown up. Its double height also allows views from the pavement outside down into the cleanroom.

The same cleanroom construction materials for the walls/ floor/ ceiling and doors will be used for the upper cleanroom (intended for industry collaborators) as the lower cleanroom. Both cleanrooms will share a primary, although restricted, gowning Area with defined protocols for cleanliness and a ‘cleaned lift’ connecting them. The supply air system utilises a clean plenum, fan filter units (FFU’s) and sensible cooling coils for flexibility and efficiency.

Professor Novoselov had two particular requirements concerning the laboratories: they should be flexible and easily adaptable to future requirements, and they should be interspersed with the offices for the researchers to make it convenient for experimental work and write up/documentation, and also so that different research teams could be holistically accommodated easily in the building.

Laboratories are set out in modules measuring approximately 6.6m wide (minus the wall construction thickness).  This is consistent across all of the laboratories and assists with the interchanging of flexible furniture. The overall typical module itself is designed to achieve the client’s brief of min 42m2, although due to the structural grid the majority of laboratories achieve an area of approximately 55m2.

First and second floors of the facility house a mix of flexible open plan laboratory space opening onto offices on one side and fixed modular laboratories on the other. This offers maximum flexibility in the use of the space being adaptable to suit many types of research. A number of laboratories are located on the external perimeter of the building allowing natural light to penetrate the space. Light and view were felt to be very important to the laboratory work environment wherever this possible. The larger, open plan labs also benefit from borrowed light and views through the glazed partitions to the adjacent offices. More demanding laboratories in terms of service requirements such as the chemistry and furnace room are located on the third floor of the building below the roof level, reducing the run of services and therefore cost.

The internal modular laboratories within each floor level are primarily located within the centre/ core of the building and are positioned back to back with a central shared grey space running the full vertical length. The laboratory walls including the walls of the grey space will be constructed using a system which will support wall mounted shelving and equipment, as requested by the users for future flexibility. Centrally located laboratories also offer vertical flexibility in terms of connection between lower and upper laboratories/ grey spaces to ease service connections and sharing of lab support equipment.

Each laboratory zone will also have provision for creating future cores within the structural floor slab to allow for interconnectivity between laboratories and grey spaces as mentioned above. Each modular laboratory will also have a double door entry for equipment move in and user access, of a suitable height (circa 2.4m). The laboratory will provide a clear floor slab to soffit height of 4m within the centre of each room to allow the location of future equipment, with all services coordinated to ensure this is achieved.

Bespoke modular benching with integrated shelving will be located around the perimeter of the room with clear height zones for larger pieces of equipment. Within the exposed concrete soffit will be recessed Halfen channels to facilitate the hanging of exposed services, lighting and Laboratory equipment/ furniture. Gas distribution and sink locations will be dependent on lab user preferences.

As requested by the users, 2 No. Radio Frequency (RF) shielded rooms will be provided within the new facility. Both RF shielded rooms will be located within separate Laboratories, and will be of a modular panel construction to allow both rooms to be dismantled, relocated to another area and re-erected or changed in size and configuration at a later date if required to cater for future flexibility.

Lastly, the project brief requires specific items of scientific equipment, which are essentially high powered magnets to be accommodated within certain laboratory areas. However, it was not possible to determine the precise specification of the equipment at the time of design. As a result the building may require further measures to be carried out by the University at a later date to ensure the selected magnets are located in suitable locations and in installed in the correct environments. As part of the design, consideration has also been taken in positioning services throughout the facility to reduce clashes with magnet gauss lines, e.g. services within the clean room plenum area.

Authors:
Tony Ling, Jestico +Whiles, with contributions from Ramboll and CH2M Hill

Design Team
Led by EC Harris as project managers and cost planners through the OGC Framework, the design team also includes CH2M Hill who is providing specialist architectural laboratory design services together with M&E consultant services, with Ramboll providing Civil and Structural services.

Amyotrophic lateral sclerosis (ALS) – also referred to as Lou Gehrig’s disease, Charcot disease, or motor neuron disease – is a neurodegenerative disease characterised by rapidly progressive weakness, muscle atrophy and spasticity, difficulty speaking (dysarthria), difficulty swallowing (dysphagia) and difficulty breathing (dyspnea). The disease occurs relatively uniformly in Western countries, with an average incidence of 1.89 per 100,000 population/year and an average prevalence of 5.2 per 100,000 people¹. The mean age of onset for sporadic ALS is about 60 years^2,3 and there is a slight male prevalence with a male:female ratio of ±1.5:1¹. Only 5% of cases have an onset before the age of 30, although juvenile sporadic onset cases are being increasingly recognised⁴. ALS is the most common adult motor neuron disease, yet the pathogenesis has not been completely unraveled. It is likely to be a complex interplay between multiple pathogenic cellular mechanisms, operating either singly or in combination^5,6.

In around 90% of the cases there is no family history of the disease (sporadic ALS, sALS) and therefore the cause for ALS remains unknown⁷. Several potential causes have been investigated, from toxic influences to free radical damage to nutritional deficiencies, but without consistent findings. The remaining 10% of patients have a known hereditary factor (familial ALS, fALS). The most common mutation is a mutation in the Copper-Zinc superoxide dismutase (SOD1) gene. Transgenic mice overexpressing mutant superoxide dismutase-1 (SOD1^G93A) display neuronal cell death in a manner that is consistent with the profile of cell death seen in human ALS patients, and can thus be used as a model for the condition⁸.

Although ALS is considered incurable, many of the symptoms are treatable, which supports efforts to improve quality of life for as long as possible⁹. Up to this moment, Riluzole (Rilutek, Sanofi-Aventis) is the only FDA approved drug on the market, however, it only has a modest effect on prolonging life in ALS patients (for up to 2-3 months when taken for a 18-month duration)^10-14. Riluzole is generally well tolerated, but has common side effects like asthenia, nausea, gastrointestinal upset and abnormal liver function.

Currently, there are 209 studies in the clinical trial register of the U.S. National Institute of Health. This shows the many attempts to find a cure for this devastating disease. Some of these attempts go beyond the testing of ‘classical’ drug molecules, for example by monitoring the effect of exercise and dietary recommendations, and more invasive such as stem cell transplantations. Not only pharmaceutical or biotechnology companies have an interest in finding new treatments; many patient organisations and research foundations, like the US-based ALS Association and ALS Therapy Development Institute, the UK-based motor neuron disease association and the international alliance of ALS/MND associations are all focusing on patient care, including finding new treatments in a not-for-profit format (Figure 1).

Looking at the classical drug treatments for ALS, edaravone is currently the most advanced option in clinical research. Edaravone was approved for acute ischaemic stroke in 2001, and an open-label phase II study demonstrated that edaravone reduced oxidative stress in ALS patients and delayed the progression of functional motor disturbances¹⁶. The results from an ongoing phase III study with edaravone are expected in 2015.

Another candidate for ALS treatment is a component of traditional Chinese medicines, Ursodeoxycholic acid (UDCA). UDCA has quite recently been subjected to efficacy and safety trials for the disease and results have indicated that is it tolerable in ALS patients, and there is evidence of efficacy.

Unfortunately, several other drugs in development for ALS have recently failed in clinical practice. The largest ever phase III trial in ALS (called EMPOWER, including more than 900 patients) with dexpramipexole failed to meet the primary outcome, a joint rank analysis of function and survival called the Combined Assessment of Function and Survival. Other endpoints, including functional decline, survival, or respiratory decline, and subgroup analyses also failed to show efficacy with treatment, overall or for any subpopulation¹⁹. Likewise, in 2011, olesoxime was discontinued, as it did not show a significant increase in survival versus placebo in ALS patients receiving riluzole²⁰. These failures, as well as other failures, were obviously very disappointing for the ALS community, including patients, physicians and researchers.

Overall, it was noted that failed results in clinical research raise key questions about enrollment, the number of people with ALS needed to participate and study duration, particularly to inform and empower phase II go/no go decisions²¹. A phase III clinical trial with minocycline showed a harmful effect on ALS disease progression²², while preclinical studies showed a survival benefit of 10-22%^23,24. The initial phase I/II feasibility studies were not designed to evaluate efficacy, but did show an acceptable safety profile²⁵. Discussing the possible reasons for yet another failure, the dose level of minocycline, the possible interaction with riluzole, the clinical trial design and end-point selection were mentioned^22,26. In addition, preclinical research results should be scrutinised before moving into the clinical research phase at all. The failure of lithium in a phase II/III study proved an example of this. A pilot study in mice and humans showed positive effects of lithium on slowing the progression of ALS²⁷. However, several follow-up clinical studies have failed to show this effect^28-30. A thorough study by researchers from ALS TDI, who were examining the use of lithium as a positive control, showed that false-positive effects in the SOD1^G93A mouse model can be avoided by using a rigorous survival study design, including sibling matching, gender balancing, investigator blinding and transgene copy number verification³¹.

In the past 10 -15 years, research into the pathophysiology of ALS has pointed towards a prominent role of neuroinflammation. Both in patients (in vivo and post mortem) as well as in the mouse models of ALS, activation or proliferation of microglia and astrocytes were observed^7,32-34. This has led to the investigation and development of treatments against neuroinflammatory components. NP001 (Neuraltus), a drug that is designed to restore the normal functioning of activated macrophages, is an example of an anti-inflammatory ALS drug. The recent phase II clinical study demonstrated a positive trend in slowing the rate of ALS disease progression and a phase III study will be started mid 2013³⁵. Another example for a neuroinflammatory target is the preclinical investigation into an anti-CD40L antibody. ALS TDI, Biogen Idec and UCB Pharma SA have entered into a collaboration to investigate the use of an anti-CD40L antibody as potential treatment for ALS³⁶.

Figure 1: Key unmet needs in ALS treatment. Many factors are unknown in ALS pathogenesis and no biomarker has been identified to diagnose patients in an early stage of the disease. This prevents interference in the earliest stages and lowers survival chances. In addition, ALS patients are in need for better drugs with higher efficacy and disease-modifying effects. The main goal for companies, foundations and patient organizations is to slow the progression of disease or even halt the disease progression, instead of symptomatic treatment. Adapted from Datamonitor15.

to-BBB has recently developed glutathione pegylated liposomal methylprednisolone (2B3-201) for the treatment of neuroinflammation. Proof-of-concept results obtained in a model of Multiple Sclerosis (MS) showed that 2B3-201 was more effective in reducing clinical signs compared to free methylprednisolone and non-targeted pegylated liposomal methylprednisolone³⁷. Together with scientists from the Departments of Pharmacology and Clinical Neurology from the University of Oxford, to-BBB has tested 2B3-201 in the SOD1^G93A model. Compared to free methylprednisolone treatment, 2B3-201 treatment resulted in more normal motor neurons, less astrocytosis and resulted in improved neuroprotection. Researchers at the ALS TDI have investigated the pharmacokinetics of brain and spinal cord uptake of 2B3-201 and, based on the positive results that were obtained so far, have recently started a long-term survival study in the SOD^G93A model. Based on these initial promising results of to-BBB’s G-Technology in the application towards ALS, ALS TDI and to-BBB agreed to investigate additional promising compounds in a joint effort³⁸.

In addition to 2B3-201, to-BBB is working on several other co

mpounds that could have a benefit for patients suffering from ALS. Since we believe that combined efforts have a synergistic effect, we are actively pursuing additional collaborations, next to the ongoing investigations with researchers from Oxford University (UK) and ALS TDI (USA). We are setting up collaborations with Utrecht University (the Netherlands), as well as special purpose companies founded by ALS patients. Such projects could for example use drugs that are approved elsewhere, but not yet available in Europe, and that can benefit from enhanced delivery to the brain and spinal cord.

to-BBB’s core competency is enhancing drug delivery to the brain, using the G-Technology, a brain-targeted liposomal platform. Through the versatility of the liposomes it is possible to encapsulate a variety of drug molecules³⁹, and while most of the efforts we ar

e currently pursuing are still too early to report, we are keeping an open and collaborative mind. For example, within this network of collaborators, we are not only looking into Western-type medicinal chemistry molecules, but also towards traditional Chinese medicine with a potential beneficial effect on ALS. From the literature, as well as general Internet sources, several such traditional Chinese medicines have been described.

For devastating diseases like ALS, for which currently only moderately effective drugs exist, it is important to find disease-modifying treatment options. Since it is a complex disease, the input of many stakeholders is required. Only by combining all efforts worldwide, a safe and efficacious treatment could hopefully be developed in the shortest time possible.

1. Worms PM (2011) The epidemiology of motor neuron diseases: a review of recent studies. J Neurol Sci 191:3-9.
2. Leigh PN (2007) Amyotrophic lateral sclerosis. In Motor Neuron Disorders and related diseases Vol 82. Edited by: Eisen AA, Sham PJ. Amsterdam: Elsevier; pp:249-268. [Aminoff MJ, Boller F, Swaab DF (Series Editor): Handbook of Clinical Neurology]
3. Haverkamp LJ, Appel V, Appel SH (1995) Natural history of amyotrophic lateral sclerosis in a database population. Validation of a scoring system and a model for survival prediction. Brain 118(Pt 3):707-719.
4. Gouveia LO, De Carvalho M (2007) Young-onset sporadic amyotrophic lateral sclerosis: A distinct nosological entity? Amyotroph Lateral Scler 8(6):323-327.
5. Shaw PJ (2005) Molecular and cellular pathways of neurodegeneration in motor neuron disease. J Neurol Neurosurg Psychiatry 76:1046-1057.
6. Cozzolino M, Ferri A, Carri MT (2008) Amyotrophic lateral sclerosis: from current developments in the laboratory to clinical implications. Antioxid Redox Signal 10:405-443.
7. Philips T, Robberecht W (2011). Neuroinflammation in amyotrophic lateral sclerosis: role of glial activation in motor neuron disease. Lancet Neurol 10:253-623.
8. Scott S, Kranz JE, Cole J, Lincecum JM, et al. (2008) Design, power, and interpretation of studies in the standard murine model of ALS. Amyotroph Lateral Scler 9(1):4-15.
9. Gordon PH (2011) Amyotrophic Lateral Sclerosis; pathophysiology, diagnosis and management. CNS Drugs 25(1): 1-15.
10. Bensimon G, Lacomblez L, Delumeau JC, Bejuit R, Truffinet P, Meininger V (2002) A study of riluzole in the treatment of advanced stage or elderly patients with amyotrophic lateral sclerosis. J Neurol 249:609-615.
11. Meininger V, Lacomblez L, Salachas F (2000) What has changed with riluzole? J Neurol 247:19-22.
12. Mitchell JD, O’Brien MR, Joshi M (2006) Audit of outcomes in motor neuron disease (MND) patients treated with riluzole. Amyotroph Lateral Scler 7:67-71.
13. Guidance on the use of riluzole for the treatment of motor neuron disease [http://www.nice.org.uk/nicemedia/pdf/RILUZOLE_full_guidance.pdf]
14. Turner MR, Parton MJ, Leigh PN (2001) Clinical trials in ALS: an overview. Semin Neurol 21:167-175.
15. Datamonitor (2007) Pipeline Insight: Orphan diseases in CNS. Part II Amyotrophic Lateral Sclerosis. Reference Code: DMHC2354
16. Yoshino H, Kimura A (2006) Investigation of the therapeutic effects of edaravone, a free radical scavenger, on amyotrophic lateral sclerosis (Phase II study). Amyotroph Lateral Scler 7(4):241-245
17. Min JH, Hong YH, Sung JJ, Kim SM, Lee JB, Lee KW (2012) Oral solubilized ursodeoxycholic acid therapy in amyotrophic lateral sclerosis: a randomized corss-over trial. J Korean Med Sci 27(2):200-206
18. clinicaltrials.gov NCT00877604
19. Medscape Medical News, 4 January 2013; www.medscape.com/viewarticle/777141
20. Press Release, Trophos SA, 13 December 2011; www.trophos.com/news/pr20111213.htm
21. http://blogs.als.net/post/2012/12/14/ALSMND-2012-Clinical-Trials-Tribulations.aspx.
22. Gordon PH, Moore DH, Miller RG, Florence JM, et al. (2007) Efficacy of minocycline in patients with amyotrophic lateral sclerosis: a phase III randomised trial. Lancet Neurol 6:1045-1053
23. Kriz J, Nguyen MD, Julien JP (2002) Minocycline slows disease progression in a mouse model of amyotrophic lateral sclerosis. Neurobiol Dis 10(3):268-273
24. Van Den Bosch L, Tikin P, Lemmens G, Robberecht W (2002) Minocycline delays disease onset and mortality in a transgenic model of ALS. Neuroreport 13(8):1067-1070
25. Gordon PH, Moore DH, Gelina DF, Qualls C, Meister ME, et al. (2004) Placebo-controlled phase I/II studies of minocycline in amyotrophic lateral sclerosis. Neurology 62(10):1845-1847
26. Swash M (2007) Learning from failed trials in ALS. Lancet Neurology 6:1034-1035
27. Fornai F, Longone P, Cafaro L, Kastsiuchenka O, Ferrucci M, et al. (2008) Lithium delays progression of amyotrophic lateral sclerosis. PNAS 105(6):2052-2057
28. Aggarwal SP, Zinman L, Simpson E, McKinley J, Jackson KE, et al. (2010) Safety and efficacy of lithium in combination with riluzole for treatment of amyotrophic lateral sclerosis: a randomised double-blind, placebo-controlled trial. Lancet Neurol 9:481-488
29. Chio A, Borghero G, Calvo A, Capasso M, Caponnetto C et al. (2010) Lithium carbonate in amyotrophic lateral sclerosis: lack of efficacy in a dose-finding trial. Neurology 75(7):619-625
30. Verstraete E, Veldink JH, Huisman MH, Draak T, Uijtendaal EV, et al. (2012) Lithium lacks effect on survival in amyotrophic lateral sclerosis: a phase IIb randomised sequential trial. J Neurol Neurosurg Psychiatr 83(5):557-564
31. Gill A, Kidd J, Vierira F, Thompson K, Perrin S (2009) No benefit from chronic lithium dosing in a sibling-matched, gender-balanced, investigator-blinded trial using a standard mouse model of familial ALS. PloS ONE 4(8): e6489 doi:10.1371/journal.pone.0006489
32. Evans MC, Couch Y, Sibson N, Turner MR (2012) Inflammation and neurovascular changes in amyotrophic lateral sclerosis. Moll Cell Neurosci http://dx.doi.org/10.1016/j.mcn.2012.10.008
33. Turner MR, Cagnin A, Turkheimer FE, Miller CC, Shaw CE, et al (2004) Evidence of widespread cerebral microglial activation in amyotrophic lateral sclerosis: an [11C](R)-PK11195 positron emission tomography study. Neurobiol. Dis. 15, 601–609
34. Anneser JM, Chahli C, Ince PG, Borasio GD, Shaw PJ (2004) Glial proliferation and metabotropic glutamate receptor expression in amyotrophic lateral sclerosis. J Neuropathol Exp Neurol 63: 831–840
35. Press Release, Neuraltus Pharmaceuticals, 29 October 2012; www.neuraltus.com/pages/news_rel10_30_12.html
36. Press Release, ALS TDI, 19 December 2011; www.als.net/Media/5407/News/
37. Gaillard PJ, Appeldoorn CCM, Rip J, Dorland R, van der Pol SMA, et al (2012) Enhanced brain delivery of liposomal methylprednisolone improved therapeutic efficacy in a model of neuroinflammation. J Control Release 164(3): 364-369.
38. Press Release, ALS TDI and to-BBB, 8 January 2013; www.als.net/Media/5427/News/
39. Gaillard PJ, Visser CC, Appeldoorn CCM, Rip J (2012) Enhanced brain drug delivery: safely crossing the blood-brain barrier. Drug Discov Today: Technol 9: 155-160.

Authors: Pieter Gaillard, Corine Visser, Manon de Waard, Marco de Boer, and Chantal Appeldoorn

to-BBB technologies BV, Leiden, the Netherlands

Phone +31 (0)71 3322255

Fax +31 (0)84 8313409