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ANTIBIOTIC RESISTANCE WILL WE SURVIVE?

HIGH ALTITUDE RESEARCH AT THE ZIHP SAFER TRIPS TO THE MOUNTAINS

ZHAW WÄDENSWIL BLAZING NEW TRAILS IN SURFACTANTFREE NANOPARTICLES

LS2 Life Sciences Switzerland Annual Meeting:

Life Sciences in the Limelight Life Sciences plus 01 I 2015

1

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EDITORIAL

opinion r u o y , s r e d a Dear re to us. is importanwt.chemieplus.ch/lifesciencesplus e link ww out the new Please use th ggestions ab views and su re ur yo on to pass magazine.

Swiss research teams make powerful contributions to solving important issues for the future.

Success Stories Speak for Themselves

F

inding answers to complex questions in life sciences

caused by antibiotic-resistant bacteria. Based on estima-

usually starts with fundamental research, thus with

tions, there is evidence that in 2050, over 10 million people

developing a scientific model and deciphering interactions

worldwide are likely to die from the effects of antimicrobi-

and processes on a molecular level. Applied sciences and

al resistance. This would make antibiotic resistance the

industrial research use research findings selectively, to

most frequent cause of death, ahead of cancer. Science

develop marketable products and technologies. Obviously,

pursues two diverging strategies to curb the problem: One

this multi-stage-model is actually much more complex

approach is the development of novel antibiotic agents

than described here. True efficiency in science can only be

which show a reduced risk of resistance. The other is in-

achieved, if an international network of research teams

tensified research on pharmaceutical alternatives to anti-

continually exchanges results and knowledge. We also

biotics. Both approaches are bringing their first promising

need an open system where anyone may breach barriers

results. Swiss Universities and their outstanding research

at any time: There is nothing wrong with a theoretical

teams are not minor agents in this success (see page 34).

scientist becoming an industrial researcher (or vice versa) over the course of his or her career. Occasionally, scientific

Another example is personalized medicine: today, many

researchers succeed in founding companies and in entre-

drugs are taken “in vain”, because they do not have the

preneurship – a development which is precisely promoted

expected effect on the patient in question. In cancer thera-

by Swiss Universities (see page 25). Switzerland, as a lead-

py for instance, many patients have to take medication

ing platform for life sciences, meets many of the require-

without obtaining good therapeutic benefits. While one

ments for a modern, vibrant and active research center –

person might be lucky and be cured by a certain drug, an-

although there is always room for further improvement.

other patient may remain uncured although both suffer

The success stories speak for themselves: Swiss research

from the same type of cancer. Great hopes are therefore

teams make powerful contributions to solving important

pinned on a different kind of medicine, tailored to the in-

issues for the future.

dividual patient. Personalized medicine uses molecular diagnostic methods like high-throughput gene sequencing

One example would be antibiotic resistance. Healthcare

to estimate the ability of a specific drug to cure a specific

organizations worldwide aim to reduce the use of antibiot-

disease in specific patient. At Zurich’s Competence Center

ics. Newly developed, expensive products have an espe-

for Personalized Medicine, forces and knowledge are sys-

cially hard time, also because physicians are afraid to fur-

tematically joined to develop new therapeutic approaches

ther increase the problem of antibiotic resistance by pre-

(see page 18).

scribing them more. In this context, the international pharmaceutical industry fears the high failure rates and

The examples mentioned above are only two of the versa-

enormous costs of antibiotic drug development. According

tile topics we have edited for you in this latest issue of

to a study by GBI Research, US and European Union au-

“Lifesciences plus”.

thorities have only approved twelve new antibiotic drugs since the year 2000. In the meantime, antibiotic resistance has become the predominant issue in many hospitals and doctors’ offices. In Switzerland alone, an estimated 1000 patients per year are diagnosed with severe infections Life Sciences plus 01 I 2015

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RALF MAYER Editor in Chief

S U M M A RY

IMMUNOLOGY

T lymphocyte

mediated immune responses play an important role in the control of viral infections and cancer. Studies of such immune response mounted in response to viral infections in mice revealed that T cells not only fight infected cells but have to protect themselves from being eliminated by activated immune cells: Interferons act as an invisibility cloak for T cells.

14

TECHNOLOGY TRANSFER

ETH’s Pioneer Programm supports innovative

25

doctoral or master students with a fellowship in order to gap the time between research at ETH and a possible later application of research results via company founding. The article about Dr Andreas Essig’s work and an interview with Marjan Kraak from the ETH Transfer Department illustrate the power of the fellowships to help turning innovation into business.

SWISS RESEARCH

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06 News

10 High Altitude Research at the ZIHP: 48 High Content in Suspension Safer Trips to the Mountains

22 Professorship at ZHAW Wädenswil: Blazing New Trails in Surfactant-Free Nanoparticles

25 ETH Transfer and Pioneer Fellowship

28 Cell Biology: Unmasking Salmonella

31

LS2 Annual Meeting: Life Sciences in the Limelight PERSONALIZED MEDICINE

34 Antibiotic Resistance: Will We Survive?

Scientists of the Competence Center for Personalized Medicine in Zurich join forces in order to develop promising therapy approaches in the field of personalized medicine, which is a great hope for patients and companies. Together, they aim to overcome difficulties such as data handling or understanding the complex molecular backgrounds of diseases like cancer of HIV.

Life Sciences plus 01 I 2015

45

Protein A chromatography: Efficient Purification of Monoclonal Antibodies from High Titer Feedstocks

38 Ironing out Oxidative Stress LAB & PROCESS

No-Wash Multiplexed Cytokine Profiling of Human Peripheral Blood Mononuclear Cells (PBMCs)

52

Generating Scalable Results in Micro-Bioreactors

PLAYERS & PRODUCTS

55 TPP TubeSpin Bioreactor in Space 56

Cleanroom and Laboratories: Integrated Solutions for Critical Environments in the Field of Life Sciences

58

Cancer Therapy: The Art of Designing Selective Inhibitors

61 Tools

40 Molecular Diagnostics:

FILTRATE

Easy PCR Optimization Using the Linear Gradient Tool

63 News

44 Cryostocks: FlexiQuot Cryotube

67 Edition Notice

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SWISS RESEARCH I News

Petr Broz receives HFSP Career Development Award

NEWS

Infection biologist

Petr Broz, SNSF Assistant Professor of the Biozentrum of the University of Basel, has received one of the highly regarded Career Development Awards from the Human Frontier Science Program (HFSP). The Career Development Award is granted to applicants who have submitted projects of a high scientific quality and furthermore show potential to significantly advance life sciences research by applying novel technology and approaches in their work. In his project, now supported with US $300,000 for three years, Broz will more deeply investigate the cellular defense strategies against bacterial pathogens inside host cells. (Please read more on page 28 of this issue.) # www.unibas.ch

How the brain “remembers” pain

Chronic pain

is a common disease state, which affects more than one million people in Switzerland. Unfortunately, in many cases proper treatment strategies are missing. The constant perception of pain severely influences the quality of life. Thomas Nevian from the Department of Physiology at the University of Bern, together with fellow researcher Mirko Santello, has discovered a cellular mechanism in the brain of mice that contributes to the development of chronic pain. The findings of the two Bernese researchers resulted in the development of a novel pharmacological treatment strategy for chronic pain.

The study was published in the scientific journal “Neuron.”

Nevian and Santello investigated the modification of neurons by chronic pain in a brain region called Gyrus Cinguli, which is associated with the emotional aspects of pain. In this context, the establishment of a “pain memory” plays an important role, as Thomas Nevian explains. The neurons are constantly activated by a noxious stimulus, thus building a memory trace for pain that becomes irreversible. Pain is perceived by electrical impulses in the neurons. Therefore, the two researchers were searching for changes in the electrical properties of neurons in the limbic system. They found that neurons were more excitable in the Gyrus Cinguli. This was attributable to a down regulation of a specific ion channel, a protein in the cell membrane that determines the electrical properties of the cell. This led to an increased number of nerve impulses in these cells and thus to an increased perception of pain.

By activating a specific receptor sensitive to the neuromodulator serotonin, Santello and Nevian succeeded in re-establishing a normal function of the neuron. This reduced the pain perception in an animal model.

The researchers were able to identify a specific subtype of serotonin receptor that reduced the perception of pain. This is an important result, which might help to treat chronic pain more efficiently in the future. # www.unibe.ch

The constant perception of pain severely influences the quality of life of the patients and represents an extraordinary emotional burden. (Picture: iStock)

Purging a virus from organ transplants

Human cytomegalovirus

(HCMV) is an extremely common herpes-family virus that infects 60% of the population in industrialized countries and almost everybody in poorer regions for life. Its symptoms are easily fought off by a healthy immune system, but can be devastating to individuals with defective immunity (babies, HIV or immune suppressed patients). After infection, HCMV hides in blood-making stem cells, occasionally reactivating as these cells mature.

The lab of Didier Trono at EPFL discovered a protein that switches HCMV between dormancy and reactivation. They found this protein to be bound to the HCMV genome in latently infected hematopoietic stem cells and, upon a variety of external stimuli, to undergo a modification that allows for viral activation. The researchers were able to control this switch with a drug called chloroquine, usually used against malaria. When they treated blood-making stem cells that contained dormant HCMV with chloroquine, the virus reactivated and became exposed, opening the door to maneuvers aimed at eliminating virus-infected cells.

Published in eLife, the study shows how HCMV could be fought in high-risk patients and purged from organs before transplantation. The study sheds light on the molecular mechanisms, possibly offering insights into similar infections by other herpes viruses. Moreover, it shows a simple method for eradicating HCMV from high-risk patients and purging it from tissue before transplantation: reacitvated HCMV can be treated with an antiviral.

Trono’s team is now testing the method’s efficiency in purging HCMV from cells to be used for bone marrow transplantation. Following that step, the group will be developing the first trials in humans. # www.epfl.ch 6

Life Sciences plus 01 I 2015

SWISS RESEARCH I News

Modeling the Brain’s Energy

The brain

uses glucose for energy in a very efficient manner. However, given the brain’s structure and metabolic constraints, the way it achieves this efficiency is a mystery. The key is the little-understood relationship between neurons, blood vessels and the other cells of the brain, the glia. Scientists at École Polytechnique Fédérale de Lausanne (EPFL) and the King Abdullah University of Science and Technology (Kingdom of Saudi Arabia), have now developed the first computer model of this relationship, which has successfully matched previous experimental in vivo and in vitro data in the field. The model, published in PLOS Computational Biology, is now being integrated into Glial cells (credit: ThinkStock) the detailed brain model of the Blue Brain Project.

Over half of the brain is actually composed by glial cells, which support and insulate neurons, supply them with energy substrates, protect them from pathogens and even clear out dead neurons from the brain. In fact, glial cells, neurons and the brain’s blood vessels form a functional unit, the neuron-glia-vascular unit (NGV), that regulates the brain’s energy management. The newly developed computer model shows how glucose is shuttled between the three elements of the unit to produce energy for activated neurons. Previous experiments show that glucose flows in the form of lactate. The model confirmed that lactate flows from astrocytes to neurons. It is also the first such model to successfully simulate the actual timeframe of this process. A better understanding of the metabolic relationships between neurons and glia has also important implications for understanding the signals detected with functional brain imaging techniques, such as fMRI and PET, which monitor glucose utilization, blood flow or oxygen consumption changes that occur in register with neuronal activity. # www.epfl.ch

Nitrogen Versus Plant Diversity

High human

atmospheric nitrogen emissions lead to a reduction of plant diversity. Researchers at the University of Basel analyzed plots all over Switzerland and report that plant diversity has decreased in landscapes with high nitrogen deposition.

The researchers compared six different measures of plant diversity on 381 randomly selected study plots in Switzerland. The plots were each one square kilometer in size and located between 260 and 3200 meters elevation. In all six measures, the researchers found a negative relation to atmospheric nitrogen emissions. “Negative effects of nitrogen deposition on plant diversity were so far known from small-scale studies conducted mostly in areas of high conservation value”, says first author Tobias Roth.

“We wanted to know whether these effects are also evident when looking at entire landscapes and different elevations”. The weakest relation was found in the traditionally measured species richness, which measures the number of plant species per plot. The biologists found the strongest effect in the so-called phylogenetic diversity, a measure that compares DNA sequences. High nitrogen deposition thus leads to plant species being more strongly related to each other. # www.unibas.ch

Emissions produced in agriculture are responsible for two thirds of the nitrogen deposition in Switzerland.

Nitrogen oxides produces by burning of fossil fuels are responsible for the other third.

Life Sciences plus 01 I 2015

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SWISS RESEARCH I News

Spinal Cord Neurons that Control Pain and Itch

50 years ago,

Sensing pain is extremely unpleasant and pain can even become chronic.

(Picture: istock)

neurobiologist Patrick Wall and psychologist Ronald Melzack formulated the so-called “Gate Control Theory” of pain, which proposed that inhibitory nerve cells in the spinal cord determine whether a pain impulse coming from the periphery, such as the foot, is relayed to the brain or not. A team headed by Hanns Ulrich Zeilhofer from the Institute of Pharmacology and Toxicology at the University of Zurich and ETH Zurich did now reveal which inhibitory neurons in the spinal cord are responsible for this control function: As the study published in the science journal Neuron shows, the control cells are located in the spinal dorsal horn and use the amino acid glycine as an inhibitory messenger.

With the aid of genetically modified viruses, the research group managed to specifically interfere with the function of these neurons in mice. They discovered that disabling the glycine-releasing neurons leads to an increased sensitivity to pain and signs of spontaneous pain. Moreover, Zeilhofer‘s team developed viruses that enable these specific pain-control cells to be activated pharmacologically. Mice treated with these viruses were less sensitive to painful stimuli than their untreated counterparts. Activating these nerve cells also alleviated chronic pain. And the surprising additional result: “Evidently, the neurons don’t just control pain, but also various forms of itch,” explains Zeilhofer.

Moreover, the pharmacologists were able to demonstrate that neurons on the superficial layers of the spinal cord, where the relay of the pain signals takes place, are primarily inhibited by glycine signals. “These findings identify for the first the neurons and connections that underlie the Gate Control Theory of pain,” sums up Zeilhofer. “However, the targeted stimulation or inhibition of particular types of neurons in humans is still a long way off and might only be possible in a few decades’ time.” # www.uzh.ch

A male rat is exposed to blue light after the injection of the gene construct into its erectile tissue.

(Photo: ETH Zurich, Prof. M. Fussenegger)

Stem cell division: two paths, one goal

As the brain

forms, a few stem cells give rise to a vast number of nerve cells.

With each cell division, they also renew themselves, an ability known as asymmetric cell division, which is unique to stem and progenitor cells. Using the fruit fly Drosophila, Prof. Clemens Cabernard’s team, at the Biozentrum of the University of Basel, investigates how neural stem cells position their cell division machinery at the correct place. This is Asymmetrically dividing important to ensure the completion of asymmetric neural stem cell (Green: cell division and consequently the development cleavage furrow; Red: of the brain in general. cell differentiation factor; In dividing cells, the spindle apparatus distributes Blue: spindle apparatus). essential cell components such as the chromosomes (Picture: Biozentrum of the University of Basel) to the two daughter cells. It was already known that that the spindle apparatus also provides information that determines the precise positioning of the cleavage furrow, so that the cell constricts and divides at the correct site.

In neural stem cells, which divide asymmetrically, there is a second pathway which functions independently of the spindle apparatus.

In their study, the scientists demonstrated that the spindle dependent pathway is responsible for cleavage furrow constriction and thus to complete cell division. “It was more exciting for us, however, to discover that the location of the cleavage furrow moves when we inhibit this spindle-dependent signaling pathway. The spindle-independent mechanism still ensures furrow positioning but in the wrong place,” explains Cabernard. This means that the chromosomes and cell fate-determining molecules are incorrectly partitioned.

These findings demonstrate a clear interplay between these two signaling pathways. Misdistribution of chromosomes and molecules determining cell identity are also often observed in cancer cells, with wide-ranging consequences, affecting the fate and behavior of the resulting sibling cells. # www.unibas.ch

From blue Pill to blue Light

In the

over-60 age group, more than half of all men are affected by erectile dysfunction. The main causes for the symptom include cardiovascular disease, diabetes, hormonal imbalance, neurological disease and the side-effects of medication or even spinal paralysis. Although Viagra as a medication for the dysfunction helps men to prolong an erection, it does not actually trigger it.

Researchers led by Martin Fussenegger, professor of Biotechnology and Bioengineering at the Department of Biosystems (D-BSSE) in Basel, have now developed a novel biotechnological solution: a gene therapy that triggers reliable erections, basing on optogenetics.

A gene construct that reacts to blue light is injected into the erectile tissue of the penis. As soon as it is exposed to the light, a precursor molecule GTP is converted into the second messenger cGMP, which exists naturally in a number of human organs. This allows voltagedependent calcium channels to close, which reduces calcium levels in the cells. Thus, muscle cells relax and blood flow to the erectile tissue increases, leading to an erection. Proceeding enzymatic degradation of cGMP later ends the erection. The researchers tested their new development in male rats with good results. In most cases, the blue light acted like a switch. Fussenegger is convinced that the gene construct will also work in humans. The ETH professor does not anticipate many side-effects from this type of gene therapy since the erectile tissue is largely insensitive to pain.

In addition it is also for the most part detached from normal blood circulation, so the probability that the gene construct could reach other parts of the body is very low. An artificially induced erection would satisfy a great need among patients suffering from erectile dysfunction, says Fussenegger. Moreover, some sufferers are not allowed to take Viagra; those for instance, with known heart diseases.

Fussenegger’s gene construct exists only as a prototype; tests in humans have yet to be conducted. # www.ethz.ch

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Life Sciences plus 01 I 2015

SWISS RESEARCH I News

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SWISS RESEARCH

HIGH ALTITUDE RESEARCH AT THE ZIHP

Safer Trips to the Mountains Many mountaineers have experienced it at least once: exhaustion, lack of appetite, sleeplessness and breathlessness. Researchers of the Zurich Center for Integrative Human Physiology (ZIHP) with different expertises came together to understand the causes of those symptoms and to find possible treatments.

CHRISTINA GIGER

an advanced state, fever and disturbances of

altitude pulmonary edema (HAPE) such ede-

consciousness and sleep appear. The oxygen

ma appear in the lung, causing a severe oxy-

deficiency at high altitude initiates the activa-

gen deficiency that may lead to death. Another

the

tion of different hormone systems in our body.

phenomenon that often occurs at high altitude

blood’s ability to take up oxygen

Some of them increase heart rate and blood

is lack of appetite. Mountaineers eat less

from the air is reduced. A quick

flow. This leads to a higher pressure on the

and are therefore weakened. But also at mod-

adaptation is necessary in order

blood vessel walls. They become leaky and liq-

erate altitude minor discomforts can occur,

to keep all body functions running normally.

uid can penetrate into the surrounding tissue

potentially affecting people who stay in the

Otherwise diseases like acute mountain sick-

thereby forming edema. If the brain is subject

mountains for leisure or work. Why do these

ness or high altitude pulmonary edema may

to these edema, as it happens in acute moun-

diseases and discomforts appear at altitude

develop. Acute mountain sickness is charac-

tain sickness, symptoms like fever or distur-

and how can they be prevented? Researchers

terized by nausea, vomiting and headache. In

bances of consciousness will appear. In high

of the Zurich Center for Integrative Human

W

ith

increasing

altitude,

10

Life Sciences plus 01 I 2015

➜

InnuPure® C16/C96 Automized nucleic acid extraction

Physiology (ZIHP) joined forces in different cooperative projects to engage in these questions.

Early Prophylaxis Important To find out how to prevent HAPE, researchers of a ZIHP cooperative project went with volunteers to the Margherita hut on the summit of the Signalkuppe in the Alps. Located at 4554 meters on the Italian side of Monte Rosa, this hut is the highest mountain hut of Europe. It was already known that dexamethasone is a good prophylactic medication against HAPE. But it was not yet clear how it would act and at which time point it should be taken. Twenty-five of the volunteers had experienced HAPE before. Half of them were given a dose of dexamethasone one day before the ascent to the Margherita hut. The rest took the medication before the second night at the hut. All of the volunteers who had taken the medication before the ascent stayed healthy and only a quarter of those who received it before the second night developed HAPE. Hence, dexamethasone does indeed prevent the disease. But to achieve the optimal effect it has to be taken one day before ascending to high altitude.

Dilatory Properties To understand how this substance acts in the body, the researchers took blood from the volunteers at three different time points: before the ascent in Zurich at 490 meters and on the second and fourth day at the hut on 4554 meters.

They observed that the blood of the dexamethasonevolunteers in the hut contained an elevated level of peptides that can cause dilation of blood vessels. This possibly led to a slowing of the blood flow reducing the pressure on the vessel walls. Therefore no liquid was able to exit into the surrounding tissue and no edema developed. However, peptides need a certain time to initiate the dilation of blood vessels. This explains why dexamethasone has to be

■

taken early enough in order to be fully effective.

■

Lack of Appetite Due to Satiation Hormones?

But also in spite of sufficient prophylaxis, other discomforts may appear at high altitude. Many healthy sum-

■

miteers complain about lack of appetite when staying in the mountains. Researchers of a succeeding ZIHP cooper-

■

ative project examined the eating behaviour of healthy

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volunteers at the Margherita hut. Indeed, the volunteers ate less on the first and second day after a fast ascent. Pro-

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Automatic transfer of eluates into separate elution vessels for direct storage

Acute mountain sickness is characterized by nausea, vomiting

➜

and headache. In an advanced state, fever and disturbances of consciousness and sleep appear. www.bio.analytik-jena.com

SWISS RESEARCH I High Altitude Research at the ZIHP

➜ portionally though, they took up the same amount of fat, protein and carbohydrates as in lowland. The volunteers also felt less hungry.

In fact, their subjective sensation of appetite directly correlated with the amount of food they consumed. Only on the third day all of them returned to a normal eating behaviour.

The researchers speculated that the initial lack of appetite may have been induced by an increase in satiation hormones or by inflammation in the gut. They analyzed blood and gut tissue from the volunteers before the ascent and on the second and fourth day at the hut. However, they couldn’t find any difference in the concentration of satiation hor-

Volunteers who developed symptoms of acute mountain sickness had a more severe lack of appetite. After dexamethasone treatment these mountaineers recovered their appetite.

at moderate altitude. They carried out tests with volunteers in Davos at 1630 meters above sea level and on the Jakobshorn at 2590 meters. Particularly the deep sleep phase was reduced, a state that is important for recovery and learning. In addition, the volunteers showed repetitive interruptions of breathing – called apnea – during the night. Some of them even woke up with shortness of breath. In extensive concentration and coordination tests during the day, however, no impairment could be detected compared to examinations in lowland. These results are of great relevance for people who stay for some time in mountain territories or under similar oxygen conditions,

mones or in the abundance of inflammatory

like ski tourists, seasonal workers or flight

cells between the samples. Additionally, they

personnel. It will now be possible to estimate

isolated rat gut cells in the lab in Zurich and

potential inconveniences and dangers for

let them grow under low oxygen concentra-

those people.

tions to simulate the conditions on the Margh-

Successful Treatment of Sleep Apnea

erita hut. Similar to the samples from the volunteers, they could find neither elevated sati-

measurement under these conditions. Inter-

The ZIHP cooperative project also looked for

ation hormones nor inflammatory mediators.

estingly though, the researchers noticed that

possibilites to prevent severe health conse-

those volunteers who developed symptoms of

quences. In an other series of experiments, pa-

acute mountain sickness – such as headache,

tients with obstructive sleep apnea syndrome

nausea, dizziness – had a more severe lack

(OSA) underwent sleep and vigilance studies

What else could be the reason for the reduced

of appetite. After dexamethasone treatment

in Davos at 1630 and 2590 meters. In these pa-

food intake? In another experiment with rats,

these mountaineers recovered their appetite

tients the upper airways are blocked in the

the researchers wanted to determine whether

until day four like all others. In future experi-

throat region during sleep due to an excessive

the digestion is slowed at high altitude. This

ments it will be determined whether dexa-

relaxation of the muscles. Untreated patients

could explain why less food was consumed by

methasone prophylaxis may help to sustain a

suffer from repetitive apnea leading to oxygen

the volunteers. They kept the experimental

normal appetite from the beginning on.

deficiency. As a consequence their sleep is less

Acute Mountain Sickness Contributes to Lack of Appetite

animals in a chamber under similar oxygen

restorative and they tend to fall asleep during

the stomach of the rats emptied more slowly

Sleep and Breathing Problems at Moderate Altitude

under these circumstances, an effect known to

A stay at high altitude can bring along other

turnal apnea as compared to lowlands and

be often associated with reduced food intake.

health consequences, such as sleeping prob-

they perform poorly in driving simulator tests

However, gastric emptying could not be meas-

lems. The consortium of another ZIHP coop-

during the day. Consequently, the researchers

ured in the volunteers at the Margherita hut

erative project found out that the quality of

treated the patients with acetazolamide, a drug

because there is no practical method for this

sleep is not only affected at high but already

that enhances ventilation and that is also used

conditions as at the Margherita hut. Indeed,

12

the day. The examination of OSA patients in Davos showed that they have much more noc-

Life Sciences plus 01 I 2015

➜ A stay at high altitude can bring along other health consequences, such as sleeping problems with reduced deep sleep phase, a state that is important for recovery and learning. Volunteers showed repetitive interruptions of breathing – called apnea – during the night. to treat acute mountain sickness. This led to a partial but not complete improvement of respiration. In a next step the acetazolamide treatment was combined with continuous positive airway pressure (CPAP): Sleepers were wearing a nasal mask that applies air with a slightly positive pressure into the nose and throat region keeping the airways constantly open. This combined therapy resulted in a significant improvement of nocturnal breathing in OSA patients. It enables the patients to spend their holidays in the mountains without massive impairment of their ventilation at night and their well-being during the day.

Altitude Research Continuously in the Focus of the ZIHP The ZIHP continues to support projects dealing with high altitude research. Many athletes make use of the oxygen conditions at high altitude to enhance the body’s production of erythropoietin (Epo). This hormone stimulates the production of red blood cells and thus the oxygen transport capacity and the performance especially in endur-

Your instruments tuned to perfection.

ance sports. Within an assistant professorship financed by the ZIHP, Carsten Lundby and his group investigate the regulation of Epo production. They also pursue the question which other mechanisms influence performance at high altitude or how the heart reacts to high altitude training. But Epo can have a variety of effects of which probably only a part is known so far. For instance, it can prevent an experimentally induced myocardial injury, as the consortium of a completed ZIHP cooperative project found out. And Epo also acts in the brain. This was discovered by researchers of a recently completed ZIHP cooperative

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project. In short, the ZIHP high altitude projects contribute to a better understanding of physiological mechanisms and thus, they provide valuable information for the further development of drugs against altitude-related health conditions.

Frankfurt a. M. , 15.–19.6.2015 Halle 11.1, Stand C27

AUTHOR Dr. Christina Giger. Scientific Coordinator at the Zurich Center for Integrative Human Physiology (ZIHP) of the University of Zurich Life Sciences plus 01 I 2015

Endress+Hauser Metso AG Kägenstrasse 2 4153 Reinach Schweiz info@ch.endress.com www.ch.endress.com

SWISS RESEARCH

IMMUNOLOGY

Interferon: Protective Suit for T Cells In the control of viral infections and cancer, immune responses play an important role, in particular mediated by the white blood cells T lymphocytes. Studies of T cell mediated immune response mounted in response to viral infections in mice revealed that T cells not only fight infected cells but have to protect themselves from being eliminated by activated immune cells.

T-Lymphocytes attack cancer cell. (Picture: istock)

ANNETTE OXENIUS

ling the highly complex host-pathogen inter-

T Lymphocyte Biology in the Context of Persistent Viral Infection

pathogenesis of infectious diseases. Unraveactions by fundamental microbiological and

T lymphocytes (a subgroup of white blood

nfectious diseases, caused by various mi-

immunological research continues to be a se-

cells) are integral components of the adaptive

croorganisms, have major medical, social

rious and rewarding scientific challenge, pav-

immune responses and are responsible for

and economic consequences. The past

ing the way for the development of future

the control of many viral and bacterial infec-

decades have seen a revolution in our

therapies and vaccines. These powerful re-

tions. In particular, cytotoxic T lymphocytes

ability to diagnose and understand infectious

search avenues, which allow the acquisition of

are able to specifically recognize altered cells

diseases and host pathogen interactions at the

a wealth of information, require that this in-

in the organism, which include virally infect-

molecular, the cellular and also at the level of

formation is integrated and evaluated in rele-

ed cells or tumor cells. This recognition pro-

the whole organism. Cutting-edge technolo-

vant and well defined in vivo experimental

cess eventually leads to the selective destruc-

gies such as ex vivo multi-parameter analyses

systems (e. g. murine models of infectious dis-

tion of the altered cells via direct cytolytic

(e. g. flow cytometry, imaging, genomics and

eases) as well as in translational approaches

mechanisms. These powerful effector mecha-

proteomics) and targeted gene technology

to human disease. These latter points are the

nisms have to be tightly regulated to prevent

(e. g. recombinant pathogens, genetically mod-

common theme and the strength of the ongo-

attack of non-infected and non-transformed

ified hosts) are powerful tools in our quest to

ing and future research in the laboratory of

cells and to prevent overt immunopathology

further increase our understanding of the

Infection Immunology [Figure 1].

in case of overwhelming infections.

I

14

Life Sciences plus 01 I 2015

SWISS RESEARCH I Immunology

Besides secreting pro-inflammatory cytokines (soluble proteins which communicate with other cells and induce functional changes

This successful translation of basic

in those), cytotoxic T lymphocytes mediate their cytolytic (i. e. the process of killing a tar-

immunological studies into clinical

get cell) function by targeted release of cytotoxic effector molecules upon contact with a specific target cell. These cytotoxic effector molecules are stored in membrane-bound cytoplasmic granules which fuse with the plasma

application illustrates in a very

➜

impressive way how basic research

membrane upon specific activation, an exocy-

was key for opening new avenues

tosis process which is termed degranulation1.

In case of persistent viral infections, the

in the treatment of cancer.

immune system is challenged by continuous exposure to viral antigens, which causes constant activation of T lymphocytes. If these activated cells would be fully functional and would continuously secrete inflammatory cytokines, this might lead to serious systemic

“programmed death 1” (PD-1). Blocking the

consequences such as organ failure and overt

inhibitory signal via PD-1 in mouse models of

T Lymphocyte Biology in the Context of Acute Viral Infection

immunopathology. We discovered that in face

persistent viral infections was shown to “reju-

When antigen-specific T lymphocytes are ac-

of prolonged in vivo exposure to antigens

venate” the function of virus-specific T lym-

tivated for the first time, either after infection

(units on viral proteins which are recognized

phocytes and thus to increase control of the

or vaccination, they need to receive three dis-

by virus-specific T lymphocytes) in chronic

infection. This strategy of improving the func-

tinct signals. Signal 1 consists of sensing the

viral infections, virus-specific T lymphocytes

tion of virus-specific T lymphocytes was later

presence of antigen via the T cell receptor

selectively silence specific effector functions

confirmed in the setting of chronic SIV infec-

(TCR), a highly variable receptor expressed

in a hierarchical manner2. In particular, they

tion in macaques.

on the surface of each T lymphocyte which

down-regulate their cytokine production ca-

➜ T lymphocytes, stressed upon antigen activation, potentially end up on the killer cell’s hit list.

In this context we have asked the question

confers specificity to the cell. Signal 2 is pro-

whether the release of virus-specific T lym-

vided by a number of co-stimulatory mole-

phocytes from PD-1 blockade might not only

cules. Among them are ligands and receptors

be beneficial for improved virus control but

which provide additional signals for activa-

might also bear risks with respect to T lym-

tion and which are expressed on the surface

phocyte inflicted immunopathology. Indeed,

of the T cell and the cell presenting the anti-

using a mouse model of chronic viral infec-

gen to the T cell. Signal 3 comes in form of in-

tion, blockade of PD-1 during establishment

flammatory cytokines – soluble molecules

of chronic infection, resulted in fatal outcome

which mediate communication between im-

of the infection. Mortality was due to severe T

mune cells and provide differentiation signals

lymphocyte-induced immunopathology which

for the activated T lymphocyte. One promi-

manifested in vascular leakage and hypo-

nent signal 3 cytokine is Type 1 interferon

volemic shock due to extensive killing of

(IFN), a prototypical inflammatory cytokine

blood endothelial cells [Figure 2]4, 5. These re-

which is induced early upon infection with

sults indicate that substantial risks might be

many viruses. Besides supporting activation

taken when interfering with the regulation of

and differentiation of antiviral T cells, Type 1

antiviral T cell function in the context of ac-

IFNs are also potent activators of “Natural

tive chronic viral infections.

Killer cells” (NK cells). The job of these NK cells is to detect and eliminate the body’s own

pacity. This selective silencing is regulated at

nize altered host cells (i. e. cancerous cells in

“stressed cells.” Such stress can be induced in

the level of nuclear activity of specific tran-

tumors) are also rendered hypo-responsive

cells by viral infection or by increased cellular

scription factors which are key in regulating

within the tumor environment, as they are –

activity for instance when cells undergo in-

cytokine gene expression3. A similar downreg-

alike T cells during chronic viral infection – al-

ulation of the functional capacities of antiviral

so constantly confronted with high amounts of

T lymphocytes in face of active chronic viral

tumor antigens. This analogous behavior has

infection also occurs in human infections

prompted researchers to test whether co-in-

such as HIV-1 or Hepatitis C virus infection3.

hibitory receptor blockade might also result in

A number of cellular and molecular parame-

improved anti-tumor control by tumor-reactive

ters which regulate this functional downregu-

T lymphocytes. Based on very promising re-

lation have been identified in recent years by

sults in experimental small animal models, this

a number of research laboratories. One key

strategy of immunotherapy using co-inhibitory

aspect of this regulation is the sustained ex-

receptor blockade has been extended to hu-

pression of co-inhibitory receptors on the

man clinical trials – with spectacular results

surface of virus-specific T lymphocytes in the

when compared to traditional chemotherapeu-

setting of chronic exposure to viral antigens.

tic treatments. This successful translation of

The function of these co-inhibitory receptors

basic immunological studies into clinical appli-

is to dampen the intracellular signals which

cation illustrates in a very impressive way how

are initiated upon antigen recognition within

basic research and the molecular understand-

virus-specific T lymphocytes. A key repre-

ing of T cell regulation was key for opening

sentative of such a co-inhibitory receptor is

new avenues in the treatment of cancer.

Life Sciences plus 01 I 2015

15

➜

Interestingly, T lymphocytes which recog-

Interferon acts as a “camouflage cloak” that renders T cells invisible to the attack by killer cells.

SWISS RESEARCH I Immunology

01

Overview about the basic research concepts of the Infection Immunology laboratory.

(Picture: A. Oxenius)

A

B

02

03

Type-I Interferons protect T cells against NK cell attack, T cells lacking the ability to directly sense type-I interferons (blue cells) are highly susceptible to NK cell meditated killing, whereas T cells that receive type-I interferons (purple cells) are protected from NK cell mediated regulation.

As shown, the ability of activated T cells to receive signals through the type-I IFN receptor prevents the expression of ligands (sweat drops) for an activating NK cell receptor, thereby protecting themselves against attack by NK cells (black). In contrast, activated T cells which are unable to sense type-I interferons are killed by NK cells in a perforin (stars) dependent manner, demonstrating an important immunoregulatory mechanism of NK cells for inappropriately activated T cells. (Picture: J. Crouse and A. Oxenius)

Pulmonary endothelial cells are dying during chronic viral infection: In situ analysis of lung-sections from naive and infected WT and PD-1-deficient mice on day 5–6 after chronic virus infection.

Representative immunofluorescence stainings for endothelial cells (green, CD31+), cell nuclei (blue, DAPI) and apoptotic (dying) cells (red, TUNEL) are shown. The extent of endothelial cell death is markedly increased in absence of PD-1 signaling on virus-specific T lymphocytes. (Picture: J. Crouse and A. Oxenius)

tense proliferation which goes along with

from the other department” during acute viral

nated by the NK cells, thereby dramatically

metabolic changes. This creates now a situa-

infection: healthy T lymphocytes are able to

weakening the antiviral immune response

tion in which activated NK cells are confront-

directly sense Type 1 IFN, which bind to spe-

[Figure 3]6, 7.

ed with stressed virus-specific T lymphocytes,

cific receptors on the surface of the T cells and

Currently, it is unclear whether the same

which undergo massive clonal expansion up-

thereby conceal their stress. In other words, it

mechanism also exists in humans but it is at-

on antigen activation, thereby potentially

acts as a “camouflage cloak” that renders them

tractive to speculate that such basic processes

ending up on the NK cells’ hit list.

invisible to the attack by NK cells. If the

of how the immune system protects its acti-

We have recently discovered what keeps

T cells lack the receptor for Type 1 IFN, they

vated T cells against NK cell attack might well

NK cells from killing off their “colleagues

reveal themselves as stressed and are elimi-

be conserved. Extending from the results ob-

16

Life Sciences plus 01 I 2015

SWISS RESEARCH I Immunology

tained in the experimental viral infection model, it is conceivable that T cells which are activated in the absence of Type 1 IFN reveal themselves as being stressed and thus become targets for NK cells. Such a situation

REFERENCES

might arise during the activation of T cells with specificity for “self” (autoimmune-reac-

1

tive T cells), which normally occurs in absence of high concentrations of Type 1 IFN. In the future, we will explore this exciting hypothe-

J Exp Med 199: 925–936.

2

sis and will hopefully be able to provide definite answers in the years to come.

Wolint, P., M. R. Betts, R. A. Koup, and A. Oxenius. 2004. Immediate Cytotoxicity But Not Degranulation Distinguishes Effector and Memory Subsets of CD8+ T Cells.

Frebel, H., K. Richter, and A. Oxenius. 2010. How chronic viral infections impact on antigen-specific T-cell responses. Eur J Immunol 40: 654–663.

3

Agnellini, P., P. Wolint, M. Rehr, J. Cahenzli, U. Karrer, and A. Oxenius. 2007.

Impaired NFAT nuclear translocation results in split exhaustion of virus-specific CD8+ T cell functions during chronic viral infection. P roc Natl Acad Sci U S A 104: 4565–4570.

4

Frebel, H., V. Nindl, R. A. Schuepbach, T. Braunschweiler, K. Richter, J. Vogel, C. A. Wagner, D. Loffing-Cueni, M. Kurrer, B. Ludewig, and A. Oxenius. 2012. Programmed death 1 protects from fatal circulatory failure during systemic virus infection of mice.

J Exp Med 209: 2485–2499.

5

Frebel, H., and A. Oxenius. 2013. The risks of targeting co-inhibitory pathways to modulate pathogen-directed T cell responses. Trends Immunol 34: 193–199.

AUTHOR

6

Crouse, J., G. Bedenikovic, M. Wiesel, M. Ibberson, I. Xenarios, D. Von Laer, U. Kalinke, E. Vivier, S. Jonjic, and A. Oxenius. 2014. Type I Interferons Protect T Cells against NK Cell

Prof. Dr. Annette Oxenius. Head of Laboratory of Infection Immunology, Institute of Microbiology, ETH Zurich

Attack Mediated by the Activating Receptor NCR1. Immunity 40: 961–973.

7

Crouse, J., H. C. Xu, P. A. Lang, and A. Oxenius. 2014. NK cells regulating T cell responses: mechanisms and outcome. Trends Immunol.

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SWISS RESEARCH

PERSONALIZED MEDICINE TO CROSS THE FRONTIER

Individual Therapy requires Multitudinous Research Networks Personalized medicine is a slogan, a challenge, a great hope for patients and companies – but where do we stand today? Scientists of the Competence Center for Personalized Medicine in Zurich join forces in order to develop promising therapy approaches. Together, they aim to overcome difficulties such as data handling or understanding the complex molecular backgrounds of diseases like cancer or HIV. 18

Life Sciences plus 01 I 2015

S W I S S R E S E A R C H I Pe r s o n a l i z e d M e d i c i n e t o C r o s s t h e Fr o n t i e r

CHRISTIAN EHRENSBERGER

P

In order to bring together sound clinical

ersonalized medicine is uniting a bunch of definitely evidence-based concepts. Particularly, the methods of high-throughput

gene

expertise, fundamental biological research

➜

sequencing,

proteomics and metabolomics are used to es-

and technological development, a network

timate the ability of a specific active pharma-

of experts is required.

ceutical ingredient (API) to cure a specific disease in a specific patient. Actually, personalized or stratified medicine is aiming at the inhibition of over activated biochemical signal transduction pathways which for example boost tumour growth or prohibit apoptosis. A

putative therapy has to be proven as clinical-

regularly. Apart from that, the CC-PM offers

precondition for the success of such a therapy

ly effective.

postgraduate education, for example at their

is to detect the signalling pathway using a

Research in this area requires even more

suitable biomarker. If it is present (or not) in a

resources: First, there is the need for biobanks

Although the CC-PM does not dispose of a

patient, one can conclude that he or she will

including tissue samples and well character-

lot of money, it can smooth the way for re-

respond to a specific treatment. So the con-

ized cell lines. They are taken from tumour

search projects by providing support in find-

cept consists of a tandem arrangement in-

patients and are then used to develop preclin-

ing appropriate sources of funding. Some

cluding a drug and a corresponding predictive

ical models describing the biosynthesis of

powerful CC-PM interfaces are constituted by

biomarker that tells us prior to the beginning

proteins in the (tumour) cells using the genet-

the technology platforms which are associat-

of the therapy whether it will result in a clini-

ic information. Second, if a preclinical model

ed with the Competence Center. They offer

cally relevant success or not.

for a disease is available, technologies to in-

specific services for the members.

PhD school for translational biomedicine.

vestigate complex biological processes and

One of these technology platforms is a

their interaction with active pharmaceutical

biobanking unit compiling an archive of

“Since 2005 this concept has become reality in

ingredients are needed. Finally, a host of data

well-characterized

lung cancer therapy,” says Professor Rolf Sta-

has to be handled and interpreted.

method is typical of oncological research: Tu-

Promising Options for Cancer Therapy

hel, a member of the Competence Center for

sample

material.

This

mour tissue is collected and subjected to a cell-

Personalized Medicine (CC-PM) in Zurich

Bundled Competences

and a founding member and the first presi-

In order to bring together sound clinical exper-

tion, respectively. Currently, this technology

dent of the Swiss Society for Medical Oncolo-

tise, fundamental biological research and tech-

platform is adding to their existing tissue & cell

gy. “We examine lung tissue for predictive

nological development, a network of experts is

archive a liquid biobanking branch comprising,

markers, such as cancer cells harbouring cer-

required. One of the best examples is the CC-

such as blood samples. This is an even more

tain mutations in the EGFR, the epidermal

PM in Zurich. It is being funded by the Univer-

challenging task because, for example, suitable

growth factor receptor. It is a simple yes-no

sity of Zurich and ETH Zurich, each contribut-

cooling systems have to be installed and main-

decision: Approximately 15 per cent of all our

ing CHF 0.5 million for the period from 2014 to

tained to conserve these samples.

patients can benefit from a specific therapy

2017. The scientists, physicians and clinicians

The Theragnostics Discovery Unit is part

which is superior to chemotherapy.”

who work together here are committed to pro-

of a second CC-PM technology platform called

based screening for their function or dysfunc-

While standard chemotherapy is per-

mote personalized medicine. At the moment

Nexus and tightly connected to the ETH Phe-

formed using the well-known cisplatin, the

they are focusing on genome-based projects

nomics Center and Biomedical Automation

personalized medicine approach employs a

including hereditary diseases. In addition,

Technology Center. Among other services,

tyrosine kinase inhibitor (TKI) of the entire

there are some research groups also dealing

state-of-the-art laboratory automation tech-

human epithelial receptor (HER) family. One

with proteomics or enzyme activity.

nology for translational research and discov-

member of it is HER-1 (EGFR), and the bio-

While the branches of the university

ery are provided. The goal is to obtain a deeper

marker for a promising therapy consists of cer-

school of medicine are defined by specific dis-

understanding of molecular disease mecha-

tain mutations in the EGFR gene, more specifi-

ease patterns, the CC-PM is dedicated to in-

nisms. Nexus also includes the ‘Personalized

cally: in the exons 19 and 21. The active agent

vestigating the fundamentals underlying the

Medicine ICT Unit’, which offers specialized

(Gefitinib or Erlotinib or Afatinib) is adminis-

concepts of personalized medicine. This net-

services relating to data managing and statisti-

trated to patients having these mutations and

work consists of formal members who meet

cal data analysis.

it has the power to give them a significantly longer cancer-progression-free survival (PFS).

This is especially related to locally advanced or

➜

metastatic non-small cell lung cancer.

As personal medicine working in the manner described above is targeted to a relatively small percentage of patients – often not even fifteen but only one to three per cent.

Thus, a lot of patients have to be recruited in order to carry out a study. Approximately 4000 patients have to be screened to identify a certain mutation in a tumour gene. Afterwards the mutation is tried to be assigned to a specific active pharmaceutical agent which

Today, tissue-based predicative molecular tests are already available for approximately 35 % of all tumours.

tients having the mutation. In addition, the Life Sciences plus 01 I 2015

03

Professor Dr. Niko Beerenwinkel.

(Picture: CC-PM, Zurich)

is then regarded to be a remedy for the pa-

19

S W I S S R E S E A R C H I Pe r s o n a l i z e d M e d i c i n e t o C r o s s t h e Fr o n t i e r

Computational Biology – the Key to Deeper Understanding

each other. So you will hardly find a specific

Beyond Cancer Treatment

physiological or biochemical variation in

A lot of effort in personalized medicine is

“This is a real bottleneck of personalized

many patients. But on the basis of mutational

driven towards oncology. However, the scope

medicine,” explains Niko Beerenwinkel, Pro-

patterns we may predict successfully if an

of application includes many other areas, such

fessor of Computational Biology at ETH Zu-

existing drug stops a tumour effectively.”

as the Acquired Immunodeficiency Syndrome

rich and Co-Director of the Competence

For this purpose the research teams do

(AIDS) caused by the Human Immunodefi-

Center for Personalized Medicine. “The hu-

not always need to develop a new API. Ideally,

ciency Virus (HIV). The situation is, to a cer-

man genome consists of over three billion

they know the target proteins of an approved

tain extent, similar to cancer: high genetic

base pairs, the raw data generated by se-

drug. In this case they will try and interpret

variability

quence analysis are primarily image data – it

the genetic changes induced by the API in a

agents and a choice of existing remedies.

all sums up to a host of huge and heterogene-

specific patient, generally within a certain

Between 20 and 25 APIs against HIV have

ous sets of data. So we are continuing our re-

molecular network. With the help of such

already been developed, but the virus has a

search on new algorithms and software for

computational network models it is possible

high rate of mutation. So its genome differs

managing and statistically analyzing clinical

to screen for a host of APIs.

from one patient to the other. But with the

in

the

causative

pathological

models available Beerenwinkel is able to pre-

and many more. However, although mere

dict not only which API will be of use to an in-

data handling and storing causes a lot of

dividual patient but also which cocktail of two,

➜

data, data from translational experiments

problems, we still do not have enough information, especially in a clinical context. DNA

three or four will be the best. Since the mutations in the viral target proteins are understood pretty well, AIDS is a good candidate for

sequencing has become a routine tool at low cost (less than CHF 5,000 for a complete individual human genome), and this has considerably accelerated the progress of personal medicine. But what we are really seeking for is linking genotypical data to phenotypical characteristics of the patient suffering from a certain disease.”

Professor

Beerenwinkel

and his

col-

leagues have a long way to go. One of the main troubles seems to be the scale running from molecular size up to visible tissue or easily palpable organs. In order to get a deeper in-

Research teams do not always

personalized medicine, whereas cancer is always related to complex signal transduction pathways, remaining difficult to cure.

need to develop a new API.

Individualized Dose and Therapy-Customized Diet

On the basis of mutational

Personalized medicine commonly results in a

patterns they may predict

yes-no decision as discussed above. The dose

successfully if an existing drug

of the API to be administered plays a minor role. Is Paracelsus’ paradigm “the dose makes the poison (or the drug)” obsolete in this area?

stops a tumour effectively.

In fact, there are some cases where personal-

sight in the phenomena of a specific disease

ized means “the suitable dose.” One example is

one may prepare an x-ray, use histological

codeine which is used as an antitussive, but

techniques or dive into the sea of 109 cells a

may also be a painkiller. The human organism

tumour may consist of. In addition, the cells of

If no suitable model is available, i.e. one

a tumour are not genetically identical and dif-

does not know how a given active pharmaceu-

fer in their biological function. In general, it

tical agent is working, a lot of data being col-

has to be taken into account that a tumour is

lected and showing its biological effect may

blood morphine level will differ from patient

also harbouring normal, healthy immune cells

help. For example, one has already investigat-

to patient depending on his predisposition:

or stroma cells, etc. Apart from that, the tu-

ed how the API acts in 200 different tumour

slow metabolizer (just below 50 %), normal

mour is interacting with the patient’s organ-

cell lines. Then the cell line of a patient suf-

metabolizer (just above 50 %), and very fast

ism in various manners – a highly complex

fering from cancer is included and data min-

metabolizer (approx. 3 %) [1 – 3]. So, the most

system requiring a lot of basic research.

ing approaches such as artificial neural net-

suitable dose is not easy to define for a phy-

works are applied to the joint set of data, or

sician. The worst scenario is: A patient is not

multivariate statistics performed. This is a

graded as fast metabolizer and dies of a mor-

If things are so complicated, which will be the

promising strategy to learn and, in the end,

phine overdose. This has already happened

most promising strategy to develop new ther-

suggest an individualized therapy.

to two children in the USA, where codeine is

Indication Extension for Existing Drugs

apies? “In my view, we have already quite a lot

One classical example is the therapy of

metabolizes codeine to afford morphine which, in effect, is the analgesic.

After

administration

of

codeine, the

administered considerably more often than

of medications which we do not use for all pa-

mammal

over-

in Switzerland, for example after a tonsillec-

tients whom they might help,” says Beeren-

expression of HER-2. The presence of this bi-

tomy. On the other hand, a codeine under-

winkel. “They have been approved by the pub-

omarker indicates whether the administration

dose will not kill the pain in a slow metabo-

lic authorities for a specific disease, such as,

of trastuzumab will be a promising option or

lizer. Fortunately, today each patient may be

for example, lung cancer, but may also be of

not. Today, tissue-based predicative molecular

assigned to one of the three groups according

help for others. One difficulty is that tumour

tests are already available for approximately

to a genetic test to result in an individually

genomes tend to be extremely different from

35 per cent of all tumours.

prepared dose.

carcinomas

involving

the

This kind of pharmacogenomics may also help to optimize the dose for patients with var-

In the end, the goal will be to find out

➜

ious

genetic

polymorphisms

which

may

influence drug metabolization. One target seems to be Cytochrome P450 2D6. This en-

if the effect of a specific API against

zyme is associated with the metabolization of approximately a quarter of all APIs which are

a tumour during a therapy may

commonly used. It is encoded by the CYP2D6 gene primarily expressed in the liver. This

be enhanced by a customized diet.

gene will be the subject of tests revealing polymorphisms relevant for the serum drug level.

20

Life Sciences plus 01 I 2015

S W I S S R E S E A R C H I Pe r s o n a l i z e d M e d i c i n e t o C r o s s t h e Fr o n t i e r

Another tool contributing to a personal-

shift the ratio of cancerous to non-cancerous

consistent and easier theoretical description

ized therapy may be the diet of the patient. At

cells towards “non-cancerous.” In the end, the

resulting in powerful practical applications.

the moment, Professor Beerenwinkel is taking

goal will be to find out if the effect of a specific

It is not clear whether biological systems

part in a research project evaluating the inter-

API against a tumour during a therapy may be

have a higher complexity and are therefore

actions between cancer drugs and food com-

enhanced by a customized diet.

more difficult to deal with or, to date, no one

ponents. In a first step, the transcriptomic re-

Of course, the future concepts set forth

has discovered the key which may open the

sponses of cancerous and non-cancerous hu-

above can only encompass a part of the lively

door to a simplified access to their nature.

man colon cells to sulforaphane and selenium

and spirited research community. To elucidate

A research group or a network of scientists

were examinated. The scientific background:

how far the art of personalized medicine may

who will deliver such a potent tool would thus

Associations between diet and carcinogenesis

extend, here is one more detail: Some re-

smooth the way for a really powerful personal-

have indicated strong negative correlations

searchers, for example, feel that the tumour

ized medicine. It might go far beyond the

between fruit and vegetable consumption

stem cell will become the target cell of cancer

months of progression-free survival which are

and tumor incidence, most consistently for

therapy in the next years4.

today often regarded as a great success.

colorectal cancer and cruciferous vegetables3.

One member of this family is Broccoli, a typi-

Physics as the Ideal

cal source of sulforaphane and selenium.

For the time being, oncological and anti-HIV

Current research results3 are based on

therapies are two of the most successful

newly generated transcriptome data in one

areas of personalized medicine. It is continu-

cancerous and one non-cancerous colon cell

ously being pushed forward by advanced tis-

line (microarray gene expression profiling),

sue biobanks, laboratory automation tech-

and their statistical analysis. This encom-

nology tailored for translational research as

passed an extraordinarily broad scale ranging

well as sophisticated data management and

from the single gene level to the network and

statistics. “However, we are far from under-

D.R.: New Evidence about an Old Drug – Risk with Codeine

to the point of the functional pathway levels.

standing biological systems in an easy way,”

after Adenotonsillectomy, New England J Medicine,

The outcomes were then integrated with pro-

says Beerenwinkel.

tein expression data.

REFERENCES 1

Dingermann, T.: OH Schmerz lass‘ nach!

PTA Professional, p. 34–35 (2014)

2

Racoosin, J.A., Roberson, D.W., Pacanowski, M.A., Nielsen,

April 24, (2013); doi: 10.1056/NEJMp1302454

He sees the success of modern physics as

3

Constantinescu, S., Hecht, K., Sobotzki, N., Erzinger, M.M.,

The results of these in vitro experiments

an ideal. Building bridges, manufacturing cars

Bovet, C., Shay, J.W., Wollscheid, B., Sturla, S.J., Marra, G.,

suggest that sulforaphane and selenium have

or designing high-end microscopes – all this is

Beerenwinkel, N.: Transcriptomic Responses of Cancerous

very different influences on cancerous and

possible because there exists a relatively sim-

and Noncancerous Human Colon Cells to Sulforaphane

non-cancerous cell lines. While sulforaphane

ple way to mathematically describe the un-

was shown to have a highly specific effect in

derlying forces and interactions. It is just a

the cancerous cell line upregulating four en-

few centuries ago that mankind was not even

of Witten/Herdecke: Wittener Wissenschaftler:

zymes of the AKR superfamily (aldo-keto re-

able to sufficiently calculate the movements of

“Die Krebsstammzelle als therapeutische Zielzelle wird

ductase), selenium had little effect on these

the visible celestial bodies surrounding the

die Tumortherapie revolutionieren.” www.uni-wh.de/

cells. In contrast, selenium turned out to be

planet Earth. For more complex systems, ad-

universitaet/presse/presse-details/artikel/neueste-

very active in the non-cancerous cell line. For

vances in calculus have allowed for a far more

forschung-zu-krebsstammzellen (accessed 2015/2/20)

and Selenium, Chem Res Toxicol 2014, 27, 377−386 4

Zänker K.S.: as cited in a press release of the University

example, it induced numerous genes involved in apoptotic, angiogenic, or tumor proliferation pathways. Here the influence of sulforaphane is very limited.

As two ingredients of our every day food administered in an every day dosage were shown to change the molecular profile of cancerous and non-cancerous cell lines in a considerably different way, the authors hope that

π

ρ2

ρ3

ρ4

Z1i

Z 2i

Z 3i

Z 4i

a specific diet may have the power to, e. g., µ1,1...K

µ 2,1...K

µ 3,1...K

ρL

...

Z Li µ L ,1...K

µ 4,1...K

H1i

H 2i

H 3i

H 4i

H Li

R1i

R2i

R3i

R4i

RLi

ε

02

Modern genomic technologies allow for detailed molecular profiles of patients‘ samples. The resulting molecular data are in general extremely high-dimensional, heterogeneous and complex. So tools of modern bioinformatics turn out to be essential in order to analyze and interpret them.

In the background of the illustration shown above, a snapshot may be recognized presenting a multiple DNA sequence alignment of NGS data (next generation sequencing). The graphics in front of it represent a statistical model for analyzing them. It allows for correcting sequencing errors and haplotype structure interference. (Picture: CC-PM, Zurich)

01

Biobanking today: state-of-the-art approach for manufacturing tissue chips. (Picture: CC-PM, Zurich)

Life Sciences plus 01 I 2015

21

SWISS RESEARCH

METROHM FOUNDATION PROFESSORSHIP AT ZHAW WÄDENSWIL

Blazing New Trails in Surfactant-Free Nanoparticles How can we achieve polymeric nanoparticles without the use of any molecular or polymeric surfactants?

Andrei Honciuc, the first endowed professor at ZHAW Wädenswil, shows how his self-developed synthetic methods can be used to produce surfactant-free nanoparticles with sizes ranging from 20 nm to 200 nm, offering good control of particle size and size-distribution.

ANDREI HONCIUC ELSBETH HEINZELMANN

als’ at ZHAW Wädenswil, a partner in the bio-

Surfactant-Free Nanoparticles on the Menu

technet network.

The first challenging task of Andrei Honciuc

of six years, a professorship for ‘new materi-

The objectives are to improve the under-

and his group was to synthesize sub 200 nm

onventional materials are a thing of

standing of interaction mechanisms between

polymeric nanoparticles without the use of

the past: what we need today are

surfaces and their environment, to develop

any molecular or polymeric surfactants. Such

smart materials that respond both

control methods for surface topography and

surfactants are chemicals which can have ad-

passively and actively to their sur-

functionality, to realize ‘smart’ materials and

verse effects when used in the form of nano-

roundings and perform a variety of program-

surfaces that react to surrounding stimuli and

particles in areas like pharma, bio-imaging,

mable functions.

to test new materials for technical applica-

in vivo applications, cosmetic formulations,

tions. “Our group is working intensively on the

medicine, etc. In just a few months, Andrei

synthesis and the design of novel intelligent

Honciuc and his team developed synthetic

These materials fulfil crucial tasks, acting as

materials in order to open up new appli-

methods yielding surfactant-free nanoparti-

self-cleaning coatings, materials with self-

cations,” explains Professor Dr Andrei Honci-

cles with sizes ranging from 20 nm to 200 nm

healing ability, chromogenic materials or fa-

uc, who received his doctorate from the Uni-

with controlled particle size and size distribu-

cilitating the controlled administration of ac-

versity of Alabama (USA) in physical chemis-

tion [Figure 1].

tive substances. To promote the development

try. “To that effect, we cooperate closely with

of solutions for the materials of the future, the

industrial partners, respecting the specific

factant-free nanoparticles is the fact that

Metrohm Foundation is funding, for a period

needs of industry.”

their behaviour as colloidal solutions or inter-

C

The Stuff of the Future

01

What

motivates

him

to

realize

sur-

02

Scanning electron microscopy (SEM) image of poly (methyl methacrylate) surfactant-free nanoparticles. (Pictures: ICBC/ZHAW Wädenswil)

SEM image of a colloidosome, Oils droplet stabilized by nanoparticles in a Pickering emulsion.

22

Life Sciences plus 01 I 2015

S W I S S R E S E A R C H I M e t r o h m Fo u n d a t i o n P r o f e s s o r s h i p a t Z H AW Wä d e n s w i l

➜

facial assemblies is predictable and reproducible. “When molecular or polymeric sur-

surfactants, due to their larger size, surface inertness and reduced mobility. Nanoparticles can also carry active molecules and are used

factants are used in excess directly from the synthesis to stabilize nanoparticles, they adsorb on their surface and completely govern their behaviour,“ explains the chemist, who has experience as a former laboratory manager at BASF Ludwigshafen. “Variations in the surfactant to nanoparticle ratio may change unpredictably during the downstream handling, affecting the stability and behaviour of the colloid or the nanoparticle surface functionality.” This could have a disastrous impact especially in formulations where batch-tobatch variations in the starting material may

as nano-carriers for drug delivery and encap-

It is a difficult task to evaluate

sulation technologies.

the true toxicology of nano-

Forging Ahead with a Pioneering Spirit Andrei Honciuc is again in the front line of re-

particles smaller than 200 nm

search with his new project, which aims to further develop his synthetic methods for

if they themselves are stabilized

making

amphiphilic

Janus

nanoparticles.

Their surfaces offer two physical properties,

by the surfactant agents having

like the Roman god Janus, who was depicted as having two heads facing in opposite direc-

their own toxicology profile.

tions. In other words: two different kinds of

lead to colloid sedimentation or even loss of

chemistry take place on the same particle. For

functionality.

example, one-half may be composed of hydro-

Provide Clarity with Scientific Studies But it is a difficult task to evaluate the true toxicology of nanoparticles smaller than 200 nm if they themselves are stabilized by the surfactant agents having their own toxicology profile. Therefore research on surfactant-free polymeric nanoparticles is urgently needed, all the more because the removal of these surfactants after synthesis is a tedious and diffi-

philic groups and the other half of hydropho-

In certain applications,

bic groups. This characteristic gives Janus nanoparticles unique properties and enor-

surfactant-free nanoparticles

mous potential in various applications, ranging from flexible solid-state displays2, mag-

could even replace some

netic and fluorescent probes3, nano-propulsion systems4, polymer blend-compatibilizers5

of the main-stream molecular

and functional coatings to smart materials and switches6, 7 including drug delivery.

surfactants.

Depending on the polarity contrast be-

cult process. Andrei Honciuc is convinced that

tween both halves, Janus particles show am-

in certain applications surfactant-free nano-

phiphilic behaviour. Nevertheless, we need to

particles could even replace some of the main-stream molecular surfactants, as he explains: “For example, the alkyl ethoxylated surfactants are often used as emulsifiers in cosmetics. Upon exposure to air or UV irradiation we can see that the products decompose and potentially can cause skin irritation1.”

Surfactant-free nanoparticles also serve as

differentiate between interfacial activity and

With our surfactant-free Janus

amphiphilic behaviour: amphiphilic particles can stabilize oil/water emulsions, whereas any

nanoparticles we aim to design

homogeneous nanoparticle may exhibit interfacial activity and stabilize such emulsions

smart colloidosome systems

due to the large interfacial attachment energy.

“The interfacial activity of Janus appears to

that respond to stimuli.

increase several fold as compared to simple

emulsions,

nanoparticle components,” declares Andrei

thus potentially reducing the amount or re-

Honciuc. “To make Janus nanoparticles largely

placing the molecular surfactant. The skin

available for applications, their synthetic

penetration of the nanoparticles can be signif-

methods should be scalable to at least gram

icantly reduced in contrast to the molecular

quantities.”

emulsifiers, creating

Pickering

04

Professor Andrei Honciuc.

03

SEM image of polymeric (PS- b-PtBA) surfactant-free Janus nanoparticles obtained by phase-separation.

Life Sciences plus 01 I 2015

23

S W I S S R E S E A R C H I M e t r o h m Fo u n d a t i o n P r o f e s s o r s h i p a t Z H AW Wä d e n s w i l

Like living organisms, colloidosomes [Figure 2] in a Pickering emulsion are an example of

compartmentalized

entities

which

can

co-exist even when they contain incompatible active substances, enzymes or reagents that would normally react or neutralize in a homogeneous medium. “With our surfactant-free Janus nanoparticles [Figure 3] we aim to build such structures and design smart colloidosome systems that respond to stimuli,” points out the active researcher. “Although many smart delivery systems have been envisioned, we believe there is plenty to do in this direction.”

In a relatively short period of time, the new group has made breakthroughs in the synthesis of nanoparticles using ultrasonic radiation and their subsequent handling, pro-

05

cessing and stabilization without any use of

Portrait of the research group of Professor Andrei Honciuc.

surfactants. These methods are scalable. “We take advantage of the energy produced during cavitation and drive our polymerization reaction, starting either from water immiscible or

A Clear Goal in Mind

need alternative delivery systems to improve

non-miscible monomers and produce a whole

His strategy is to use Janus nanoparticles for

their bioavailability. Why not mimic nature?

array of polar to non-polar polymeric parti-

the encapsulation and delivery of drugs. The

“Natural living organisms protect their interi-

cles.” – We look forward to seeing further posi-

fact is that drugs and other active substances

or by compartmentalization. In this way cells

tive results!

are often not soluble in water. Also, the next

are able to synthesize vital active substances

generation of drug formulations makes it nec-

in different compartments and release the

essary to avoid mixing the substances well in

products on demand for the next process that

advance or combining active agents shortly

takes place in another compartment,“ ex-

before release of the drugs. In such cases, we

plains Andrei Honciuc.

AUTHORS Professor Dr. A. Honciuc. Endowed Metrohm Foundation Professor, andrei.honciuc@zhaw.ch, www.zhaw.ch/icbc/nanotechnology Elsbeth Heinzelmann. Journalist science + technology

REFERENCES 1

Bergh, M. et al. Contact allergens from surfactants. Atmospheric oxidation of polyoxyethylene alcohols, formation of ethoxylated aldehydes, and their allergenic activity. J. Pharm. Sci. 87, 276–282 (1998).

2

Yin, S.-N. et al. Versatile Bifunctional Magnetic-Fluorescent Responsive Janus Supraballs Towards the Flexible Bead Display. Adv. Mater. 23, 2915–2919 (2011).

3

Kaewsaneha, C., Bitar, A., Tangboriboonrat, P., Polpanich, D. & Elaissari, A. Fluorescent-magnetic Janus particles prepared via seed emulsion polymerization. J. Colloid Interface Sci. 424, 98–103 (2014).

4

Gao, W., Pei, A., Feng, X., Hennessy, C. & Wang, J. Organized Self-Assembly of Janus Micromotors with Hydrophobic Hemispheres. J. Am. Chem. Soc. 135, 998–1001 (2013).

5

Walther, A., Matussek, K. & Müller, A. H. E. Engineering Nanostructured Polymer Blends with Controlled Nanoparticle Location using Janus Particles. ACS Nano 2, 1167–1178 (2008).

6

Yoshida, M. & Lahann, J. Smart Nanomaterials. ACS Nano 2, 1101–1107 (2008).

7

Lee, K. J., Yoon, J. & Lahann, J. Recent advances with anisotropic particles.

Curr. Opin. Colloid Interface Sci. 16, 195–202 (2011).

8

Casagrande, C., Fabre, P., Raphael, E. & Veyssie, M. Janus Beads – Realization and Behavior at Water Oil Interfaces. Europhys.

Lett. 9, 251–255 (1989).

9

De Gennes, P. G. Soft matter. Rev. Mod. Phys. 64, 645–648 (1992).

10

Binks, B. P. & Fletcher, P. D. I. Particles Adsorbed at the Oil−Water Interface: A Theoretical Comparison between Spheres of Uniform Wettability and ‘Janus’ Particles. Langmuir 17, 4708–4710 (2001).

11

Kumar, A., Park, B. J., Tu, F. & Lee, D. Amphiphilic Janus particles at fluid interfaces. 6604 (2013).

Life Sciences plus 01 I 2015

SWISS RESEARCH

ETH TRANSFER AND PIONEER FELLOWSHIP

Encouraging Innovative Minds

The step from research to application of a technology can be full of obstacles and pitfalls for scientists without education in business development or business administration. At ETH Zurich, future entrepreneurs are coached by experts of the field and financially supported by foundations in order to bridge the time gap between research and incorporating their company.

SONJA BICHSEL-KÄSER

E

responsible for the money. The amount is

selected depending on the project. Their opin-

thought to pay salaries and equipment, con-

ion is highly valued, since they bring in know-

sumables or external studies such as animal

how related to what the market requires and

TH Zurich is known for its mission of

studies for the duration of the Fellowship

which ideas have the highest potential for an

technology transfer. Moreover, defined

of 18 months. In addition, the ieLab offers a

industrial application.

in the Swiss Federal Law on the Swiss

lot of benefits for entrepreneurs-to-be. Be-

Federal Institutes of Technology, one of

sides workspaces (laboratories and offices)

How successful is the programme so far?

the purposes of ETH is “to exploit their re-

the ieLab includes networking among the pio-

Since 2010 we could assign the grant to 48 ap-

search findings.” In order to transpose this

neers and existing spin-offs, coaching of the

plicants, which already resulted in 17 ETH

task, ETH community is supported by the

pioneers, preparing for further financing of

spin-offs. In 2014 alone, 35 researchers ap-

technology transfer office “ETH transfer” – a

their business idea or seeking for industrial

plied for the grant and 13 fellowships were se-

staff unit which provides support to all ETH

partners. The ieLab has two different sites: at

lected. The latest projects of the ieLab are cur-

researchers and employees.

ETH Zentrum for e. g. the mechanical and

rently looking for further financial support to

Dr. Marjan Kraak is staff member of ETH

electrical engineering and ICT projects (since

continue their spin-off after the grant is fin-

transfer and manager of the “Spin-off Group,”

2012) and at ETH Hönggerberg for the life

ished. Partners for this can be e. g. CTI, the

which supports potential company founders

sciences projects (since 2013) in the newly

Zürcher Kantonalbank, Business Angels or

among the ETH members. In addition, she is

constructed HPL building. There, the labora-

Venture Capitalist. Examples of promising

responsible for the ieLab (Innovation and En-

tory workspaces are equipped for work at Bi-

ETH spin-offs coming out of the Pioneer Fel-

trepreneurship Lab) in life sciences and the

osafety Level 1 and 2.

lowship Programme are: Glycemicon who develop a novel treatment for diabetes type 2 or

Pioneer Fellowship Programme which promotes ETH students who aim for turning their

Who are the founders and sponsors

Versantis who develop a detoxification process

research findings into an industrial application.

of the programme?

with a versatile antidote. Whether the spin-

The programme was founded in 2010 by

offs resulting from the Pioneer Programme

Mrs Kraak, what is the meaning of the Pioneer

Roland Siegwart who was Vice President Re-

will result in successful companies can’t be

Fellowship in the context of technology transfer?

search and Corporate Relations at ETH until

predicted. But there are successful older spin-

Dr. Marjan Kraak: Technology transfer is

the end of 2014. Sponsors are the ETH Zurich

offs, as for instance Covagen, founded in 2007,

supported by ETH transfer via different ap-

Foundation, ETH Zurich and the Hasler

which was sold to Johnson and Johnson for

proaches. First, we assist ETH researchers in

Stiftung. The list of donors comprises compa-

more than 200 million Swiss francs last year.

working with third parties when it comes to

nies, organisations and foundations as well as

Finally, the success of the programme is multi-

research contracts or cooperation with the in-

private persons, several of them ETH alumni.

plied by spin-off entrepreneurs coming back to ETH to assist and coach the next generation

dustry. Second, we consult researchers in inventions, patenting and licensing. And third,

Who can profit from the Pioneer Fellowship

we support researchers in starting their own

Programme and what selection procedure

company based on an idea or technology de-

do applicants have to face?

veloped at ETH Zurich.

The grant is given to Master or PhD Students

For each of these process steps from re-

from ETH Zurich after finishing their degree

search to market – financial support is need-

to support their further research on the actual

ed. And sponsors are often hard to find. Espe-

topic. New research ideas are therefore not

cially when the research findings are still at

accepted. The researchers must apply for the

the very beginning, it is necessary to bridge

grant with the consent of their thesis supervi-

the gap between the patent proceeding and

sor, which must be an ETH professor. These

the incorporation of a spin-off. With the Pio-

prerequisites lead to an early stage selection,

neer Programme, we directly support re-

thus reducing the eligible applicants.

searchers in this intermediate step.

The application then passes an expert

of entrepreneurs.

DR. MARJAN NIENKE KRAAK

opinion and selected applicants can present What does the Pioneer Fellowship comprise?

their projects to a jury consisting of ETH Pro-

Dr. Marjan Nienke Kraak is a member of ETH transfer,

Financially, an overall amount of 150,000

fessors, members of ETH transfer and finally

Manager of the Spin-off Group, responsible for the ieLab

francs is granted to the fellow who is solely

industry partners. The industry partners are

Life Sciences and the Pioneer Fellowship Programme.

Life Sciences plus 01 I 2015

25

SWISS RESEARCH

ETH PIONEER FELLOWSHIP

Turning Innovation into Business How to turn a scientific discovery into a profitable business? ETH researchers are assisted in bringing their inventions from research to market. Andreas Essig is one of the ETH Pioneers, who benefits from a programme which offers money and coaching for entrepreneurs-to-be.

SONJA BICHSEL-KÄSER

D

ed and fractionated by several chemical sepa-

in patent law and intellectual property, a sci-

ration procedures such as dialysis or precipi-

entist can get easily lost in the world of para-

tation. Thus, fractions with antimicrobial ac-

graphs,” remarks the researcher.

igging in the dirt can pay off. This is

tion could be identified. Finally, the compound

In this context, Essig was nominated for

demonstrated by the story of re-

was identified and its gene sequence could be

the ETH Spark Award 2014, which awards the

searcher Dr. Andreas Essig from the

found since the model organism’s genome had

most promising patent of the year. Essig was

Institute of Microbiology at ETH Zu-

been fully sequenced before. Copsin could

among the top 5 of the nominees. The nomina-

rich. In the course of his doctoral thesis, he

then be recombinantly expressed in the yeast

tion was of great interest for the press world-

cultivated a fungus on horse dung – its natural

model

Pichia

Higher

wide. ETH Transfer finally suggested him to

substrate – and was finally rewarded by find-

amounts of the protein were needed for fur-

apply for the ETH Pioneer Programme. Which

ing a novel antibiotic substance.

ther analysis and were yielded by cultivation

he did – successfully.

organism

pastoris.

While studying the proteome of the model

of the yeast in a bioreactor system. “For

organism Coprinopsis cinerea he noticed that

NMR-structure analysis of the protein and for

Grants for Innovative Ideas

the fungus inhibits growth of several bacterial

identification of the mode of action I got help

The ETH Pioneer Fellowships are an instru-

strains. In fact, many fungi or other micro-

and support of researchers from the group of

ment awarded to people intending to inde-

biologic organisms produce compounds with

Gerald Wider at ETH and from the group of

pendently develop a highly innovative prod-

antimicrobial effects. They do so for example

Hans-Georg Sahl in Bonn,” Essig acknowl-

uct or service to be exploited commercially

to overcome food competitors. “The antibiotic

edges. Copsin acts by inhibiting the peptido-

and/or for the benefit of society. They are the

compound of Coprinopsis, called Copsin, is

glycan synthesis of the bacteria, which is es-

result of a competitive process to ensure the

probably also part of a competition machin-

sential for building of the cell wall.

innovation potential of the successful applica-

ery,” says Essig. But Copsin is – unlike other

In order to secure the rights of a possible

tion. (Read more about it in the interview with

known antibiotic proteins – highly stable. “The

industrial application of Copsin, Essig finally

Marjan Kraak.) “Unlike Grants from the Swiss

unique structural properties of Copsin make it

applied for a patent of the substance. An or-

National Fond (SNF), which supports funda-

a possible scaffold for new antibiotics.” The

ganisation within ETH called ETH Transfer

mental research, Pioneer Fellowships are

compound might therefore also be a solution

assists researchers who want to patent their

dedicated to scientists with projects on ap-

for increasing antibiotic resistance of patho-

findings. The support mainly consists of con-

plied research. Therefore, the selection for the

genic bacterial strains.

sulting by patent lawyers, “which was the big-

grant starts already during candidacy. Only

gest benefit so far. Without the help of experts

about 20 % of the applicants could be consid-

Instead of proceeding fundamental re-

ered in 2014.” In Andreas Essig’s case, the Fel-

other microorganisms and pursuing an aca-

lowship offers him the possibility to proceed

demic career, Essig switched focus to applied

with his studies, which includes of course fi-

research and entrepreneurship: “Finding a possible industrial application for my discov-

➜

search on the interaction of the fungus with

ery and maybe taking a step further and founding a company, is very attractive to me,”

“I feel very privileged

Spark Award

for having obtained such

could confirm that Copsin was still produced when the fungus was grown in vitro on glass beads in a liquid culture medium instead of the natural horse dung substrate. While grow-

lated equipment. Professor Markus Aebi, Essig’s doctoral thesis supervisor, offered him

Essig looks ahead.

During the proceedings of his studies, he

nancial resources for salaries and project re-

to stay at the institute and make use of the lab infrastructure. “Otherwise, I could have profited from the workstations of the ieLab, which is included in the Pioneer Programme’s services,” Essig explains. For biologists, the term

a scholarship.”

of the Pioneer Fellowship is rather tight. “If

DR. ANDREAS ESSIG. Researcher at ETH Zurich at the Institute of Microbiology

you work on drug development or drug research, you must be prepared for a long way,” Essig says. “Unlike informatics, where soft-

ing, the fungus secreted proteins in the cul-

ware applications for instance can be brought

ture medium. In order to find out which com-

to market quite fast, in drug research you

pound of the secretome was the antimicrobial

need great powers of endurance. From re-

substance, the secreted proteins were extract-

search to market, about 10 years may pass.”

26

Life Sciences plus 01 I 2015

S W I S S R E S E A R C H I E T H P i o n e e r Fe l l o w s h i p

Potential new drugs undergo a long time of complex preclinical and clinical testing, before they can be registered as a drug. Since the beginning of 2015, Essig is now supported by the Pioneer grant. He is currently sounding out, in which field Copsin could be applied. “The advantage of Copsin is that the protein is quite small and has, compared to its size, rather many disulfide bonds, which make it highly stable.

Thus, the protein can be treated with high temperatures or extreme pH values without losing its activity. This is important when processing the compound. And hence, a great variety of applications are imaginable: Copsin could be used in food industry – where high temperatures are often applied, or as pharmaceutical for the treatment of humans or animals. The substance might even be interesting as a novel antibiotic against multi- resistant hospital pathogens.” In fact, studies on the activity of Copsin against the most abundant hospital pathogens are already in process.

Don’t Give up – Start-up!

Besides the financial support, the Pioneer Programme includes education in business administration as well. “Once a week I attend a seminar with other Pioneer Fellows,” Essig explains. “Pioneers make their first awkward steps in the process of company founding. And they need assistance and inputs.” In the seminars they learn how to elaborate a business model and how to found a start-up company. There are several consulting companies coaching the Pioneers.

Whether Copsin will make it to market still remains uncertain. For sure, the goal of Essig’s work is to further investigate the substance and enter preclinical trial studies. But there is as well the researcher’s sword of Damocles: “Every experiment you make might go wrong and thus put an end to your studies,” Essig ponders. But as a researcher he focusses on his work, not on the outcome. “Of course, scientists always bear in mind that their findings could change something, like for example healing the sick. But in the meantime one has to proceed with the daily business.” And to do so, motivation is needed: “One has to be passionate about this kind of work, since it’s not a nine-to-five job.” In addition, there is the responsibility towards the sponsor. “I feel very privileged for having obtained such a scholarship. For sure, I want to seize this opportunity and make something of it.”

Dr. Andreas Essig works as researcher at ETH Zurich at the Institute of Micro-biology. He discovered a novel antimicrobial substance by analysing the secretome of the fungus Coprinopsis cinerea.

Thanks to the Pioneer Fellowship he is able to pursue applied research in order to exploit his findings.

Life Sciences plus 01 I 2015

27

SWISS RESEARCH

CELL BIOLOGY

Unmasking Salmonella Salmonella are able to manipulate cells of the innate immune system in order to replicate inside them. Cell biologists from the University of Basel recently elucidated a host cell’s defense mechanism uncovering Salmonella hiding inside macrophages. The results cast a new light on complex inflammatory mechanisms and even sepsis.

BEATE PEISELER-SUTTER

ologist Petr Broz, Professor at the Centre for Molecular Life Sciences of the University of

H

Basel, together with his five-man team, is reigher vertebrates display two types

searching the innate immune system of mice,

of host response to infection: The ef-

which is nearly identical to that of humans.

ficient but relatively slow adaptive

The group uses Salmonella enterica serovar

immune system, which reacts to

typhimurium as a bacterial pathogen model

pathogens by producing specific antibodies

inducing immune responses in mice and their

and second, as “first line of defense” the evo-

immune cells. S. typhimurium is the most com-

lutionarily conserved innate immune system.

mon pathogen which causes diarrheal illness

Latter includes the anatomical and physiolog-

in humans but is harmless in patients with an

ical barriers of the skin with its sebum and

intact immune system. In mice, S. typhimuri-

sweat, as well as lysozyme containing tears

um triggers typhoid-like disease.

and saliva, gastric acid and the commensal gut flora, all work to keep pathogens in check. A

Boarding the Gut Epithelium

central component of the genetically deter-

“Salmonella infections most often occur due

mined innate immune system are pattern rec-

to intake of contaminated food. The pathogens

ognition receptors (PRRs) on the surface of

enter the epithelial layer of the small intestine

immune and other cells. These receptors de-

with the help of molecular injection systems,“

tect highly conserved pathogen specific struc-

explains Petr Broz. The type III secretion sys-

tural motifs, so-called PAMPs (Pathogen-

tem of Salmonella and other Gram-negative

associated Molecular Patterns). Molecular bi-

bacteria acts as such an injection system and

iStock

28

Life Sciences plus 01 I 2015

SWISS RESEARCH I Cell Biology

consists of over twenty proteins. It spans the inner and outer

vacuole take place. But we were able to show

bacterial membrane up to the plasma membrane of the host

that the process activates the inflammatory

cell. In order to prepare host cells for pathogen uptake, Sal-

enzyme caspase-11,” summarizes Broz in his

monellae inject a cocktail of effector and signalling proteins

findings.

into the host cell’s cytoplasm. Following the infection of epi-

rected proteases, in short caspases, cleave spe-

thelial cells, Salmonellae then move to the underlying layer of

cific peptide bonds. Eight of the twelve known

connective tissue (lamina propria). The lamina propria is in-

caspase enzymes play important roles in pro-

terspersed with immune cells including scavenger cells (mac-

grammed cell death, termed apoptosis. Pro-

rophages) as part of the innate immune system, which recog-

grammed cell death also occurs for instance

nize the pathogen.

when tissue has to be reduced during embry-

Cysteine-dependent

aspartate-di-

onic development for removal of webs from

Salmonellae stay hidden in the cellular

➜

phagosome in the macrophage, where they replicate until they escape from the host cell and later infect more cells.

the interdigital space between fingers and toes. Caspase-11 has been discovered only three

years

ago

in

mice.

It

resembles

caspase-4 in humans and can trigger death of Salmonellae infected host cells so that the pathogen cannot replicate anymore. In order to distinguish this kind of cell death as a consequence of bacterial infection from apoptosis in general, the process is named pyroptosis.

“Host cell mediated breakup of pathogen occupied vacuoles is an essential immune function and necessary for efficient recognition of

Detection works via PRRs of the Toll-like receptor (TLR) family, supervising the extracellular space. The receptor ver-

pathogens

sion TLR-4 detects lipopolysaccharides (LPS) occurring in

states Broz. „As soon as Salmonellae show up

by

the outer membrane of Gram-negative bacteria such as Sal-

in the cytosol, their LPS activate caspase-11,

monella. 13 highly conserved TLRs have been identified in

leading to cell death. Research on sepsis

mice and humans so far. All are homologues of the toll pro-

mouse

tein, which was first discovered in the mid-1990s in the fruit

caspase-11-mediated cell death quickly re-

fly Drosophila. TLRs activate signal pathways regulating bio-

sults in the death of the animal and that this

synthesis of cytokines, which are involved in inflammatory

process is independent of TLR-4, unlike previ-

processes. Furthermore, they interconnect the adaptive and

ously anticipated. With regard to the treatment

innate immune systems. In 2011, French biologist Jules Hoff-

of sepsis, trials with TLR-4 inhibitors have

mann und American immunologist Bruce Beutler received

proven unsuccessful until now. It may be more

the Nobel Prize for their work on the TLR-mediated innate

promising to inhibit caspase-4,” reflects Broz.

models

inflammasome

has

recently

complexes,“

shown

that

immune response.

Hide and Seek Subsequent to detection, the macrophage engulfs the Salmonella bacterium, resulting in its uptake into a membrane-

➜

enclosed cell organelle (a vacuole called phagosom) inside the macrophage. Usually, such vacuoles fuse with lysosomes, cellular organelles with content of low pH containing hydrolytic enzymes. The resulting phagolysosomes then digest the pathogen. However, Salmonellae can prevent the fusion of

Interferon induced GTPases

vacuole and lysosome, probably by way of bacterial proteins inhibiting intracellular vacuole transport. Salmonellae stay

recognize Salmonellae-contain-

hidden in the cellular organelle where they replicate until they escape from the host cell and then infect further cells.

ing vacuoles and attach to them

Petr Broz and his team were able to show that host cells are not defenseless against this hidden pathogen, but spring into

causing them to burst open.

action against the invaders. Due to the initial detection of the pathogen by TLR-4, cells would have produced type 1 interferons, immune-stimulating proteins which trigger the biosynthesis of small guanylate binding proteins, (GTPases), in

Inflammasome complexes

infected cells and in nearby cells. These interferon induced GTPases recognize Salmonellae containing vacuoles and at-

form when bacterial

tach to them causing them to burst open. The result is that Salmonella cannot replicate anymore and are detected by in-

components bind to NLRs

ternal PRRs again.

“The cell recognizes only about one of thirty infected vac-

in the cytoplasm.

uoles. We do not know yet how recognition and breakup of the

Life Sciences plus 01 I 2015

29

SWISS RESEARCH I Cell Biology

Inflammasome Enhances Signal

external stimuli are transduced into the inner

the two mechanisms that are mediated by

The fact that there are not only pattern recog-

cell, signal cascades often heavily enhance the

caspase-1, the maturation of IL-beta and IL-18

nition receptors monitoring the extracellular

incoming signal. The remarkable structure of

and the cell death of macrophages, are geneti-

space but also PRRs monitoring the cytosol

canonical inflammasomes seems to be of sub-

cally separated and proceed in different in-

has only been known for ten years now. These

stantial significance for amplifying the signal

flammasome complexes. In the process of py-

cytosolic sensors are called NOD-like recep-

after PAMP recognition by just a few NLRs,“

roptosis, caspase-1 is activated without auto-

tors (NLR, NOD means nucleotide binding oli-

reasons Broz.

proteolytic cleavage in ASC-independent in-

gomerization domain). TLRs and NLRs trigger

flammasomes. At present it is only possible to

interconnected signal cascades. NLRs are a

Fast Reaction to Pathogen Infestation

central element of high molecular weight cy-

Inflammasome integrated pro-caspase-1 en-

masomes under the microscope by labelling

tosolic protein complexes, so called inflam-

zymes tetramerize and start cleaving each oth-

their components with different fluorescent

masomes, which have been discovered and

er. This autoproteolytic cleavage produces ac-

dyes. “Such ASC-dependent inflammasomes

first described in 2002 by the research group

tive caspase-1 enzyme. Caspase-1 converts in-

assemble within twenty to thirty minutes. Their

of Jürg Tschopp at the University of Lausanne.

active inflammatory mediators into their active

diameters are about one micrometer, correlat-

Different inflammasomes are distinguished

form, which had been made in response to ac-

ing to half of the diameter of a bacterial cell.

by their NLRs. So far, only few of the 23 differ-

tivation of surface receptor TLR4: Precursor

Usually, only one inflammasome per cell is

ent NLR variants in the human genome are

cytokines pro-IL-1beta and pro-IL-18 become

built,“ illustrates Broz. Caspase-11 is likewise

well characterized. The receptors NLRC4 and

active IL-beta and IL-18. Their release from

activated within an inflammasome, which, to

NLRP3 are involved in salmonellosis. NLRC4

the cell causes inflammation and attracts cells

better differentiate from caspase-1 activated

recognizes bacterial flagellin and the rod-like

of the innate immune system such as neutro-

inflammasomes, is called “non-canonical“ in-

sub-unit of certain type III secretion systems.

phil granulocytes, macrophages and natural

flammasome. Caspase-11 is not able to cleave

The NLRP3 ligand has not been identified so

killer cells to the site of infection. Both

pro-IL-1beta und pro-IL-18 and has to activate

far. Inflammasome complexes form when

caspase-1 as well as caspase-11 induce pyrop-

caspase-1 for this purpose. The underlying

bacterial components, toxins, xenobiotics etc.

tosis. Broz and co-workers discovered that of

mechanism isn’t yet understood.

visualize the bigger ASC-dependent inflam-

bind to NLRs in the cytoplasm. A few receptors dimerize under the ligand’s influence and recruit several thousand molecules of the inflammasome adapter ASC (apoptosis-associated speck-like protein containing a CARD) plus several thousand non-functional pro-

02

caspase-1 molecules form the cytosol. In co-

GTPases (green) attack Salmonella cells (red).

operation with Sebastian Hiller, Professor at the Center for Molecular Life Sciences of the University of Basel, Broz and co-workers have conducted structural biology studies on casome deeper insights into the complex. “The adapter proteins ASC consist of two domains.

One of them builds long fibril-like aggregates

01

by oligomerization, while the other one is holding the whole structure together and participates in activation of pro-caspase-1. When

Petr Broz is professor at the Biozentrum of the University of Basel since 2013. Together with his team he investigates how bacterial pathogens are detected by the host and how they circumvent being detected by the innate immune system.

Lausanne Beaulieu Lausanne

06 - 07 May 2015 The show for laboratory technologies & services

Keynote speech

Benoit Dubuis Campus Biotech

INVITATION CODE FOR A FREE ENTRANCE:

4802

Register at: www.easyfairs.com/laboteclausanne

30

2015

nonical inflammasomes and thereby gained

Life Sciences plus 01 I 2015

SWISS RESEARCH

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LS2 ANNUAL MEETING

Life Sciences in the Limelight The annual meeting 2015 of the Life Science Switzerland coalition was guided by the motto “light.” Researchers within Switzerland and international guests presented their recent work.

Methods, techniques and possible commercial applications were discussed in a tight schedule.

SONJA BICHSEL-KÄSER

T

his year’s Life Science Switzerland (LS2) annual meeting took place in the

promote networking among scientists, to show

In order to detect light, vision is necessary.

possibilities of career development to young

Still, there are several pathological aberra-

researchers and finally to reach the broad

tions affecting the vision in humans. In a sym-

public beyond the academic experts.”

posium entitled seeing the light, visual neuroscientists presented their work on under-

premises of University Zurich Irchel

Mixed Bag of Symposia

and included plenary lectures, sympo-

In life sciences, light is an important physical

sia, poster exhibitions and an industry exhibi-

force. It is a tool, which can be helpful to make

Optogenetics

tion. In the context of the event, the Friedrich

things visible or to control genes but it also has

Related to the meeting’s motto, many scientists

Miescher Award 2015 was granted to Martin

the power to cause severe problems in living

nowadays work with light-responsive proteins

Jinek, assistant professor at the Biochemical

organisms, when for example damaging DNA

in order to investigate the function of genes or

Institute of the University of Zurich. Jinek was

resulting in carcinogenesis or even worse, cell

to study complex neuronal networks in tissue

awarded by the Swiss Society for Molecular

death.

cultures or even in the brain of living animals.

standing the early visual processing.

and Cellular Biosciences for his work on the

Light as a potent modulator of processes in

The technique is called optogenetics and

bacterial CRISPR-Cas9 system, which plays an

biology and medicine was discussed in a sym-

makes use of light-sensitive proteins which

important role in phage defence in bacteria.

posium. Light determines our days in terms of

allow to turn neurons on or off selectively.

The LS2 Meeting 2015 was guided by the

biological rhythm and the day-night cycle and

The reaction to the light stimulus is immediate

motto Light: From the Basis of Life to Life Sci-

affects sleep and alertness and well-being.

and can be detected almost in real-time.

ence Technologies. Thierry Soldati, president

This can be investigated on molecular level,

Usually, light responding channelproteins

of LS2, explained the main targets of the

since DNA methylation plays a role in circadi-

– such as Channelrhodopsin 2 (ChR2) or

meeting in his welcome address. “We aim to

an behaviour or on behavioural level.

Halorhodopsin (NpHr) are used. These mem-

Life Sciences plus 01 I 2015

31

RUBRIK VERSAL I Unterrubrik

brane proteins were originally found in algae

(a) EEG head-set

and can be recombinantly expressed in ani-

eSense value plot

Electroencephalogram

mals. ChR2 for instance is activated by blue light, and its opening leads to a Na2+influx,

Bluetooth eSense value

resulting in a membrane depolarisation and induction of an action potential as a neuronal

(b) BCI, Field generator interface

signal. Conversely, the NpHr is a chloride

Arduino single microcontroler with time-relay device

Transmiter coil

pump which leads to hyperpolarisation of the membrane upon illumination with yellow light.

Introducing these proteins into cultured cells or the brains of live animals, the side effects of

(c) Field generator Reciever coil Membrane

classical neuronal mainipulation techniques

Cultivation chamber

(drugs, electrical stimulation) can be circumvented. The introduction of the Rhodopsins

RC

(d) Inductive power link

can be achieved by either generating transgenic mice or by transfection with prepared lentivirus or adeno associated virus. The readout of an experimental set up can be made with

Receiver coil (RC)

voltage-sensitive dye imaging. The technique monitors the electrical activity of neurons ex

measurement as in vivo detection methods.

Oxytocin Attenuates Fear Response

Designer cells

(e) Wireless-powered optogenetic implant

vivo and in vivo. A second class of readout involves electrical recording and behavioural

NIR-LED

Fig.5

Scheme of the experimental set up for mind-controlled gene expression: Mental power is measured by electro-encephalogram EEG, transmitted via the interface and field generator to the implant and then translated into a light stimulus. NIR light-sensitive cells in the implant then release alkaline phosphatase SEAP. (Picture: Marc Folcher)

Professor Ron Stoop, Team Leader at the Centre for Psychiatric Neurosciences of the University of Lausanne presented his studies on the effects of oxytocin on the behaviour of mammals during fear reaction. Oxytocin is a hypothalamic neuropeptide, known for its role during lactation and childbirth in humans and

he and Marc Folcher, both from the Depart-

The system consists of an EEG headset

mammals and acts as a neuromodulator in a

ment of Biosystems Science and Engineering

that captures brain activity. The activity is

fear reaction circuits. Oxytocin containing

ETH Zurich, published their findings on

then transferred wirelessly to a brain-com-

axons originating from the hypothalamus

mind-controlled gene expression. In the ap-

puter interface (BCI) which is linked to an

reach to the central amygdala, which is in-

proach of controlling genes with mind power,

optogenetic implant containing a cultivation

volved in fear response. In order to investigate

the scientists combined optogenetic and cy-

chamber harbouring designer cells sensitive

how oxytocin is released in the central amyg-

bernetic tools. The latter are electromechani-

to near-infrared light. Thus, when brain activ-

dala, optogenetic tools were used.

cal man-machine interfaces that allow brain

ity starts, the BCI translates the EEG signal

By means of recombinant viruses, light-

activities to control bionic prostheses. Opto-

into a near-infrared light signal. The light

sensitive Channelrhodopsin was introduced

genetics provides the techniques for light-

then activates a modified light-sensitive pro-

in the hypothalamic neurons. Upon exposure

controlled therapeutic interventions and con-

tein within the genetically modified cells. This

to blue light, the oxytocin axons activated a lo-

trol of brain and gene activity.

starts a signal cascade, which finally results in

cal GABAergic circuit in vivo. GABA inhibits

target gene expression and thus production

neurons in the output region of the central

of SEAP, a secreted alkaline phosphatase. The

amygdala. In vivo, the blue light stimulus led to an oxytocin release which remarkably decreased the freezing response in fear condi-

secreted phosphatase then diffuses out of the

About Life Sciences Switzerland

precise results and better animal welfare,” Stoop emphasizes. In his studies, he could measure that stress was also less, if social buffering was applied during blue light fear induction. An individual accompanied by a second individual shows a reduced fear response upon light stimulus. The results of Ron Stoops work show that oxytocin release from local axonal endings influence fear behaviour.

“These data could serve as a basis for further research in psychophysiology of breast-feeding women and the development of postpartum depression.”

Controlling Genes with Mind Power

bloodstream of the mouse where it can be measured. This groundbreaking approach

tioned rats. “With the means of optogenetics, we provide better science in terms of more

chamber and out of the implant into the

The coalition

Life Sciences Switzerland – the union of Swiss Societies for Experimental Biology – is a non-profit organisation dedicated to advancing biological sciences within the Swiss academic community.

The union focusses on the exchange among scientists and the dialog between the scientific community and the society at large. www.naturalsciences.ch/ organisations/ls2

works in the human-mouse combination: Humans generating different mental states such as concentration (focussing), meditation (relaxation) or biofeedback control could influence the SEAP production in the subcutaneous implants in mice.

“We hope that our findings can be used in treatment of epilepsy or chronic pain,” said Fussenegger. “Thanks to our system it would be possible to automatically release drugs if an illness-triggered intentional or unconscious brain activity occurs.” This means that drugs could be released before an epileptic attack and thus preventing it. Another advantage of the system is its reversibility, which is needed for applications in humans. With the

Close to science fiction is the work of profes-

fast and local release of drugs, dosages could

sor Martin Fussenegger. In November 2014,

be reduced to a minimum.

32

Life Sciences plus 01 I 2015

SWISS RESEARCH I LS2 Annual Meeting

Professor Ron Stoop from the University of Lausanne explains the role of oxytocin in the fear response.

(Picture: Shem Johnson)

Networking and knowledge exchange during poster exhibition.

(Picture: Shem Johnson)

Thomas Egli, former professor at EAWAG Dübendorf, presents the findings on UVA disinfection of drinking water. (Picture: Shem Johnson)

Implant for mind-controlled gene expression: The implant consists of a receiver coil, a NIR-LED and cultivation chamber. In order to show the implant’s size, a Coin of one Swiss Franc is displayed.

(Picture: Marc Folcher)

Solar Disinfection of Drinking Water

sunlight treated cells,” says Egli. “We are sure

“UV lamps are abundant for the disinfection

that the effect of solar stress affects the pro-

of water in chemical and pharmaceutical in-

teins. Protein damage can’t be repaired fast

dustry,” says Thomas Egli, former professor at

enough,” he further explains. “In order to re-

Eawag Dübendorf. But instead of UV lamps,

pair and resynthesize the proteins, time and

simple sunlight can do: Solar disinfection of

resources are simply too short.” Luckily, Sodis

• Zhang, F. et al.: Optogenetic interrogation of neural

drinking water is promoted by the Eawag’s

technique also works for bacteria growing in

circuits: technology for probing mammalian brain

Sodis project which wants to help people in

anaerobic conditions. And last but not least,

structures, Nature Protocols, Vol. 5 No. 3, 439–456, 2010

countries with poor hygienic conditions.

even viruses get inactivated by solar disinfec-

• Stroh, A. and Diester, I.: Optogenetik: Eine neue Methodik

By incubating water in a PET bottle in the

tion.

REFERENCES • Folcher, M. et al.: Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant, Nature Communications, 5:3592, DOI 10.1038, 2014

zur kausalen Analyse neuronaler Netzwerke in vivo,

plain sun for 5 hours, the amount of viable

Neuro Forum 4 280–289, 2010

bacteria decreases below pathogenic levels.

• Knobloch, H. S.: Evoked Axonal Oxytocin Release in the

“Researchers of the project still examine how

Central Amygdala Attenuates Fear Response, Neuron,

this works.” Heat cannot be the reason for the

Vol. 73, Issue 3, 553–566, 2012

reduction of bacteria, since the water only

• Berney, M.: Flow-cytometric study of vital cellular functions

reaches 50 °C during treatment and is there-

in Escherichia coli during solar disinfection (Sodis),

fore not boiled. E. coli bacteria were exposed

Microbiology, Vol. 152, No. 6, 1719–1729, 2006

to artificial sunlight in order to investigate cellular functions. It could be observed that exposure had different effects. First, efflux pump activity and thus ATP synthesis decreased due to a lower proton motive force.

The loss of membrane potential, glucose uptake activity and cultivability could be detected and finally the cytoplasmic membrane be-

PEOPLE AND PROJECTS MENTIONED IN THIS ARTICLE

came permeable. The exposure to UVA at a flux comparable to global sunlight damaged E. coli cells which were no longer able to repair the damages and finally died. Researchers therefore suggest that UVA interferes with the respiratory chain of the bacteria. The effect of solar stress on proteins is higher than UV-induced DNA damages, as might be supposed when thinking of UV damage and mel-

Prof. Dr. Martin Jinek. University of Zurich, Institute for Biochemistry. www.bioc.uzh.ch Prof. Dr. Martin Fussenegger and Prof. Marc Folcher. ETH Zurich, Department of Biosystems Science and Engineering. www.silva.bsse.ethz.ch Prof. Dr. Ron Stoop. University of Lausanne, Centre for Psychiatric Neurosciences, Unit for research on the Neurobiology of Anxiety and Fear. www.chuv.ch/psychiatrie/ en/dp_home/dp-recherche/dp-recherche-centres/dp-cnp/dp-anxiete.htm Prof. Dr. Thomas Egli. Former Head of Drinking Water Microbiology, Swiss Federal Institute for Environmental Science and Technology (EAWAG). www.sodis.ch

anoma development in human skin. “It is evident that DNA synthesis is still functioning in Life Sciences plus 01 I 2015

33

ANTIBIOTIC RESISTANCE

Will We Survive?

Dangerous antibiotic-resistant bacteria and other pathogens have now emerged in every part of the world and threaten to roll back a century of medical advances. The search for and the production of new antibiotic substances is of major importance.

CLAUDIA BORCHARD-TUCH

I

are of crucial importance in fighting resistant

are now hardly damaged by the new substanc-

and multiresistant pathogens. A research

es, while the antibiotic effect against patho-

team including Andrea Vasella, Professor

gens is sustained, as shown in trials with mice

n Switzerland, each year a severe infec-

Emeritus at the Laboratory of Organic Chem-

infected with Staphylococcus aureus. Toxico-

tion with antibiotic-resistant bacteria is

istry at ETH Zurich, and Erik Böttger, Profes-

logical experiments shall follow as the next

diagnosed in about 1000 hospital patients.

sor at the Institute of Medical Microbiology,

step, in which the effectiveness and the anti-

At least 80 cases are fatal. World Health

University of Zurich, modified aminoglyco-

bacterial potency are optimized, before the

sides and thus improved effectiveness3.

actual development of the medication can

Organization (WHO) drew on drug-resistance

take place3.

data in 114 countries and concluded that dan-

Aminoglycosides fight bacteria in a sensi-

gerous antibiotic-resistant bacteria and other

tive spot: their protein producing ribosomes.

pathogens have emerged in every part of the

Human cells are also equipped with ribo-

Teixobactin: A group of researchers led by

world and threaten to roll back a century of

somes that look deceptively similar to those of

Professor Kim Lewis of Northeastern Univer-

medical advances1 [Figure 1].

the bacteria, which is why the aminoglyco-

sity in Boston, USA, developed a novel anti-

The main cause of antibiotic resistance is

sides also sometimes latch onto them, leading

biotic, which the scientists named Teixobac-

antibiotic use in animals and humans. Bacte-

to damage: Ototoxic side effects may cause

ria are able to develop resistance, not just as

loss of hearing or even deafness and may be

the consequence of mutations in the targets of

traced back to irreversible damage of the sen-

antibiotics, but by acquiring genes conferring

sory cells in the inner ear3.

resistance to antimicrobials .

2

Novel Antibiotics

Aminoglycosides fight

The Swiss research team modified the structure of aminoglycosides so that they can distinguish between human and pathogenic

Various antibiotics with low risk of resistance

ribosomes far better. 40-O-substituted amino-

are under development.

glycosides possess increased selectivity towards bacterial ribosomes and show little ac-

Modified Aminoglycosides: WHO upgraded

tivity for any of the human drug-binding

aminoglycosides into the group of drugs that

pockets of ribosomes3. Human auditory cells 34

➜

bacteria in a sensitive spot: their protein producing ribosomes.

Life Sciences plus 01 I 2015

SWISS RESEARCH I Antibiotic Resistance

in a single step with hundreds of individual

➜

tin. They used an important new tool – the Isolation Chip (ichip) – to grow non-culturable organisms in their natural environment4. Ap-

cultures incubating on a single chip4.

In a screen of non-cultivable bacteria, Lewis and his team discovered the new antibiotic

proximately 99 percent of all species in external environments are non-cultivable, which means that they do not grow under laboratory conditions. Apparently, the natural environment of bacteria holds key ingredients that have not been discovered. A solution of the problem is to bring the natural environment of the bacterium into the laboratory4. For this, a diffusion chamber was developed which sandwiches bacterial samples between a breathable material with pores that are big enough to let nutrients in and waste out, but small enough to contain the bacteria. The dif-

CALO2 is able to sequester and neutralize bacterial toxins.

Without toxins, the bacteria are rendered defenseless and can be eliminated by the cells of the host’s own immune system.

Teixobactin. It inhibits cell wall synthesis by binding to a highly conserved motif of lipid II (precursor of peptidoglycan) and lipid III (precursor of cell wall teichoic acid). The researchers did not obtain any mutants of Staphylococcus aureus or Mycobacterium tuberculosis resistant to Teixobactin4.

Alternatives for Antibiotics It can be assumed that each antibiotic triggers resistance sooner or later. Therefore alternatives for antibiotics are sought.

fusion chamber typically produces a mixed culture that requires considerable time to iso-

CALO2: A research team from the University

late, purify and re-inoculate individual colo-

of Bern including Dr. Eduard Babiychuk and

nies4. To streamline this process into a

Isolation Chip consists of hundreds of minia-

Professor Annette Draeger developed a novel

high-throughput system, a variant of the dif-

ture diffusion chambers that can be loaded

substance for the treatment of severe bacterial

fusion chamber for massively parallel micro-

with an average of one cell per chamber. The

infections without antibiotics, which can pre-

bial isolation was developed. This so-called

ichip enables microbial growth and isolation

vent the development of antibiotic resistance.

03

Chemical Structure of Teixobactin.

(Source: Wikipedia)

01

Deaths attributable to antimicrobial resistance every year compared to other major causes of death. (Source: Wikipedia)

02

The iChip. a–c, The iChip (A) consists of a central plate (B) which houses growing microorganisms, semi-permeable membranes on each side of the plate, which separate the plate from the environment, and two supporting side panels (C). The central plate and side panels have multiple matching through-holes.

When the central plate is dipped into suspension of cells in molten agar, the through-holes capture small volumes of this suspension, which solidify in the form of small agar plugs. Alternatively, molten agar can be dispensed into the chambers. The membranes are attached and the iChip is then placed in soil from which the sample originated. (Picture: Originally published in [4], adapted by C. Borchard-Tuch)

Life Sciences plus 01 I 2015

35

iStock

SWISS RESEARCH I Antibiotic Resistance

The treatment is being developed as a new

to bacterial cells based on their shape and

energy is converted into heat. This effect has

medicine named “CAL02” by Lascco SA5.

size, which was used for their selective killing

been utilized in the so-called photothermal

Bacterial toxins bind to phospholipids in

by photothermal therapy. The shape of target

therapy to heat and consequently kill cancer

the membrane of the host cell and destroy it.

cells was recognized by fabricating silica shell

cells; it has also been used to kill bacteria and

Inspired by the principles that govern natural

fragments templating the cell surface. Such

damage viruses6.

toxin-host interactions, the Bernese scientists

shell fragments were produced by depositing

The scientists demonstrated that frag-

engineered CALO2 – an artificial liposome

silica by a sol – gel process onto the surface of

ments of these composite AuNP/silica shells

consisting of two lipidic nanoparticles exclu-

target cells, forming core − shell particles. This

can bind to yeast cells of the same shape and

sively composed of ubiquitous dietary lipids

was followed by their fragmentation by mild

size and deliver AuNPs directly onto their

constituents of the mammalian membranes.

ultrasonic treatment and further removal of

surface. After laser irradiation, the localized

CALO2 is able to sequester and neutralize

the cells’ cores by a bleaching process6.

heating around the AuNPs kills the microbial

bacterial toxins. Without toxins, the bacteria

The cell shape recognition by such shell

cells of matching shape. The cell shape-spe-

are rendered defenseless and can be elimi-

fragments is due to the increased area of sur-

cific killing by photothermal colloid antibod-

nated by the cells of the host’s own immune

face contact between the cells and their

ies was confirmed in a mixture of two bacteri-

system. Since the bacteria are not targeted di-

matching shell fragments which resembles

al cultures of different cell shape and size.

rectly, the liposomes do not promote the de-

antibody − antigen interaction. The colloid par-

This approach opens a number of avenues for

velopment of bacterial resistance5.

ticle recognizes the cell of matching shape,

building powerful selective biocides based on

hence the term “colloid antibody.” This strategy

combinations of colloid antibodies and cell

allows for selective killing of bacterial cells

killing strategies which can be applied in new

with matching colloid antibodies by combining

antibacterial therapies6.

➜

the cell recognition with the delivery of bio-

WHO and other international agencies believe that antibiotic resistance is a serious threat to future medical treatment.

Administration

of

artificial

liposomes

cidal agent directly to the target cell surface.

The way forward

The latter can be achieved, for example, by

There is a need for an improved effort. The

loading the colloid antibody with antibacterial

combination of increasing incidence of resist-

payload. Paunov’s team demonstrated selec-

ance and the lack of new substances is why

tive killing of target microbial cells by delivery

WHO and other international agencies believe

of gold nanoparticles (AuNPs) onto their sur-

that antibiotic resistance is a serious threat to

faces by photothermal treatment6.

future medical treatment. For the development

When AuNPs are irradiated with light at a frequency which is resonant with their sur-

of new classes of substances sufficient financial resources must be made available.

face plasmon resonance, the absorbed light

REFERENCES

within ten hours after infection rescues mice from septicemia caused by Staphylococcus aureus

and

Streptococcus

1

pneumoniae,

whereas untreated mice die within 24 – 33

2

hours. Furthermore, liposomes protect mice

Front Microbiol. 2012;3:1.

3

lipids, tailored liposomes are not bactericidal and could be used therapeutically either alone

Henry BD, et al. Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice. Nat Biotechnol. 2015;33(1):81–8.

6

The first clinical study, conducted on pa-

Ling LL, et al. A new antibiotic kills pathogens without detectable resistance. Nature.

2015;517(7535):455–9.

5

duced tissue damage that occurs during bacterial clearance5.

Perez-Fernandez D, et al. 4’-O-substitutions determine selectivity of aminoglycoside antibiotics. Nat Commun. 2014;5:3112.

4

or in conjunction with antibiotics to combat bacterial infections and to minimize toxin-in-

Martínez JL. Natural Antibiotic Resistance and Contamination by Antibiotic Resistance Determinants: The Two Ages in the Evolution of Resistance to Antimicrobials.

against invasive pneumococcal pneumonia.

Composed exclusively of naturally occurring

WHO. Antimicrobial resistance: global report on surveillance 2014, http://apps.who.int/iris/bitstream/10665/112642/1/9789241564748_eng.pdf, 25.02.2015.

Borovička J, et al. Photothermal colloid antibodies for shape-selective recognition and killing of microorganisms. J Am Chem Soc. 2013;135(14):5282–5.

tients suffering from severe streptococcal pneumonia, is scheduled for 20155.

Photothermal

colloid

antibodies. A re-

searcher group around Professor Vesselin Paunov, University of Hull, United Kingdom, found another approach – one that bacteria would be unable to elude by mutating into drug-resistant forms. Their inspiration was the antibodies that the immune system produces when microbes invade the body. Those antibodies patrol the body for microbes and bind to their surfaces, triggering a chain of events in which the body’s immune system attacks and destroys the microbes6.

Paunov’s team developed a new class of antibiotic agents based on colloid particles, which are capable of recognition and binding 36

Life Sciences plus 01 I 2015

w

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SWISS RESEARCH

IMMUNOLOGY

Ironing out Oxidative Stress Oxidative stress damages the immune system. Manfred Kopf and his team of ETH research scientists have now shown for the first time that higher doses of vitamin E can reduce the stress on immune cells.

PETER RÜEGG

Y

ou’re up in the mountains, the snow is blindingly white, and the sun is blazing down from the sky: ideal skiing conditions – but any skiers carry-

HO

ing the herpes virus might also have to reckon with the onset of cold sores after their day out.

Increased exposure to UV radiation releases

O

free radicals in the body. These put the body under oxidative stress, which weakens the immune system. And that in turn allows the herpes virus to prosper.

Oxidative stress has become a major topic; not only is it implicated in many diseases, it may even be one of their causes. Other environmental influences besides UV radiation can also increase oxidative stress on the body, including air pollution, smoking and the consumption of alcohol, and not least in-

Structure of the α-tocopherol form of vitamin E. Vitamin E has many biological functions, the antioxidant function being the most important and best known (picture: Wikimedia)

fections. Again and again, the talk is of fighting these free (oxygen) radicals by supplementing our diet with the appropriate vitamins.

T Cells divide after Contact with the Enemy Researchers working with Manfred Kopf, a professor at ETH Zurich’s Institute of Molecular Health Sciences, took these questions as their starting point and have now identified a

Increased exposure to UV radiation releases free radicals in the body. These put the body under oxidative stress, which weakens the immune system.

And that in turn may allow herpes virus to prosper.

phenomenon that explains the effects of oxidative stress on immune cells.

Whenever a foreign body such as a virus or other pathogen enters our bodies, a certain class of immune cells – the T cells – jump into

A white blood cell, also known as a T cell, carries special structures on its surface with which to recognise specific pathogens. (Grapics: Blausen.com, CC BY 3.0 via Wikimedia Commons)

action, proliferating rapidly. One sub-class of these cells, the CD8+ T cells, eliminate the virus by killing cells it has infected. Other T cells, known as CD4+ T cells, coordinate the immune response to all kinds of pathogens.

These are the generals in the immune system’s army.

But a week can pass before these T cells start to take their toll on a virus, because in the early stages of an infection too few T cells are able to recognise the specific pathogen. Only once they have had “enemy contact,” these few “scout” cells begin to divide, forming “clones” of 38

Life Sciences plus 01 I 2015

SWISS RESEARCH I Immunology

themselves. With cells dividing every eight to

Kopf considers it significant that immune

twelve hours, it takes a few days to gather a

cells suffer the same type of death as certain

strike force of cells in the hundreds of thou-

kinds of cancer cells when treated with a cyto-

sands: enough to overwhelm the infection.

static drug, and judges this fact to be of scien-

No Immune Response without a Repair Kit

tific value. Known as ferroptosis, this type of programmed cell death was first described in a scientific publication in 2012. “We are the

However, this immune response does not

first to demonstrate that oxidative stress

work if significant oxidative stress is damag-

causes immune cells to suffer the same type

ing the T cells and depriving the body of the

of death as cancer cells,” he says.

tools it needs to repair them, as Kopf’s re-

What Kopf isn’t yet sure of is what impact the

search group with doctoral student Mai Mat-

results of their study will have on human

sushita as lead author have now shown in

health. He finds that people with a normal

their new publication in the Journal of Experi-

level of health and a balanced diet shouldn’t

mental Medicine.

need vitamin supplements. But a supplement

If the immune cells lack repair enzyme Gpx4

of vitamin E or other liposoluble antioxidants

(or it is defective), the T cells die off as they

could well make sense in the event of oxida-

divide, which means the immune system can-

tive stress, which can arise in everyday situa-

not eliminate the pathogen and the infection

tions such as an infection or exposure to UV

becomes chronic. This is the enzyme respon-

light. Patients with certain neurodegenerative

sible for repairing oxidative damage to the

diseases or diabetes suffer massive oxidative

cell membrane.

stress, and in these cases antioxidants could

Vitamin E comes to the Rescue

be a worthwhile addition to their treatment.

How high the dose should be is something

To their astonishment, when testing mice

Kopf cannot say on the basis of the results of

whose immune cells lacked the repair en-

this study. It is not something they analysed,

zyme, the researchers were able to save the

given that the research was conducted using a

immune cells from cell death by mixing a high

mouse model.

ready-to-use

AUTHOR

REAGENTS and CHEMICALS

dose of vitamin E into the animals’ food. That was enough antioxidant to protect the T cells’ cell membranes from damage, so they could multiply and successfully fend off the viral infection. At 500 milligrams per kilogram of

Peter Rüegg. Hochschulkommunikation

mouse feed, this quantity of vitamin E was ten

ETH Zürich

times higher than was present in the standardised normal food.

The researchers demonstrated this by way of

REFERENCES

a mouse model using animals in which the Gpx4 gene can be deactivated either cell-spe-

• Matsushita M, Freigang S, Schneider C, Conrad M,

cifically or at a chosen point in time. These

Bornkamm GW, Kopf M. T cell lipid peroxidation induces

mice were developed by researchers at the

ferroptosis and prevents immunity to infection.

Helmholtz Zentrum München. The ETH sci-

Journal of Experimental Medicine, March 30th 2015.

entists then altered the mouse line so that the Gpx4 gene was inactive only in T cells or cer-

for everyday use and special applications.

All products can also be found in our web shops!

doi: 10.1084/jem.20140857 • www.ethz.ch/en/news-and-events

tain phagocytes.

www.carlroth.ch

Vitamin Supplements disputed “The benefit of vitamin tablets is a controver-

LABWARE

sial topic,” says Manfred Kopf. With little scientific evidence as yet making a strong case

LIFE SCIENCE

for vitamin supplements, he felt their study was all the more interesting because it proved the effectiveness of vitamin E: “Our

CHEMICALS

work shows that even a genetic defect in a major part of a cell’s antioxidative machinery can be compensated for by delivering a high dose of vitamin E. That is new and surprising.”

Life Sciences plus 01 I 2015

39

ROTH AG Fabrikmattenweg 12 · 4144 Arlesheim Tel. 061/712 11 60 · Fax 061/712 20 21 info@carlroth.ch · www.carlroth.ch

LAB & PROCESS

MOLECULAR DIAGNOSTICS

Easy PCR Optimization Using the Linear Gradient Tool

The Biometra Linear Gradient Tool saves the user time and effort when optimizing new primer pairs. The optimal primer annealing temperature (Ta) can be found in a single PCR experiment. The Linear Gradient Tool allows to enter the calculated primer annealing temperature and to define the temperature increment. By the Linear Gradient Tool it is easy to obtain gradients with even temperature values from lane to lane and to transfer the gradient step results into a standard PCR protocol.

iStock

LAB & PROCESS I Molecular Diagnostics

The Linear Gradient Tool allows entering the calculated Ta value and offers to set

➜

integer temperature values with constant difference (increment) from lane to lane.

HOLGER DENSOW, MARTINA PICK

T

of the nucleotides in the primer sequence are

experiment. However, the calculated mean Tm

considered and thus the algorithms are not

value and the optimal annealing temperature

meant to be used for sequences longer than 14

can differ by more or less than 5 °C and using

he base composition and length of

nucleotides or give questionable results for

a too low annealing temperature can lead to

PCR primers generally determine

longer primers. By salt adjusted Tm calcula-

the

the annealing temperature of primer

tions3 at least the salt concentration is consid-

whereas if the annealing temperature is set

formation

of

non-specific

products,

pairs. New primer pairs are designed

ered. The best methods to calculate primer Tm

too high, the PCR yield may be reduced or no

to anneal at a specific temperature by calcu-

values are base-stacking Tm calculations4, 5, 6.

PCR products are formed.

lating the primer melting temperature (Tm).

The base-stacking formulas use the nearest

By using the gradient function of a PCR

The melting temperature (T m) is the temper-

neighbor algorithm and calculate the primer

thermal cycler, the optimal primer annealing

ature at which one-half of a particular DNA

Tm value based on the nucleotides position

temperature (Ta) can be determined, which is

duplex will dissociate and become single

and salt concentration.

then used as integer temperature value (Ta

strand DNA. The stability of a primer-tem-

value) in routine PCR protocols. Traditionally

plate DNA duplex can be measured by its Tm

Linear Gradient Tool

and new primers are usually supplied with

Primers are usually supplied inclusive infor-

ature values for the left and right side of the

an additional data sheet providing informa-

mation on the calculated theoretical melting

sample block (e. g. 55 °C – 65 °C). Due to techni-

tion on the calculated theoretical melting

temperature (Tm). However, the different Tm

cal reasons, the temperature gradient of most

temperatures (Tm).

calculation methods give widely varying re-

PCR instruments has a sigmoid shape what

There are a number of different algo-

sults and the precise optimum annealing tem-

means that the temperature difference from

rithms to calculate the estimated melting tem-

perature (Ta) has to be determined experi-

lane to lane differs across the sample block.

perature (Tm) of a primer that may give widely

mentally. In general the annealing tempera-

The traditional way of gradient programming

varying results. The simplest methods are the

ture (Ta) is set 5 °C below the mean Tm value of

and the unequal temperature differences be-

basic Tm calculations according to Marmur

the primer pair. From experiences using the

tween the lanes lead to disadvantages when

and Doty1 or Wallace et al.2 For these methods

equation Ta = mean Tm -5 °C most often leads

trying to find the optimal primer annealing

neither the salt concentration nor the position

to formation of a PCR product in the first PCR

temperature (Ta):

Life Sciences plus 01 I 2015

41

gradients are programmed by setting temper-

LAB & PROCESS I Molecular Diagnostics

• Non-integer temperatures in sample block lanes are obtained • Temperature differences amongst lanes increase towards the middle of the sample block

• It is impossible to use the calculated Ta value for gradient programming

ple block 12 different lanes (temperatures)

nealing temperature of 56 °C (lane 6) and a

are available in total.

temperature increment of ±3.0 °C. Additional-

Finding the optimal primer annealing temperature (Ta)

ly, the forward and reverse primer has been used at concentrations of 1:1 (A), 2:1 (B) and 1:2 (C). The best results were obtained using a

As mentioned above, usually the primer an-

primer concentration of 2:1 and an annealing

nealing temperature is calculated from the

temperature of approximately 60 °C [Figure 2].

mean primer melting temperature using the

The results were transferred to a standard

Some thermal cycler models use thermal

following equation: Ta = Tm -5 °C. In practice,

PCR program to demonstrate the specificity

zones. Here it is possible to set a defined tem-

the optimal primer annealing temperature

and robustness of the results. When using a

perature for a specific zone. However, for each

cannot be calculated using a formula but has

primer concentration of 2:1 and an annealing

temperature zone the temperature value has

to be determined experimentally. As a starting

temperature of 60 °C, the PCR yields a single

to be set individually and the overall number

point, the calculated primer annealing tem-

product in all 12 lanes of the sample block

of temperatures is reduced compared to gra-

perature (Ta) and a temperature increment of

[Figure 3].

dient enabled thermal cyclers.

±1.0 °C should be used to program the gradi-

The mentioned disadvantages are over-

ent. If necessary, the increment can be re-

Conclusion

come by the Biometra Linear Gradient Tool

duced for fine-tuning in later experiments. In

The Biometra Linear Gradient Tool is a useful

[Figure 1]. The Linear Gradient Tool allows

the gradient step, different primer concentra-

feature to find the optimal primer annealing

entering the calculated Ta value and offers to

tion combinations can be used to find the best

temperature (Ta) in a single PCR experiment.

set integer temperature values with constant

annealing temperature and primer mix simul-

It allows to enter the calculated primer anneal-

difference (increment) from lane to lane. For

taneously.

ing temperature and to define the temperature

example, a calculated primer annealing tem-

In the following example, a 210 bp β-glo-

increment. With the Linear Gradient Tool it is

perature (Ta) of 60.0 °C can be programmed

bin fragment has been amplified from human

easy to obtain gradients with even temperature

for lane 6 of the 96 well sample block and the

cDNA. To demonstrate the effect of the an-

values from lane to lane and to transfer the

temperature difference between the sample

nealing temperature on the PCR results, the

gradient step results into a standard PCR pro-

block lanes set to ±1.0 °C. For the 96 well sam-

gradient has been programmed using an an-

tocol. Thus, the Linear Gradient Tool saves the user time and effort when optimizing new primer pairs.

Methods and Materials For PCR the Analytik Jena innuTaq HOT-A DNA-Polymerase (845-EZ-3000500) has been used according to the user manual. As template, Promega human DNA has been used at a final concentration of 0.5ng/ul. The human β-globin fragment was amplified using forward primer RS41 and reverse primer RS42 at

01

Diagram showing the sample block temperatures with gradient programmed from 55 °C to 65 °C using a temperature difference (increment) of 1 °C per lane. Other than for the competitors, for Biometra thermal cyclers the temperature difference from lane to lane is exactly the same from lane 3 to lane 10 as indicated by the dotted grey line.

44.9 45.5 47.3

50

53

56

59

62

65 67.7 69.5 70.1

44.9 45.5 47.3

50

53

56

59

62

65 67.7 69.5 70.1

A

B

02

Gradient PCR using different primer concentrations. A human β-globin fragment was amplified using primers RS41 and RS42 at concentrations of 1:1 (A), 2:1 (B) and 1:2 (C).

42

Life Sciences plus 01 I 2015

LAB & PROCESS I Molecular Diagnostics

Table 1

varying concentrations of 1:1, 2:1 and 1:2 (0.5 µM to 1.0 µM).

For gradient PCR (A – C), the Biometra TAd-

Temperature and time protocol

vanced thermal cycler (846-2-070-101) was used with following PCR program [Table 1].

For standard PCR Biometra TAdvanced

Step

Cycle

1

1

thermal cycler was used with the following PCR program [Table 2].

2

www.bio.analytik-jena.com

3

www.huberlab.ch

30

4 5

1

Profile

Temperature

Holding time Ramp rate

Initial denaturation

96 °C

2 min

max

Denaturation

96 °C

10 sec

max

Annealing

56 °C ± 3.0 °C 10 sec

max

Elongation*

72 °C

20 sec

max

Final Elongation

72 °C

1 min

max

Profile

Temperature

Holding time Ramp rate

Initial denaturation

96 °C

2 min

max

Denaturation

96 °C

10 sec

max

Annealing

60 °C

10 sec

max

Elongation*

72 °C

20 sec

max

Final Elongation

72 °C

1 min

max

Table 2 Temperature and time protocol Step

Cycle

AUTHORS

1

1

Holger Densow, Martina Pick. Analytik Jena AG, Business

2

Unit Lifescience, Biometra Product Line, 37079 Göttingen,

3

Germany

4 5

REFERENCES 1

30

1

Marmur J and Doty P (1962) J Mol Biol 5:109–118.

2

Wallace et al. (1979) Nucleic Acid Res. 6: 3543.

3

Rychlik, W. and Rhoads, R.E. (1989) Nucl. Acids Res. 17, 8543.

4

Breslauer et al. (1986) Proc. Natl. Acad. Sci. USA 83: 3746.

5

SantaLucia, J. (1998) Proc. Nat. Acad. Sci. USA 95, 1460.

6

von Ahsen N. et al. (1999) Clin. Chem. 45, 2094.

44.9 45.5 47.3

50

53

56

59

62

04

Analytik Jena, PL Biometra thermal cycler TAdvanced.

65 67.7 69.5 70.1

60

60

60

60

60

60

60

60

60

60

60

60

C

03

Results of gradient PCR (A – B) were transferred to a standard PCR reaction at 60 °C using a primer concentration of 2:1.

Life Sciences plus 01 I 2015

43

LAB & PROCESS

CRYOSTOCKS

FlexiQuot Cryotube: fill it – freeze it – snap it

P

rogress in life sciences comes with

searcher can snap off and thaw the quantity of

tiveness of 5 ml cryotubes. Life sciences ex-

such a fast pace that it is impossible

the sample that is required for an analysis,

perts predict that it will bring a true revolu-

to predict which biomarkers or mole-

without needing to thaw the whole sample.

tion in cryogenic storage.

cules a researcher will be able to de-

Using the specific snapping and holding tools

tect or wishes to measure in the future. That

for FlexiQuot tubes, any contact with the fro-

is why researchers need to make sure that bi-

zen sample surface is avoided, to ensure that

omaterial used today will be exactly the same

there is no contamination at all. Thanks to

when used at a later stage. Storage in multi-

multiple caps that can cover both sides of the

ple smaller tubes or in larger tubes that are

snapped aliquot and the remaining sample,

repeatedly thawed and refrozen limits the

the aliquots are safely sealed The remaining

chances of success. The first option is much

sample is kept in the freezer, without any im-

more time-consuming and increases the pos-

pact on its quality that a continuous proce-

sibility of human error, the second option di-

dure of thawing and freezing could bring.

FlexiQuot is distributed in Switzerland by

minishes the sample quality over time.

Blood proteins and other sensitive plasma

Faust Laborbedarf AG.

Increased usage of biomarkers in combi-

biomarkers can thus be preserved for a long

For more information or a free sample, please

nation with the aforementioned fundamental

time, available for accurate future testing. This

visit the producer’s website.

problems of the existing cryotubes has re-

ensures the best results for the laboratories at

sulted in the need of a flexible cryotube. This

any time. FlexiQuot combines the flexibility of

www.faust.ch

was what sparked Dr Ove Andersen’s, Head

standard 1 ml cryotubes with the cost-effec-

www.1cryobio.com

Value creation through

• Long-term sample handling flexibility • Higher sample quality and stability • Contamination and error reduction • A more efficient workflow • Optimized freezer storage • Sample tracking efficiency

of the Clinical Research Centre at Hvidovre University Hospital in Denmark, genius idea for a cryotube that could be easily divided into subsections. A team of life science experts and entrepreneurs from Denmark and Switzerland materialized Dr Andersen’s idea, created 1Cryobio AG, an innovative start-up company in Technopark Lucerne, and the result of their researches was FlexiQuot.

FlexiQuot is the first, patent protected, dividable 5 ml cryotube in the world that can be broken into five 1 ml aliquots. Thus, the re-

FlexiQuot: A revolutionary solution to cryogenic storage 1Cryobio’s dividable vials offer the solution to all of these issues. FlexiQuot features pre-defined breaking points that can be used to divide the frozen sample into parts for individual thawing and analysis. This important advancement enables FlexiQuot to improve labor and storage efficiency as well as sample quality, stability and flexibility. The invention is protected worldwide by two patent families and a trademark.

44

Life Sciences plus 01 I 2015

iStock

LAB & PROCESS

PROTEIN A CHROMATOGRAPHY

Efficient Purification of Monoclonal Antibodies from High Titer Feedstocks

JUDITH VAJDA, ANGELIKA WACKER

I

n Protein A chromatography, remaining host cell proteins (HCP) of the expression cell line flow through the column

Protein A chromatography has become a widely used platform in monoclonal antibody (mAb) purification. It makes use of the specific interactions that take place between the Fc regions of immunoglobulin G and immobilized Protein A. Its high specificity typically generates more than 95 % purity in one step. Recently, a new generation of high capacity Protein A chromatography resins has been introduced.

and are usually reduced by two to three

orders of magnitude. Subsequent mAb elution requires a pH shift of the mobile phase which at the same time serves as an acidic virus inactivation step but may cause mAb

focused on increasing the expression rate.

mAb Adsorption

aggregation. One example of a recently

MAb titers higher than 5 g/L have been re-

The dynamic binding capacity (DBC) of a sta-

introduced new generation of high capacity

ported3 and 10 g/L seem close to reach. Today,

tionary phase is influenced by the contact

Protein A chromatography resins is Toyo-

the purification of the target molecules, the

time between the sample and the ligand, the

pearl AF-rProtein A HC-650F. Herein, we

so-called downstream processing, has be-

so-called residence time, which decreases

describe the impact of comparably higher

come the bottleneck of the production pro-

when increasing the flow rate. Good mass

mAb loadings on aggregation during Protein

cess. Coping with higher titers seems to be a

transfer properties enable a resin to reach a

A chromatography, as well as the benefits of

challenge, especially with regards to the ex-

high binding capacity even at high flow rates.

high capacity Protein A resins for high titer

pensive Protein A chromatography step. The

The binding capacity also depends on the

feedstocks.

new Toyopearl AF-rProtein A HC-650F car-

feed concentration. The capacity of the new

MAbs are a unique and versatile class of

ries a recombinant ligand that is stable in al-

resin was tested at various residence times

molecules. They combine high target affinity

kali solutions applied in industrial cleaning

and mAb titers. Dynamic capacities are typi-

and specificity with various different effector

procedures. It benefits from superior mAb

cally calculated from so-called breakthrough

functions, such as complement activation and

capacity and increases capturing productivi-

curves. A 6.6 mm ID glass column was packed

opsonization1. MAbs are increasingly used in

ty. Besides higher capacities, the mAb uptake

to a bed height of 2 cm. A mAb stock solution

diagnostics and therapy. While the number of

behavior is a major driver in process eco-

was diluted to 5 and 10 mg/ml in 100 mM so-

approved mAbs for pharmaceutical purposes

nomics. High capacities at high feed concen-

dium phosphate buffer, pH 6. After bypassing

has grown tremendously 2, mAb production

trations will lead to concerting effects when

the column until the corresponding maxi-

processes have improved, too. Recent ap-

it comes to fast and efficient capturing solu-

mum absorptions of the mAb solutions were

proaches to reduce cost of mAb production

tions.

reached, the column was switched into the

Life Sciences plus 01 I 2015

45

LAB & PROCESS I Protein A chromatography

flow line and loaded. The column starts to absorb the antibody until the capacity of the stationary phase is reached and UV signal is in-

A

creasing because the antibody solution is ‘breaking through’ the column. The DBC of the column is typically calculated from the volume that generated 5 % or 10 % of the maximum UV adsorption of the sample.

Figure 1 shows the breakthrough curves for Toyopearl AF-rProtein A HC-650F at the two feed concentrations. The resin shows complete mAb adsorption until breakthrough occurs. This remains unaffected for the comparably short residence times of 1 min. The measured capacities of more than 100 mg/ml exceed the DBCs of all other known base stable Protein A resins.

B

MAb Elution from High Capacity Resins A purified humanized monoclonal IgG was diluted in 100 mM sodium phosphate buffer, pH 6.5, to a final concentration of 4.75 g/L. Simulating high HCP density in the Protein A feed, these solutions were spiked with concentrated cell culture fluid. Protein A chromatography with Toyopearl AF-rProteinA HC-650F was conducted in 5 mm ID × 1 cm columns using a robotic chromatography station. The total loaded mass was varied from 10 to 50 mg/ml resin. A residence time of 2 minutes (linear velocity of 30 cm/h) was applied.

Subsequently,

the

columns

were

washed for 20 column volumes with binding buffer. For mAb elution, 100 mM sodium acetate buffer, pH 3.25 was used. More than 95 %

01

mAb could be recovered. Due to the acidic pH

Breakthrough curves of mAb A on TOYOPEARL AF-rProtein A HC-650F packed into a 6.6 mm ID × 2 cm L column for a mAb. A: 5 g/L mAb. B: 10 g/L mAb.

applied for Protein A elution, mAbs are prone to aggregation. Naturally, high capacity Protein A resins adsorb large amounts of mAb. This might enhance mAb aggregation due to higher target protein concentrations in the elution pool. Thus, special attention was paid to the aggregate content after elution. Size exclusion chromatograms of two mAb elution pools are shown in Figure 2. The elution pools of 10 mg/ml and 50 mg/ml mAb load were injected, respectively. Although the SEC chromatograms seem to show aggregates for the higher loading only, a closer look reveals similar aggregate contents when referring to the corresponding total protein amount detected by SEC. Both pools contain 0.6 % aggregates.

Protein A leaching In Protein A chromatography used in biopharmaceutical manufacturing, ligand leaching is a major safety concern. Low ligand leaching is crucial and needs to be proofed

by Elisa testing. Host cell proteases con-

02

tained in the cell culture fluid, contribute to

Size exclusion chromatogram of two Protein A elution pools. 50 µl were injected on a TSKgel SuperSW mAb HR U(HPLC) column, 7.8 mm ID × 30 cm L at a flow rate of 1 ml/min, 100 mM sodium phosphate buffer, pH 6.7 + 100 mM sodium sulfate.

UV @ 280 nm.

46

ligand leaching 4. The longer the resin is in contact with the HCP proteases contained in the feed, the more probable seems ligand leaching. From this perspective, high titers Life Sciences plus 01 I 2015

LAB & PROCESS I Protein A chromatography Supporting your great ideas

Expertise in pressure sensors

are favorable, as they reduce contact time.

Further, mAb molecules have been shown to have a ligand detaching effect 4. Altogether, one would expect increased Protein A leaching for low mAb titer feedstreams and higher absolute loadings. Protein A leaching was analyzed for 2.5 g/l, 4.75 g/l and 7 g/L concentrated feed streams. Spiking and residence time were kept constant. Figure 3 exemplarily depicts the contour plot for the highest load.

According to these results, the absolute load has little influence on numeric Protein

AUTHORS

A leaching. Overall, Protein A leaching does not exceed 45 ppm for any of the tested pH

Judith Vajda. Tosoh Bioscience GmbH

and load conditions. Higher absolute mAb

Angelika Wacker. University of Applied Sciences Mannheim

loadings seem to be advantageous, since the relative Protein A content of the mAb pool

AG/AP(B)/AL series From 0 – 20 mbar to 12 bar Analogue or digital ± 1.5 % precision FS

REFERENCES

decreases.

Considering the obtained results regarding Protein A leaching, aggregate content

1

and protein adsorption, high titers seem favorable for Protein A chromatography. This mAb seemed unaffected with regards to aggregation, and

was

efficiently

7th Edition 2009, Spektrum Akademischer Verlag: 38–49 2

adsorbed,

edited by An, Z., John Wiley & Sons, 2009: 3–50.

3

plying higher titers. Thus, ultra-high capacity

Kelley, B., Industrialization of mAb production technology – The bioprocessing industry at a crossroads. mAbs 2009, 1(5):

Protein A resins offer additional benefits besides reducing costs because less resin vol-

Strohl, W.R., Therapeutic Monoclonal Antibodies: Past, Present and Future. In: Therapeutic Monoclonal Antibodies,

which reduces Protein A cycle time. Further, Protein A leaching was even lower when ap-

Murphy, K., Travers, P., Walport, M., Effektormechanismen der adaptiven Immunität. In: Janeway Immunologie,

443–452 4

CCD54/CCD53 series From 0 – 2.5 mbar to 10 bar Analogue or digital ± 1.8 % to ± 2.2 % precision FS

Carter-Franklin, J.N., Fragments of protein A eluted during

ume is needed to purify a given amount of

protein A affinity chromatography. J. Chromatogr A. 2007,

monoclonal.

1163(1-2): 105–111

www.sebio.ch

HPSD 3000/4000 series

From 0 – 10 mbar to 7 bar Analogue and digital Integrated temperature sensor ± 0.7 % precision FS

Stainless steel and ceramic pressure sensors From 0 – 20 mbar to 600 bar Analogue or digital Piezoresistive or capacitive

Visit us at SENSOR + TEST Hall 12 . Stand 310

03

Protein A leaching contour plot for a high load concentration of 7 g/L. Protein A leaching is plotted against pH and absolute load.

Life Sciences plus 01 I 2015

47

Click and buy

www.pewatron.com

LAB & PROCESS

SCREENING SOLUTIONS FOR LIFE

High Content in Suspension No-Wash Multiplexed Cytokine Profiling of Human Peripheral Blood Mononuclear Cells (PBMCs) Researchers in therapeutic areas like cancer, immunology or toxicology often focus on secreted proteins which provide key information about many diseases. However, common methods to analyze secreted proteins are very limited in multiplexing capability. IntelliCyt’s QBeads assays detect multiple proteins simultaneously. In addition, an easy workflow without washing steps delivers faster screening results.

M.D. SJAASTAD, R. NARANG, K. LUU

T

he detection and quantification of

proteins. Profiling of secreted proteins is ide-

pensive. Importantly, many bead-based assays

proteins in solution provides key in-

ally achieved by the detection of multiple pro-

require multiple wash steps, which limits the

formation for researchers in thera-

teins simultaneously. This maximizes the con-

ability to automate the assay and causes bead

peutic areas ranging from cancer and

textual and correlative value of the data. Tra-

loss, increasing variability in the results.

immune function to aging and regeneration.

ditional technologies like plate-based Elisas

The MultiCyt QBeads PlexScreen leverag-

The ability to perform high content screens

are limited in this regard as they are inherent-

es the benefits of IntelliCyt screening systems,

for cytokine secretion on suspended cells has

ly single-endpoint readouts. Newer technolo-

such as the iQue Screener, to provide a unique

been hampered by the lack of speed, automa-

gies, such as bead-based Elisas, offer the abil-

no-wash protocol that is ideal for medium to

tion and high cost. Current high content imag-

ity to multiplex the detection of multiple

high throughput screening. Here, we demon-

ing approaches struggle with non-adherent

proteins simultaneously, however, the assay

strate

cells and also cannot easily measure secreted

protocols are laborious and ultimately too ex-

QBeads are assembled into user-specified

fast,

affordable

cytokine

profiling.

01

QBeads Assay Principles. QBeads function by performing a sandwich Elisa on the surface of beads. (A) QBeads are coated with analyte-specific capture antibodies. (B) QBeads capture analyte that binds to the analyte-specific antibodies on the bead surface. (C) A fluorescently-labeled detection antibody is added, causing the beads to fluoresce in proportion to the analyte concentration in the sample and providing a signal detectable on an IntelliCyt screening system.

48

Life Sciences plus 01 I 2015

LAB & PROCESS I Screening Solutions for Life Supporting your great ideas

Expertise in gas sensors The detection and quantification of proteins in

➜

solution provides key information for researchers in therapeutic areas ranging from cancer and immune function to aging and regeneration.

multiplate kits that can contain up to 30 ana-

stimulating agents. The stimulation methods

lytes. Compared to other bead-based protein

selected were:

detection assays, QBeads provide an easier

bodies (top dose = 5 μg/mL for each anti-

read times, and lower sample volume require-

body), which activates T-cells through

ments. Combining these advantages allows the

binding of CD3 and CD28 on the cell

practical screening of large libraries for the

surface.

QBeads Assay Principles QBeads are used to detect proteins in solution by performing a sandwich Elisa on the surface

Measuring range 0 % to 25 % or 0 % to 95 % High precision and fast response time Analogue or digital interface

1. Co-treatment with CD3 and CD28 anti-

screening workflow, lower cost, faster plate

ability to modulate secreted protein profiles.

FCX-MC25/MC95 series (oxygen)

2. Co-treatment with PMA (top dose = 10 ng/mL) and ionomycin (top dose = 10 μg/ mL), which activates T-cells by entering cells and directly activating protein ki-

EC4/SGX-4 series

Electrochemical 4 series (20 mm diameter) Measurement cells for industrial gases Suitable for portable measuring devices Long lifespan and low drift

nase C.

of a bead. In the example of a single analyte

3.

Phytohemagglutinin (PHA; top dose =

[Figure 1], capture beads coated with anti-

5 μg/mL), which activates T-cells by

bodies directed against an analyte of interest

cross-linking T-cell receptors (TCRs) at

are directly combined with sample, typically

the cell surface.

cell culture supernatant or serum/plasma.

Soluble analyte is bound by the capture beads and a fluorescent detection antibody is added to the reaction which binds the analyte fluorescence signal is now associated with the bead complex, and the fluorescence intensity

➜

to form a sandwich-complex [Figure 1C]. The

directly correlates to the concentration of analyte in the sample.

QBeads: No-Wash Screening Workflow QBeads enable a simplified, no-wash workflow on IntelliCyt screening systems [Figure 2]. First, 10 μL of sample and 10 μL of QBeads are combined and incubated for 1 hour. After incubation, 10 μL of the detection antibody is added and incubated for 2 hours. The plate is read on an IntelliCyt screening system without the need for additional processing. The entire protocol requires less than 3.5 hours and minimal hands-on time. Plate reads typically take less than 20 minutes, regardless of

Many existing technologies

Carbondio-ppm series (carbon dioxide) Measuring range 0 – 500 ppm up to 0 – 5000 ppm ± 2.0 % precision Response time (T90) adjustable from 1 s to 30 s Analogue or digital interface

for the detection of proteins in solution suffer from limitations and trade-offs: Often they are fast but not multiplexed or multiplexed but not cost-effective.

the number of QBeads multiplexed for the detection of multiple proteins.

MiCS MOS gas sensors

Low power consumption Ammonia, carbon monoxide, nitrogen oxide, ozone and VOC Available with evaluation electronics

Visit us at SENSOR + TEST Hall 12 . Stand 310

PBMC Stimulation Model and Cytokine Detection with QBeads In three independent experiments, performed on three consecutive days, cryopreserved PBMCs from a single donor were thawed and treated in 384-well plates with known T-cell Life Sciences plus 01 I 2015

49

Click and buy

www.pewatron.com

LAB & PROCESS I Screening Solutions for Life

02

QBeads enable a simplified, no-wash workflow on IntelliCyt screening systems. (1) 10 μL of beads are incubated with 10 μL of sample for 1 hour. (2) 10 μL is added and then incubated for 2 hrs. (3) The plate is read directly on an IntelliCyt screening system. Plate reads typically take less than 20 minutes, regardless of the number of analytes multiplexed for detection.

03

Day-to-Day Reproducibility of Standards. Standard curves for IL-2 across multiple days are shown.

Serial titrations of purified IL-2 were independently performed on three days and quantified using QBeads.

Error bars represent the standard deviation of the mean (N=3).

QBeads were used for the multiplexed detection of 7 cytokines: IL-2, IL-4, IL-6, IL-10, IL17a, TNF, and IFN-g. Cytokines were considered responsive to treatment if their median fluorescence intensity (MFI) was greater than 3 standard deviations above the mean (n = 96) MFI of unstimulated controls.

Assay Robustness The performance of the QBeads reagents were assessed by comparing the standard curves generated for each analyte on three different days. Representative data for IL-2 is shown in [Figure 3].

The additional 6 analytes measured in the experiment performed similarly (data not shown). The table summarizes the inter-day variability of the standards for all analytes. In general we found excellent reproducibility (CV range 0.7–15.9 %) between the median fluorescence intensity (MFI) at each dose, across every analyte. As expected, lower concentrations of analyte trended towards higher CV values.

04

Day 1 cytokine profiles for 3 different PBMC stimulation methods. The dotted line represents 3 standard deviations of the mean response for unstimulated controls (N=96). All dose response series are 1:2 serial titrations. The top doses for the treatments were: CD3/CD28, 5 μg/mL/5 μg/mL, PMA/ionomycin, 10 ng/mL / 10 μg/mL, and PHA-P, 5 μg/mL. 50

Life Sciences plus 01 I 2015

LAB & PROCESS I Screening Solutions for Life

The MultiCyt QBeads PlexScreen and the benefits of IntelliCyt

➜

screening systems, such as the iQue Screener, provide a unique no-wash protocol that is ideal for medium to high throughput screening.

QBeads Detect Distinct Cytokine Profiles Between Different Stimulation Methods

IntelliCyt products are available in Switzerland at Bucher Biotec AG.

Cytokine secretion profiles were observed

www.bucher.ch

that differed depending on which PBMC stim-

www.intellicyt.com

ulation method was used. A subset of the cytokines assayed (IL-4, IL-10, and IFN-g) highlights some of the most significant differences

AUTHORS

between the three stimulation methods. PMA/ Ionomycin stimulation caused no observable

Michael Sjaastad. Ph.D. Director of Business Development,

IL-4 or IL-10 secretion at any dose, while in-

IntelliCyt Corporation, Albuquerque, NM, USA

ducing a strong IFN-g response [Figure 4].

Robbie Narang. R&D Scientist, IntelliCyt Corporation,

CD3/CD28 and PHA produced a similar cyto-

Albuquerque, NM, USA

kine profile: IL-4 is negative at all doses while

Kim Luu. Director Assay Development, IntelliCyt Corporation,

both IL-10 and IFN-g are positive.

Albuquerque, NM, USA

Summary To perform an effective screening campaign, the screening technologies and reagents utilized must not only have the speed and sensitivity to support high-throughput, but also be cost-effective and be able to provide meaningful data. Many existing technologies for the detection of proteins in solution suffer from limitations and trade-offs on cost and/or context: technologies that are fast are often not

Nucleic Acid Isolation System

multiplexed, and multiplexed technologies are typically not cost-effective. IntelliCyt’s MultiCyt QBeads portfolio presents an opportunity to elevate the screening of secreted

QuickGene

proteins to an unprecedented level of effi-

Only 6 Minutes to prepare DNA/RNA

ciency by offering a streamlined, multiplexable workflow at significantly lower cost than many existing technologies. QBeads allow users to capitalize upon:

• Streamlined, no-wash assay workflow • Significantly lower cost per data point than other multiplex methods • Design your custom panel from 50 validated human secreted protein analytes • Multiplex up to 30 analytes in one kit • Custom generated acquisition and analysis templates facilitate data analysis • ForeCyt Control and Analysis Software

– Lightweight, benchtop, uses minimal space – Special Filter Membrane for high yield – Easy and simple isolation operation – Available for tissue, whole blood, cultured cells and plasmids

offers 5-button operation and rapid data analysis

For more information: www.igz.ch IGZ Instruments AG Räffelstrasse 32 CH – 8045 Zürich Tel. +41 44 456 33 33 www.igz.ch

Life Sciences plus 01 I 2015

51

LAB & PROCESS

MICRO-BIOREACTORS

Generating Scalable Results in Micro-Bioreactors Micro-bioreactor systems are supposed to be realistic scale down models for the larger laboratory scale systems. The performance of micro-organisms in different scales of cultivation systems is shown in a study with Pichia Pastoris in different cultivation systems. Results show that the micro-bioreactor generates results comparable with the miniBio and 3 liter bioreactors.

ERIK KAKES, STEFAN BERGER

M

timized process will be easily transportable to

processes that can be run in a short time due

the larger volumes and relations found on

to availability of laboratory scale bioreactors.

small scale are present in the larger volumes

The overcome this bottleneck, smaller volume

icro-bioreactors are gaining mar-

as well. The organism selected in the micro-

bioreactors

ket share in R&D laboratories.

bioreactor needs to be the best producer in the

micro-bioreactor is the most recent develop-

Common applications are strain

laboratory and pilot scale fermenter as well.

ment and is (arbitrary) defined as a bioreactor

have

been

developed.

The

with a volume less than 10 ml.

selection, medium optimization

The aim of this study is to show the per-

and optimization of cultivation conditions.

formance of micro-organisms in different

Challenges in the scale down to these

The advantage of the micro-reactors is that a

scales of cultivation systems. Therefore culti-

small volumes are accurate measurement and

large number of cultures can be run on limit-

vation results of Pichia Pastoris are compared

control of basic process parameters (pH, tem-

ed space on a laboratory bench. This results in

with different cultivation systems.

perature and dissolved oxygen) and mimick-

faster process development and shorter time to market.

ing the fluid dynamics (mass transfer and

Micro-Bioreactors

mixing time) of the larger volume bioreactors.

The Applikon micro-Matrix is developed to

One of the key demands of these micro-

Conventional laboratory bioreactors are me-

bioreactor systems is that they are realistic

chanically complex systems of between 1 and

overcome

scale down models for the larger laboratory

15 liter volume. Early stage development of

24 micro-bioreactors of 1 – 5 ml working volume

scale systems. This will guarantee that the op-

new drugs is usually limited by the number of

in standard SBS format micro-titer plate. The

these

challenges

and

to

offer

The Next Generation in Micro-Bioreactors Applikon’s micro-Matrix offers an integrated, easy-to-use and costeffective technology platform for the rapid handling and growth of large numbers of microbial strains, clone libraries, mutant banks, and cells.

The system offers 24 independent bioreactors in a cost effective microtiter plate footprint. pH and dissolved oxygen can be controlled in each individual bioreactor via gas and liquid addition. Temperature is controlled individually in each bioreactor by the integrated cooling and heating system. The micro-Matrix offers a true scale down of smallscale bioreactors. The bioreactor’s square well cassette design is based upon the popular SBS-format microtiter plates, which seamlessly integrates into lab automation robots. The PC-based human interface offers simple, intuitive interaction for advanced process control in each of the 24 bioreactors. www.applikon-bio.com Official distributor for Applikon in Switzerland is ReseaChem GmbH. www.reseachem.ch 52

Life Sciences plus 01 I 2015

LAB & PROCESS I Micro-Bioreactors

standard SBS format allows an easy integration in the automated liquid handling systems.

The micro-Matrix uses fluorophor sensors for pH and Dissolved Oxygen, temperature is measured with two contact sensors in each well while proper mixing is achieved by placing the bioreactor cassette on the integrated orbital shaker plate. Every micro-bioreactor has up to four individual controlled gasses for control of oxygen (Nitrogen, Air and Oxygen) and pH (via CO2 gas and optional via NH3 gas). Every well can be equipped with an individual liquid feed line. These liquid addition lines allow nanoliter droplets to be added to the culture. The liquid addition can be used for pH control or for fed batch medium addition.

To prove that the cultivation conditions in the micro-Matrix mimic these of the standard laboratory scale systems, we have cultivated Pichia Pastoris in different volume cultivation systems.:

• Orbital shaker, previously the gold

standard for cultivating cultures in parallel for strain selection

• RAMbio , an enhanced system for

cultivating in standard shakeflasks

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2015

Sept. 2 1–24

KeyNotes

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Sept. 2 2–24

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Life Sciences plus 01 I 2015

53 © Basel Tourismus

LAB & PROCESS I Micro-Bioreactors

• micro-Flask, a micro-bioreactor with

only temperature control, scale down of a shaker system

• micro-Matrix, a micro-bioreactor with

the oxygen concentration in the RAMbio is

peated with different orbital shaker speeds

above the setpoint of 50 %.

(100, 200, 250, 300 350 and 400 rpm). The re-

Inoculation volume is set to 10 % (volume) in all systems. During the growth phase, the

sults of the experiments are shown in table 1.

pH, Temperature, Dissolved Oxygen

glucose in the medium will be consumed so a

Conclusion

control and active gas and liquid addition

glucose bolus is added to allow the growth to

Cultivation of Pichia pastoris in different cul-

continue. When this glucose bolus is con-

tivation systems show that the micro-Matrix

sumed, the growth will flatten out. OD 600 is

generates results comparable with the min-

measured during the growth phase and is

iBio and 3 liter bioreactors. The results in the

used as an indication of the biomass concen-

shake flask and micro-Flask systems are re-

tration. The maximum oxygen transfer rate in

sulting in the lowest OD600 values. This is due

the different cultivation systems will deter-

to the lack of control of essential process pa-

Pichia pastoris is cultivated using YEPD medi-

mine the maximum achievable biomass con-

rameters in combination with the lower mass

um. Yeast extract peptone dextrose, also often

centration.

transfer in these systems. The RAMbio shows

• miniBio, a 250 ml scale down model of the standard 3 liter bioreactor • 3 liter bioreactor, the gold standard for process development.

Experimental results

abbreviated as YPD, is a complete medium for

Figure 1 shows the results of the Pichia

a high OD600 value in a short time. This is due

yeast growth. It contains yeast extract, pep-

pastoris cultivation in the different cultivation

to the high mass transfer in this system. The

tone, distilled water, and glucose or dextrose.

platforms.

lack of control of basic parameters will result

The yeast extract typically contains all the

in high dissolved oxygen values.

amino acids necessary for growth.

KLa measurements in the micro Matrix

Cultivation conditions were where possible:

To determine what the oxygen transfer in the

Matrix show that the values achieved in this

The KLa measurements in the micro-

• DO 50 % • pH 6 • Temperature 24 °C

micro-Matrix is, we measured the KLa in this

micro-bioreactor is in-line with the values ex-

micro-bioreactor system. The KLa measure-

pected for small scale stirred tank bioreactors.

ments of the micro-Matrix were performed

The influence of the orbital shaker speed is as

with a PreSens oxygen micro-optode. This

expected. The higher the orbiter speed, the

In the micro-Bioreactor, miniBio and 3 liter

measurement system was selected since the

higer the KLa. The influence of the gas flow

bioreactors cultivation conditions can be con-

integrated oxygen sensors in the micro-Ma-

rate shows that up to 200 rpm shaking speed,

trolled accurately. The orbital shaker, the

trix have a t90 of app. 2 minutes which is too

the influence of the gas flow is limited. This is

RAMbio and the micro-Flask cultivations only

slow to measure the rapid change in oxygen

probably due to the limited mass transfer at

allow temperature control. The YPED medium

concentration expected in this experiment.

these lower mixing speeds. From 300 rpm up-

is buffered to make sure that pH=6 for the or-

Measurements were done in demineralized

wards the mass transfer is in line with small

bital shaker/shake flask, the RAMbio and the

water, a coalescenting medium. KLa values of

scale stirred bioreactors and higher gas flow

micro-Flask cultivations.

regular stirred tank bioreactors are in the 200/

rates result in higher KLa values.

Oxygen is not measured or controlled in

hr range.

Based on the measured KLa values and the

these systems. The orbital shaker/shake flask

The micro-optode was mounted in the top

Pichia pastoris cultivation results, it can be

and micro-Flask systems will be oxygen limit-

plate, and the tip of the sensor was fully sub-

seen that the micro-Matrix is a good scaledown

ed during growth of the Pichia culture due to

merged into the liquid. Oxygen was removed

model for standard laboratory bioreactors.

poorer mass transfer in these systems. The

from the bioreactors by flushing with nitrogen

oxygen concentration in the RAMbio will not

gas. When all oxygen was removed, the air

be limiting since the mass transfer in this sys-

supply was opened at gas flows of 9 ml/min

tem is much higher and is at the level of a

and 18 ml/min (2 vvm and 4 vvm). Gas was

stirred bioreactor. Since the oxygen is not

supplied to the headspace of the micro-bio-

Erik Kakes. Applikon Biotechnology B.V.

controlled in this system, it is expected that

reactors (gas overlay). Experiments were re-

Stefan Berger. ReseaChem GmbH

AUTHORS

Table 1 5 ml volume

Controller output (gas flow)

RPM

100 % 50 % (18 ml/min) (9 ml/min)

100

3.2

200

26.8

250

57.7

300

122.2

350

172.7

400

204.9

27

97.1

147.3

KLa (1/hr) measurements in the micro Matrix

01

OD 600 measurement of Pichia pastoris cultures in different cultivation systems.

54

Life Sciences plus 01 I 2015

P L AY E R S & P R O D U C T S

TPP TubeSpin Bioreactor in Space One more application area for cell culture bioreactors can be provided by spaceflight practice: Fruit flies went in a two-month space travel, withstood the test and returned back to earth alive and unharmed. Good sterile gas exchange was very important for the sustenance of the insect.

W

hen performing studies with bio-

tion.

with

in duration. At the return to the Earth, the fruit

logical objects in long-term un-

Drosophila melanogaster fruit fly considerable

In

long-duration

experiments

flies showed manyfold population growth thus

manned space flight, we face the

inner space for actively moving stages of de-

confirming efficiency of gas transfer in micro-

problems such as the risk of mi-

veloping insect are also essential.

gravity conditions and high strength properties

cro-bial contamination of the housing for liv-

In the Drosophila experiment at Foton-M #4

of the TubeSpin Bioreactor 600.

ing organisms, microgravity-induced decrease

satellite flight, July – August 2014, the TPP

in gas exchange, which impair the habitability

TubeSpin Bioreactor 600 was a key element of

www.tpp.ch

conditions, and substantial mechanical im-

the device developed by the Russian Federation

Noykem Ltd., the TPP dealer in Novosibirsk (Russia)

pacts on construct elements caused by linear

State Research Center Institute of Biomedical

has generously supported the experiment in space.

and impact accelerations during spacecraft

Problems RAS (IBMP), Moscow, Russia. Air en-

In Switzerland, the TPP Bioreactor 600 is available

launch and recovery.

tered the device via gas-permeable membrane

through www.faust.ch

These suggest the use of high-performance

mounted in the bioreactor cap. Jelly nutrient

Thanks to the sales force of Noykem and Olga Larina with

gas-permeable membranes in experimental

medium was molded on the bioreactor bottom

her scientists of the FGBUN SSC RF Institute for Biomedical

devices, which enable sufficient oxygen influx

side, leaving approximately 400 cm3 of free

Problems (Moscow) for kindly providing information and

along with effective microorganism intercep-

space for flying. The Foton flight was 1.5 month

photos of the experiment!

THE VERY BEST FOR INSTRUMENTAL TLC Good sterile gas exchange was very important for the sustenance of the insect.

Drosophila melanogaster flies successfully withstood the test and returned back to earth alive and unharmed.

Flying apparatus immediately after completion of the experiment. The bioreactors were experiment equipment, as well as house and dining room for flies (Pictures: Sales force of Noykem and the scientists of the FGBUN SSC RF Institute for Biomedical Problems, Moscow).

Life Sciences plus 01 I 2015

TLC VISUALIZER PROFESSIONAL VISUALIZATION, DOCUMENTATION AND EVALUATION OF HPTLC AND TLC PLATES

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P L AY E R S & P R O D U C T S

CLEANROOM AND LABORATORIES

Integrated Solutions for Critical Environments in the Field of Life Sciences Cleanrooms and labs pose immense challenges especially in relation to running-cost optimization, quality standards, availability, and safety. Considerable sums are invested behind the processes that run in these environments, as illustrated by the life sciences industry’s high research costs. It is therefore only logical that solutions are implemented in these environments that are based on smart building technology which meet quality assurance requirements and guidelines.

ROBERTO FUMAGALLI

C

Where ambient conditions are critical,

integrating particle counters which allow fans

the protection of people and products takes

to be optimized. The speed of supply and ex-

top priority. It must be ensured at all times

traction fans is adjusted to suit actual demand

leanrooms and labs are cutting-edge

that the product is not contaminated through

taking into consideration the minimum air-

scientific

environments

cross contamination. Here, moving air is the

exchange rate and particle concentration

which pose the highest demands on

key protective barrier. Its temperature, hu-

levels in the cleanroom. This noticeably im-

systems that protect people, assets

midity and particle concentration also influ-

proves the facility’s energy efficiency.

working

and data. Owing to hazardous substances,

ence product quality.

cross-contamination risks and exceptionally

Owing to the extremely high air-exchange

stringent regulations, it is essential that all

rates demanded in cleanrooms for such a pro-

ambient conditions are controlled. Building

tective-barrier function, HVAC systems ac-

Integration of Assorted Building Technology Solutions

management solutions play a key role here.

count for around 65 % of energy costs. To re-

Different risks are present in each room of a

These can increase safety, optimize procedur-

duce particle concentrations, in most areas

life sciences facility. In a lab, it is essential,

al and process efficiency and ensure regulato-

the air needs to be exchanged up to 100 times

for example, that explosions are prevented

ry compliance – all while keeping life-cycle

per hour. Here, an integrated monitoring sys-

and people are protected against poisonous

costs down.

tem to track critical GxP parameters such as

substances. By contrast, high air-flow rates in

pressure, humidity, temperature and particle

cleanrooms demand rapid and reliable fire

concentrations achieves savings such as by

detection. In addition, countless safety chal-

Energy-Efficient Working Environment in Demanding Areas

lenges need to be tackled – from perimeter protection, visitor and personnel manage-

when facilities are equipped with the best

ment and entry control of specific areas such

technology. This is made possible with

as labs, isolation rooms, blood banks, and

building automation which allows heating, cooling, ventilation and lighting to be ad-

➜

Significant energy savings can be achieved

pose specific challenges. Ambient lab conditions have a direct impact on people’s safety and research quality in critical areas of the life sciences industry. With integrated building automation solutions for lab buildings, demanding workplace conditions can be managed safely, conveniently and economi-

risks, an increasing number of companies are demanding safety strategies that contrib-

justed to suit requirements. Labs and cleanrooms are demanding working areas that

cleanrooms. Given the ever-changing safety

Significant energy savings can be achieved when facilities are equipped with the best technology.

ute toward protecting business continuity and viability.

As such, integration is synonymous with greater safety, improved oversight, greater investment protection and process optimization. For instance, entry systems can be combined with building automation solutions to achieve additional efficiency gains by ensuring areas are suitably lit and venti-

cally at all times.

56

Life Sciences plus 01 I 2015

P L AY E R S & P R O D U C T S I C l e a n r o o m a n d L a b o r a t o r i e s

➜ One integrated, seamless platform allows cross-system interaction and enables the individual systems to be combined in a smart way.

integrated, such as elevators and technical lab systems. Total Building Solutions also ensure integrated systems function reliably throughout their entire life cycle – with no

➜

Where ambient conditions are critical, the protection of people and products takes top priority.

fire protection solutions to optimize processes in the event of an incident. The entry system enables a central department to watch one or more areas. This makes it easier to deal with events, ensures safety in critical situations and is key to standardization and

first update is installed during live operation.

This security is based on the rigorous application of internationally standardized inter-

Convenience, efficiency, and lated depending on use – or combined with

unpleasant surprises, for example, when the

safety can be increased across the board by integrating building technology solutions in labs and cleanrooms.

optimization.

faces such as BACnet, LON, OPC and M-Bus. The second essential requirement is met through the systematic certification of the integration interfaces in the TBS certification center from Siemens.

Events in Zug and Lausanne in Cooperation with Swiss Cleanroom Concept GmbH To delve deeper into the challenges posed by critical environments in life sciences, Siemens

In an emergency, standardized processes

Switzerland together with Swiss Cleanroom

can be of incalculable value. If, for example,

Concept GmbH is holding two Events on Sep-

toxic gas concentrations rise, the ventilation

tember 23, 2015 in Zug and on December 2,

system’s extraction rate increases thereby

2015 in Lausanne. In fascinating presenta-

accelerating the venting of harmful vapors. In

individual systems support each other and

tions, guests will learn more about integrated

the event of a fire, shutters lift automatically

generate an added value which separate, iso-

solutions for safe, economical, and easier-

thereby improving visibility and giving emer-

lated systems are unable to achieve.

to-manage labs in the R&D field, cost-opti-

gency services easier access.

mized project implementation, and compre-

Value-Add through Integration

Total Building Solutions for Critical Environments

The examples given illustrate that conve-

With its Total Building Solutions (TBS),

panels will be exhibited covering each disci-

nience, efficiency, and safety can be increased

Siemens offers products and solutions for

pline, such as fire detection, entry control, and

across the board by integrating building tech-

smarter and more productive buildings. Solu-

video surveillance, where attendees can test

nology solutions in labs and cleanrooms. To

tions for critical environments are part of the

out the particular systems live. In Zug, the

pool all the know-how and operate multiple

Siemens TBS offering. These ensure that all

program will be rounded off by a guided tour

facets of a building with ease from one loca-

the building’s technical systems are perfectly

of the fire and TBS lab.

tion, a powerful building management system

coordinated. Total Building Solutions are

is used such as Desigo CC from Siemens. Com-

based on a standard IT infrastructure – and

For further information please visit

bining building automation (HVAC, room auto-

offer secure operation from any location. All

www.swisscleanroomconcept.ch

mation, energy efficiency), building safety (fire

the building’s technical infrastructure sys-

detection, intrusion detection, entry systems,

tems are integrated – from heating, ventila-

video surveillance, evacuation systems, gas de-

tion, air

tection) and power distribution on one inte-

equipment, entry control, video surveillance,

grated, seamless platform allows cross-system

fire detection, alarm signaling and evacua-

interaction and enables the individual systems

tion to lighting. Using standardized inter-

Roberto Fumagalli. Sales and Market Manager

to be combined in a smart way. In doing so, the

faces, additional building technology can be

Life Science at Siemens Switzerland

Life Sciences plus 01 I 2015

hensive building technology solutions – and lots more besides. In addition, interactive

conditioning, cooling, electrical

57

AUTHOR

P L AY E R S & P R O D U C T S

CANCER THERAPY

The Art of Designing Selective Inhibitors

SWISS START-U Ps

Cancer, inflammation, and autoimmune diseases often involve the activation of the PI3KPKB/AKT-mTOR pathway. Numerous enzymes – lipid and protein kinases – work hand in hand in this highly complex signaling network, which is key to cell growth and survival. PIQUR Therapeutics AG, a recent spin-off company of the University of Basel, is within the vanguard of pharmaceutical developers of dual PI3K/mTOR inhibitors for cancer therapy.

cine at the University of Basel, discovered that

Beyond academic research, Basel is a center

Wortmannin, a steroid metabolite of the fungi

of industrial research on the highly conserved

Penicillium funiculosum, is a highly potent

PI3K-PKB/AKT-mTOR

inhibitor of phosphoinositide 3-kinase (PI3K).

Roche, Genentech (a subsidiary of Roche

Too toxic to become a drug candidate, the mol-

since 2009), and Novartis are all developing

ecule evolved into a useful tool for studying

inhibitors for mTOR, isoform-specific PI3K,

signal transmission of PI3K. While Wymann

pan-class I PI3Ks (inhibiting all 4 isoforms α,

became an expert on PI3K and phosphoino-

β, γ, δ), PI3K and mTOR (dual PI3K/mTOR

sitide signalling, biochemist Brian Hemmings,

inhibitors) and/or AKT. Recently, the newly

who retired in 2014, lead one of the three re-

established Piqur Therapeutics AG, a spin-

search groups in which the enzyme Protein

off from the University of Basel, founded in

asel is an important center of academ-

Kinase B (PKB), also known as AKT, was dis-

2011, emerged in the midst of these pharma

ic research in the field of the highly

covered in 1991. Hemmings worked at the

giants. Newcomer Piqur possesses a dual

conserved PI3K-PKB/AKT-mTOR sig-

Friedrich Miescher Institute for Biomedical

PI3K/mTOR inhibitor, PQR309, which is un-

naling pathway, crucial for cellular

Research in Basel, which belonged to the No-

dergoing phase I clinical multicenter studies

balance. Over 25 years of intense investigation

vartis Research Foundation until 2012. Finally,

since January 2014, and already employs

in this area have shaped the image of a highly

in 1993, molecular biologist Michael Hall, Pro-

about 25 people (18 full time equivalents), in-

complex network whose activation is tightly

fessor at the Biozentrum University of Basel,

cluding scientists as well as economic and fi-

controlled by a multistep process. Multiple

discovered the kinase enzyme mTOR (mam-

nancial professionals.

players participate in its activation and prompt

malian Target of Rapamycin). Hall is a leader

“Clinical phase I studies with PQR309 are

regulation. Many of them are kinase enzymes

in the field of mTOR signalling.

nearly completed, results will be published

BEATE PEISELER-SUTTER

B

signalling

pathway.

that transfer a phosphate group from adenosine triphosphate (ATP) to specific substrates.

Others regulate the localization of their downstream targets, mostly by promoting their at-

Successful Fund Raising

tachment to the inner side of the cell membrane. The pathway includes several internal feedback loops and interacts with other signal-

According

to CEO Vladimir Cmiljanović, Piqur raised about 40 million Swiss francs in the last two years. While seed money was brought together by private investors, investment fund Versant Ventures, specializing in life sciences investments, joined the company bringing 11 million Swiss francs in April 2014. Versant Venture partner Guido Magni is member of Piqur’s Board of Directors, another Versant Venture member, Gianni Gromo, serves as an observer. “Both Versant Venture experts bring a lot of professionalism to our company and keep us in line,” state Cmiljanović and Matthias Wymann, member of the Board of Directors of Piqur. The Swiss Commission for Technology and Innovation (CTI) provides further important support (see article above).

ing cascades (see box). If one or more kinases or regulatory proteins are deregulated, e. g. due to gene mutations, cells may become unbalanced and enter into a phase of unabated growth or chronic inflammation. It is likewise harmful if proteins are on hand in too high concentration or if they are overactive due to failure of inhibitory mechanisms. The result is serious health problems and diseases such as cancer, allergies, cardiovascular illness, diabetes, and adiposity.

In the early 90’s, biochemist Matthias Wymann, today Professor at the Department of Biomedi58

Life Sciences plus 01 I 2015

soon. Phase II clinical studies are under preparation,” informs Piqur’s CEO Vladimir Cmiljanovi . Cmiljanovi

was trained as a

chemist in Serbia and Switzerland. He entered the field of PI3K-PKB/AKT-mTOR signaling after his master thesis in the group headed by chemist Bernd Giese in Basel, now Professor emeritus still pursuing his work at the University of Fribourg since 2010. During his master thesis, Cmiljanovi

The chemistry team of the University of Basel and PIQUR united to target the PI3K/mTOR pathway.

From left to right: Emeline Teillet, Technician (Piqur), Dr. Denise Rageot (UniBas), Stephanie Bünger, Technician (UniBas), Fatime Imeri, Master student (UniBas), Dr. Alexander Sele (UniBas), Alix Dall Asen, student (Chemical Engineering School of Mulhouse). Missing: Dr. Florent Beaufils, lab head chemistry PIQUR; Mathieu Berthelot, Master student University of Burgundy.

came into contact with

PI3K expert Matthias Wymann. “We didn’t initially plan to design drugs, but slid into inhibitor synthesis by proof-of-concept experiments

PI3K-PKB/AKT-mTOR signaling pathway

for another project. Soon Vladimir and his sister Natasa came up with a synthetic route yielding very powerful PI3K inhibitors and we could derive a recipe for a very high hit rate,” remembers Wymann. Through earlier work with PI3K/mTOR inhibitors from pharmaceutical companies, they were thus able to estimate the potential of their discovery and filed a patent application. Cmiljanovi continued to improve physicochemical properties of his small molecule inhibitors during doctoral and postdoctoral research studies.

Dream Team “on Board” “I contacted Roche and Novartis several times in order to see whether they were interested to take over the project but was told to pursue until phase I clinical studies. After some hard times, I finally had to decide whether to give up the project or to start up my own business,” he tells. On the advice of family members, including chemists, business people, and team play experienced professional handball players, Cmiljanovi (who has already worked as a professional handball player too) co-founded Piqur Therapeutics AG with Giese and Wymann. Since 2011, he brought what he tells

➜ “We didn’t initially plan to design drugs, but slid into inhibitor synthesis by proof-of-concept experiments for another project.”

PROF. MATTHIAS WYMANN. Piqur, University of Basel

Life Sciences plus 01 I 2015

Signal

transmission in the PI3K-PKB/AKT-mTOR pathway starts when an external ligand (e. g. a growth factor, chemokine, cytokine) binds to a cell surface receptor, e. g. a receptor tyrosine kinase or a G protein-coupled receptor, which causes phosphorylation of a multitude of intracellular proteins. Receptor tyrosine kinases are transmembrane proteins. They possess an extracellular binding site for ligands and a cytosol-directed catalytic kinase center. Binding of the external ligand induces formation of receptor dimers. Due to that, the enzymatic moieties come closer to each other and start mutual phosphorylation, which makes PI3K move from the cytosol to the cytoplasmatic membrane. PI3K is composed of a regulatory and a catalytic subunit. Class I PI3 kinases are associated with chronic disease. They comprise a regulatory subunit p85 and a catalytic subunit p110. There are different p110 isoforms, which are differently distributed in differing tissues. p110alpha and p110beta influence cell growth, p110gamma and p110delta are involved in the immune system and inflammation. All class I PI3Ks bind via p85 to the ligand-activated receptor tyrosine kinase, whereupon p110 transfers a phosphate group to membrane phosphoinositides. These phospholipid components of eukaryotic cell membranes act as signal mediating secondary messengers. Lipid phosphatidylinositol 4,5-bisphosphate (PIP2) is phosphorylated and becomes phosphatidylinositol(3,4,5)-trisphosphate (PIP3). Increased PIP3 levels stimulate protein biosynthesis, cell growth and cell mobility.

PIP3 has a binding site for the kinase PKB/AKT, which moves to the cell membrane where it gets in contact with other kinases. PKB/AKT becomes phosphorylated and thus activated. Activated PKB/Akt in turn participates indirectly in the activation of mTOR complexes (mTORC). mTORC1 and mTORC2 regulate cell growth, proliferation, and cell survival. The activation of mTOR complexes and resultant feedback loops is part of a highly complex signal cascade, which does not proceed in a linear manner and is not yet fully understood. The situation becomes critical when protein biosynthesis and cell growth run out of control. This may be the case when regulatory proteins, which usually slow down or switch-off the PI3K-PKB/AKT-mTOR signal pathway, do not function. PTEN (phosphatase and tensin homologue deleted on chromosome 10) is one such regulatory protein. This lipid phosphatase enzyme acts as an opponent of PI3K. While kinases catalyze phosphorylation reactions, phosphatases catalyze dephosphorylation reactions. In healthy tissue, PTEN is able to abolish PIP3, but in many advanced tumors it is inactivated due to mutations, gene deletion, or epigenetic silencing gene mutations. PI3K and PIP3 play important roles in inflammation and allergies too. Matthias Wymann and co-workers were able to demonstrate that mice, which are lacking the PI3K gamma gene (PI3K gammaknockout mice), are protected against some chronic inflammatory reactions.

Such basic know-how can help adapting therapy to each patient and their individual tumor in order to receive best results in apoptosis and tumor shrinking. “Phenotypic and functional heterogeneity arise among cancer cells within the same tumor.

Besides, cancer cells display compensatory plasticity. Cells may switch to other signaling pathways when under inhibitory attack. The result is primary resistance, which can become reinforced owing to mutations acquired through radio- or other therapy,” as PI3K expert Wymann knows well.

59

P L AY E R S & P R O D U C T S I C a n c e r Th e r a p y

sites for ATP. It is an art to design an inhibitor

Bernd Giese serves as Chairman of the Board

that recognizes distinctly only one or two of all

of Directors, co-founder Matthias Wymann is a member of the Board of Directors, and Michael

➜

“a dream team” to the company. Co-founder

these kinases and to fine-tune its inhibitory effects,” emphasizes Piqur`s CEO Vladimir

Hall is a member of the Scientific Advisory

Cmiljanovi . PQR309 shows strong inhibition

Board. Doriano Fabbro, until 2012 head of ki-

of PI3K and weaker inhibition of mTOR. “Its

nase biology at Novartis, became Piqur’s CSO. “When Novartis shut down Doriano’s research department, he joined our team,” rejoices Cmiljanovi .

The University of Basel supports Piqur with

“We came a long way with

preclinical anticancer activity against solid tumors and lymphomas is comparable to Novar-

very limited resources.”

tis dual panPI3K/mTOR inhibitor BEZ235,” says Cmiljanovi , “but it is best in class with

VLADIMIR CMILJANOVIĆ. CEO, Piqur

regard to its physiochemical and therefore

patent applications and by providing labora-

drug-like properties.”

tory space. Chemical research and develop-

Well-Filled Pipeline

ment activities are carried out in a university’s lab in the Biopark Basel-Rosental next to the

Commission for Technology and Innovation)

Physicians often notice hyperglycemia as a side

Department of Biomedicine, the Friedrich

projects. The first was designated to expand

effect of PI3K inhibition because the PI3K-

Miescher Institute, and ETH Zurich’s Depart-

the company’s portfolio, the second served to

PKB/AKT-mTOR pathway is involved in cellu-

ment of Biosystems Science and Engineering.

sharpen sub-specific inhibitor selectivity, and

lar metabolism and glucose/insulin regulation.

Biological PI3K research and assays are exe-

the third project is still ongoing. “We came a

“This effect is reversible and can be managed

cuted by Wymann’s research group. The man-

long way with very limited resources,” states

by administering oral hypoglycemics. We see it

ager’s offices are located in the Technology

the CEO. He is especially proud to have man-

as a biomarker predicting that PI3K inhibition

Park Basel, from where outsourced activities,

aged fine-tuning of PI3K versus mTOR inhibi-

works,” declares PIQUR’s CEO. The company

accomplished by contract research organiza-

tion of PQR309 thanks to structure biological

has some more inhibitors of the PI3K-PKB/

tions, are coordinated. “The Technology Park

approaches in cooperation with Roger Wil-

AKT-mTOR pathway in its pipeline, including

offers the possibility to rent additional labora-

liams from the MRC Laboratory of Molecular

a selective mTOR inhibitor (preclinical stage;

tory space,” praises Cmiljanovi , who plans to

Biology in Cambridge, UK. “The human ge-

indication cancer therapy) that is able to pass

go through phase II clinical trials autono-

nome displays more than five hundred differ-

through the blood-brain barrier and molecules

mously.

ent kinases genes. We distinguish between

suited for non-oncological applications (pre-

In conjunction with the University of Basel,

protein kinases, lipid kinases, and carbohy-

clinical stage; indication Inflammation, oph-

Piqur was already awarded three CTI (Swiss

drate kinases, all of them possessing binding

thalmology, dermatology and CNS).

Competence in Process and Laboratory Technology

Save the Date

20 to 23 September 2016 Messe Basel www.ilmac.ch 60

Life Sciences plus 01 I 2015

P L AY E R S & P R O D U C T S

TOOLS EchoMRI – Whole Body Composition Analyzer

FCX-MP1000Extern-FH-CH: Oxygen sensor module for ppm range Robust design for demanding applications: Pewatron has released a new oxygen sensor module that can be used in demanding applications where oxygen concentrations below 1000 ppmO2 are measured. The key element of the sensor module is the oxygen sensor from Fujikura; it is an amperometric solid-state sensor (zirconia) with high output accuracy, a short response time to gas concentration changes and ruggedness against pressure change, humidity and other gases. The non-amplified signal from the oxygen sensor is in the range of micro amps and linear.

The FCX-MP1000 sensor module is a signal-conditioning unit that amplifies the sensor current output and gives an analogous, low-noise linearized current output (4-20 mA). As the total power consumption is low (< 2W), the FCX-MP1000 oxygen sensor module is suitable for mobile applications. The oxygen sensor is configured in a flow configuration with connectors for hosing. The ppm sensor is encapsulated in a pressure proof aluminium flow housing, enabling analysis of oxygen concentrations from small sample extracts.

The output oxygen concentration is highly linear and the accuracy is better than +/- 50 ppmO2 with a response time for gas concentration changes of below 10 seconds. Concentration ranges are from 0 to 1000 ppmO2.

Pewatron AG | www.pewatron.com

➜

Life Sciences plus 01 I 2015

EchoMRI Quantitative Magnetic Resonance Body Composition Analyzers for animals and humans take direct measurements of total body fat, lean mass, free water and total body water. The measured values depend on the density of hydrogen nuclei and the physical state of the tissue. More rapid, accurate and precise than other methods, the EchoMRI technology allows for fast measurements (0.5 – 3.2 minutes, depending on the precision option) in-vivo without anesthesia or sedation. Unlike DXA, EchoMRI measurements are radiation-free, do not require the subject to remain still, and in a peer-reviewed study (Taicher et al., 2003) yielded 24 times DXA‘s precision facilitating convenient, low-stress repeated tracking of small changes in body composition. Moreover, EchoMRI instruments measure fat and lean mass as independent characteristics, unlike DXA, which measures the fat to lean ratio and is, therefore, prone to error propagation from lean to fat. The systems are less expensive than conventional MRI, are completely silent and do not require advanced training to use.

Zinsser Analytic GmbH | www.zinsser-analytic.de

AL4: Customised, high precision, digital low pressure sensors from Fujikura Fujikura officially released the new digital AL4 series of gauge low pressure sensors for volume production. The AL series has a very small footprint (11.36 mm × 10.32 mm) in comparison with other low sensors, SMT pin configuration and a high total accuracy (+/-1.5 % FSO) within a compensated temperature range of 0 °C to 50 °C. The digital AL4 series is a two-chip system consisting of a piezoresistive pressure sensing chip with a very high maximum load pressure of 100 kPa and an ASIC signal conditioning chip. The ASIC allows digital signal conditioning and communication via the I2C protocol. The digital AL4 series low pressure sensor product follows the successful introduction of the analogue, high precision A2 series pressure sensor and the digital, high precision A4 series pressure sensor. The heart of the ASIC is a 14-bit ADC, which achieves the equivalent sensitivity of up to 0.15 Pa/LSB for a 2 kPa full-scale sensor. Six slave address codes can be assigned to the pressure sensor for individual communication with the micro-controller master. The sensors are delivered in the usual high quality Fujikura packages comprising tray or tape & reel, according to customer specification. Standard measurement ranges are between 0 kPa to 2 kPa and 0 kPa to 10 kPa; non-standard ranges are available on request.

The pressure range is configurable as positive, negative or bidirectional gauge.

Supply voltage is configurable at 3.0, 3.3 or 5.0 VDC. Pewatron AG | www.pewatron.com 61

P L AY E R S & P R O D U C T S I To o l s

WAKO Highlights QuickGene-Mini80 is a compact system requiring no centrifugation in the isolation process, giving less strain to samples and enabling rapid nucleic acid isolation. DNA/RNA can be easily isolated from various samples including whole blood / tissue / cells / plants / virus and others. The QuickGene-Mini80 shares a common isolation mechanism and kits with our semi-automated nucleic acid isolation system, Quick-Gene-810. QuickGene-Mini80 offers high performance comparable to Quick-Gene-810 at a reasonable price.

Phos-tag is a functional molecule that specifically binds all phosphorylated forms of Ser / Thr / Tyr. It is applicable for the specific separation of phosphorylated proteins as well as for the western blot detection, purification by agarose gel chromatography and Maldi-TOF/MS analysis. The following five kinds are available: • P hos-tag Acrylamide recognizes and separates all phosphorylated forms of Ser / Thr / Tyr and can be used by applying almost the same procedure as that of a conventional SDS-PAGE with any electrophoresis tank.

• SuperSep Phos-tag Ready-to-Use Precast Gels have a long-term stability (Stable for 6 months).

• Phos-tag Biotin for Specific Detection of Phosphoproteins on PVDF membrane with similar procedures as those in ordinary western blotting. No detection without any anti-phosphorylated antibodies on western blot.

• Phos-tag Mass Analytical Kit MALDI-TOF/MS with High Sensitivity. Before use, Phos-tag Mass Analytical Kit is mixed with samples for MALDI-TOF/Mass analysis. Phosphorylated molecule-Phos-tag complex is detected in a positive mode, and phosphorylated molecule usually difficult to detect can be detected with improved sensitivity.

• Phos-tag Agarose Purification of Phosphorylated proteins by Affinity Chromatography.

No reducing agent or surfactant is used.

IGZ Instruments AG | www.igz.ch

LIFE SCIENCE Brenntag Schweizerhall Inc. Business Unit Life Science Elsässerstrasse 231, CH-4013 Basel Tel.: +41 (0) 58 344 80 00 Fax: +41 (0) 58 344 82 08 www.brenntag.ch Business Manager Life Science Beatrice Del Principe Tel.: +41 (0) 58 344 86 68 beatrice.delprincipe@brenntag.ch

www.brenntag.ch

From producing food that serves as a vital source of nutrition and energy to manufacturing pharmaceuticals that cure illnesses and keep us in good health, companies in the life science industry manufacture products and provide services that are of fundamental significance to modern society.

Brenntag fully understands the importance of making sure that these offerings are undertaken with the highest quality and most effective products on the market.

Over the years, Brenntag Life Science has emerged as the specialty chemical industry’s number one partner when it comes to the distribution of products and services that have an indispensable impact on a company’s performance in the life 62 science industry. Life Science at Brenntag

encompasses a variety of business units that offer our customer high-quality ingredients, tailor-made supply chain solutions and an unrivaled degree of technical and market expertise. Partnering with Brenntag Life Science, one of three divisions at Brenntag along with Material Science and Environmental, means that your company is aligned with a true industry leader, one with a presence in every European market who understands the importance of always staying one step ahead of the curve.

Our division at a glance Brenntag is the distributor of choice for companies and suppliers in the specialty chemicals industry. The Life Science division at Brenntag incorporates our renowned Food & Nutrition, Animal

Nutrition, Cosmetics as well as Pharma business units. Brenntag Life Science is made up of an experienced team of sales representatives and technicians who consistently rank amongst the best in their field for expertise and professional service. Brenntag has a distinguished presence in every European market, with over 210 distribution centers in more than 30 countries and an extensive network of specialized application centers across Europe. We aim to create value for every one of our customers and suppliers, irrespective of the application or business challenge at hand. Brenntag Life Science is committed to fulfilling all of your requirements by offering you the widest range of products, most innovative and sustainable solutions and best technical Lifethe Sciences plus 01 I 2015 expertise on market.

F I LT R AT E I N e w s

NEWS

Comparing the Genomes of the Leprosy Bacteria

Leprosy

is a chronic infection of the skin, peripheral nerves, eyes and mucosa of the upper respiratory tract, affecting over a quarter of a million people worldwide. An infection with the bacteria can lead to reduced sensitivity in the body, resulting in skin lesions, nerve damage and disabilities. Although the disease could be pushed back with antibiotics, leprosy remains endemic in many developing countries today. Until recently, leprosy was attributed to a single bacterium, Mycobacterium leprae. Today, it is suspected that its close relative, Mycobacterium lepromatosis, might cause a rare but severe form of leprosy. Scientists of the lab of Stewart Cole at EPFL’s Global Health Institute have analyzed for the first time the complete genome of M. lepromatosis, and compared it to that of the major leprosycausing bacterium. Published in PNAS, the study reveals the origin and evolutionary history of both bacteria. In collaboration with researchers of the University of Tübingen, the Max Planck Institute for the Science of Human History, and the Universidad Autónoma de Nuevo León Cole’s studies found that the two species of bacteria are very closely related and have a common ancestor. The study showed that M. lepromatosis is relatively scarcely spread and seems to be restricted to patients in Mexico, whereas M. leprae is widespread across the world. M. leprae is able to infect neuronal Schwann cells, which produce protective sheaths of the axons of peripheral neurons. The presence of related genes in M. lepromatosis means that this bacterium could also be attacking these cells. This genomewide comparative study offers new and deeper insights into the biology of M. lepromatosis. By uncovering the evolutionary history of the two leprosy bacteria, it can open up new ways for developing therapeutic strategies. # www.epfl.ch

Mycobacterium leprae (red). (Picture: CDC)

Mental Disorders and Physical Diseases Co-occur in Teenagers

Every third

Every third teenager has suffered from one mental disorder and one physical disease. (Picture: Wikimedia)

Life Sciences plus 01 I 2015

teenager has suffered from one mental disorder and one physical disease. These findings were reported by researchers from the University of Basel and the Ruhr-Universität Bochum. Their results based on data from 6,500 U.S. teenagers, have been published in the scientific journal “Psychosomatic Medicine.”

A research team led by PD Dr. Marion Tegethoff from the Faculty of Psychology at the University of Basel analyzed how often and in what manner these associations already occur in children and adolescents.

The researchers analyzed data from a national representative cohort of 6,482 U.S. teenagers aged 13 to 18. And found that 35.3 % of children and adolescents reported at least one mental disorder and one chronic physical disease. The strongest correlation was found between affective disorders (e. g. depression) and diseases of the digestive system. Adolescents with anxiety disorders were also suffering above-average from arthritis, heart disease and diseases of the digestive system. Similar correlations occurred between eating disorders and seizures (epilepsy).

Factors such as age, gender or socioeconomic status of the adolescents did not account for these associations. Due to the cross-sectional design of the study, the results do not show if and how mental disorders and physical diseases are also connected causally. Future studies should identify risk factors as well as the biological and psychological mechanisms responsible for these associations. Treatment should take into account both the physical disease as well as the mental disorder.

This would lead to better health care for children and adolescents and would prevent unfavorable long-term effects for individuals as well as for the health care system in general. # www.unibas.ch

63

Seminaragenda 2015 06. Mai 15 Sicherer Umgang mit Zytostatika Rheinfelden 07. Mai 15

Single Use im Reinraum Bereich Rheinfelden

20. Mai 15

HACCP versus GMP in der Lebensmittelindustrie ein Widerspruch?

Rheinfelden

24. Juni 15 Reinraummesstechnik für den Anwender Wattwil 10. Sep. 15 GMP Aufbauwissen Rheinfelden 16. Sep. 15 Anforderungen an die GMP Zonen E und F Rheinfelden 23. Sep. 15 Critical Environment im Life Science Umfeld Zug 21. Okt. 15 Train the Trainer Rheinfelden 29. Okt. 15 GMP Basiswissen Rheinfelden 03. Nov. 15

9. Swiss Cleanroom Community Event Muttenz

12. Nov. 15

Basis Seminar für Reinraummitarbeiter Rheinfelden

18. Nov. 15

Anforderungen an die Quality Oversight Rheinfelden

19. Nov. 15

Lean Manufacturing / Six Sigma im Pharma Bereich Rheinfelden

24. Nov. 15

Anforderungen an die Spitalpharmazie Rheinfelden

25. Nov. 15

Qualifizierung / Validierung im GMP und Reinraum Umfeld Rheinfelden

Informationen und Anmeldung: www.swisscleanroomconcept.ch

F I LT R AT E I N e w s

No Gold without Microbes

Due

to its high densitiy, gold ought to have been moved to the Earth’s core in the course of the creation of the Earth.

But in actual fact it also accumulated in the Earth’s crust. Geologists do not agree on the reasons for this phenomenon.

Professor Hartwig Frimmel, chairman of the Department of Geodynamics and Geomaterials Research at the University of Würzburg is now adding a new theory: He published his idea, that prehistoric microbes are responsible for binding gold to the Earth’s crust, in the the journal “Mineralium Deposita.”

The Würzburg scientist is regarded as the world’s leading expert in gold deposits. He spent a long time as a professor at the University of Cape Town conducting research in South Africa’s Witwatersrand region, among others. This is where the world’s largest concentration of gold in the Earth’s crust can be found.

“Wherever there are large quantities of gold, there are also layers of stratified carbonaceous substances that are of biological origin,” says Frimmel. He supposes that these are the remains of cyanobacteria which lived three billion years ago and produced oxygen by performing photosynthesis.

At that time, a lot of gold had to be accumulated and solubilized in water due to acid rainfalls and high quantity of hydrogen sulphide.

Where this water came upon colonies of cyanobacteria arranged into mats, the gold was chemically adhered to the surface of the microbes immediately by the oxygen.

Over time and in this manner, for example, the huge gold deposits were created that can be found in such places as the Witwatersrand region of South Africa. # www.uni-wuerzburg.de

Carbonaceous layer of sediment rocks, essentially consisting of kerogen, from the Witwatersrand basin in South Africa – an extremely rarely well preserved remnant of mats of the first microbes performing photosynthesis. (Photo: Hartwig Frimmel)

How proteins find the right path

Two competing

activities ensure that proteins safely arrive at their intended destination – in cell organelles like the mitochondria and the endoplasmic reticulum (ER), in particular.

The team around the Konstanz-based biologist Professor Elke Deuerling, who also is the speaker for the Collaborative Research Centre Chemical and Biological Principles of Cellular Proteostasis (SFB 969), succeeded in uncovering that, in contrast to the prevailing view, successful protein transport requires not only the signal recognition particle (SRP), but also the nascent polypeptide-associated complex (NAC). The elucidation of this fundamental cellular process may have far-reaching implications for research on age-related defects and diseases, Wild-type C. elegans hermaphrodite with stained nuclei. (Picture: Wikimedia) such as Alzheimer’s. The study’s results were published 10 April 2015 in Science.

Elke Deuerling compares NAC with ticket checkers who let people into football games, concerts or the cinema based on the kind of ticket. This controlling function is crucial because ribosomes that produce proteins tend to bind the membrane of the endoplasmic reticulum unspecifically.

“Without NAC, a part of the proteins being produced by ribosomes mistakenly end up in the ER. The transport only works correctly when the balance between SRP and the NAC complex is right,” explains Deuerling.

Proteins ending up in the wrong location, not only disturb the homeostasis in the endoplasmic reticulum, but also in mitochondria, because proteins specific to them don’t arrive in the mitochondria, but rather in the endoplasmic reticulum. “This creates an enormous stress in the organism and leads to a drastically shortened lifespan of the model organism C. elegans, used in the study,” says the molecular biologist. # www.uni.kn 64

Life Sciences plus 01 I 2015

F I LT R AT E I N e w s

Driving Tumour Cells to their Death

B cell

acute lymphoblastic leukaemia (B-ALL) is the most common tumour disease in children and also occurs in adults. It develops when signalling pathways in immature B cells, or pre-B cells, are dysregulated.

Prof. Dr. Markus Müschen from the University of California in San Francisco and his team worked together with the researchers Prof. Dr. Hassan Jumaa and Prof. Dr. Michael Reth from the Centre for Biological Signalling Studies University of Freiburg to find a new approach for treating the B-ALL tumour disease and published their research in the journal Nature.

B cells and white blood cells produce antibodies against antigens. Normal B cell development and maturation is regulated by kinase and phosphatase enzymes. These enzymes phosphorylate or de-phosphorylate the signalling subunits of the B cell antigen receptors (BCR). Phosphorylation of BCR activates the B cell. Thus, kinases and phosphatases affect the receptor’s capacity to send signals. In B-ALL tumour cells, certain kinase enzymes, such as the Abelson tyrosine kinase (ABL), are altered and act as oncogenes, spurring the growth of tumours independently of the BCR. The B cells then continue to divide although they do not function. Established treatments with agents that inhibit the ABL kinase are not effective, if resistant ABL mutants develop.

The American and German team discovered that the signalling subunits of the BCR in B-ALL tumour cells are hardly phosphorylated and that there is a higher number of inhibiting receptors on the cell’s surface. Because these receptors bind phosphatases, they prevent the BCR from becoming active. When the researchers shut off the inhibiting receptors or the associated phosphatases, the B-ALL tumour cells died instantly.

Future ALL treatments could aim at inhibiting the phosphatases instead of the ABL kinases and thereby strengthen BCR signals. # www.uni-freiburg.de

Cells of a B cell tumour. (Picture: Reth Group, University of Freiburg)

The metabolism influences the manifestation of respiratory diseases. This was shown in the mother-child study (LiNA), following the medical and environmental development of more than 620 children.

(Picture: André Künzelmann, UFZ)

Metabolic imbalance triggers respiratory diseases in childhood

An alteration

in metabolic balance can be the trigger for an inflammatory immune response. In such cases, forces are mobilised to combat an enemy that does not actually exist. Researchers of Helmholtz-Zentrum für Umweltforschung (UFZ) have been able to show that this applies even to newborns and children under one year of age, and is correlated with the development of respiratory diseases in early childhood. UFZ-researcher Dr Gunda Herberth and her colleagues examined blood samples and made a surprising discovery: Elevated concentrations of specific sugars – hexoses – in the blood were accompanied by elevated concentrations of inflammatory immune parameters. Conversely, a high concentration of other metabolites, such as protein components (amino acids) or degradation products of certain fats, inhibited the development of inflammatory parameters. “Increased concentrations of sugars in the blood therefore do actually lead to the development of an inflammatory immune response, even in newborns. In turn, this is directly correlated with the development of respiratory diseases in early childhood,” explains Dr Herberth.

In-vitro tests carried out by the researchers have confirmed the findings of her epidemiological investigation: In cell cultures, immune cells exposed to hexoses showed elevated concentrations of inflammatory parameters, while those exposed to amino acids inhibited the production of inflammatory components. # www.ufz.de

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Batteries for Microbes

Some bacteria

The Fe(II)-oxidizing bacteria remove electrons from the magnetite battery (discharging, left) whereas the Fe(III)reducing bacteria deposit electrons onto the magnetite battery (re-charging, right side).

are known to use iron for energy generation in nature. Now researchers from the University of Tübingen have shown that many of these microbes can use tiny magnets (magnetite nanoparticles that occur in many soils and sediments) in order to share electrons in the same way that we might use a re-chargeable battery, i. e. for energy storage and use.

Iron is usually only thought to be available as an electron source to microbes as dissolved Fe(II) ions, or as an electron sink in form of poorly crystalline Fe(III) oxides. Fe(II)-oxidizing bacteria have been shown to extract electrons from the dissolved Fe(II) to form orange Fe(III) minerals (i. e. rust). Other bacteria known as Fe(III)-reducers can reverse this process and transfer electrons to Fe minerals but only when the Fe(III) is poorly crystalline. The new results show that to some organisms the iron is also available as an electron source and sink when it is present in a crystalline magnetic mineral (magnetite), which is present in many different environments on Earth and even on Mars. In a study, researchers from the University of Tübingen, Dr James Byrne, Dr Nicole Klueglein, Prof. Erwin Appel and Prof. Andreas Kappler, together with Carolyn Pearce (University of Manchester, UK) and Kevin Rosso (Pacific Northwest National Laboratory, USA), incubated bacteria with magnetite and controlled the amount of light the cultures were exposed to. Using magnetic, chemical and mineralogical analytical methods, the team showed that in light conditions which replicated day-time, phototrophic iron-oxidizing bacteria removed electrons from the magnetite, thereby discharging it.

During night-time conditions, the iron-reducing bacteria took over and were able to dump electrons back onto the magnetite and recharge it for the following cycle. # www.uni-tuebingen.de

From Moss to Mouse

The Chair

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of Plant Biotechnology from the University of Freiburg, Germany, and the biopharmaceutical company Greenovation Biotech GmbH in Heilbronn, Germany, have started Moss bioreactor producing a cooperation to explore in mice human proteins. (Source: the effectivity of human proteins Plant Biotechnology, that have been biotechnologically University of Freiburg) produced in moss. The moss Physcomitrella patens can be cultivated in closed containers such as bioreactors with a volume of up to 500 litres. Complex proteins such as glycoproteins, which are needed as biopharmaceuticals for the treatment of human diseases, can be produced in these Moss Bioreactors.

The advantage is that moss cultures do not contain animalderived components, or pathogens that can affect humans, nor antibiotics that may cause resistance in patients. Further, products from moss have a superior purity. Scientists from the Department of Plant Biotechnology were able to produce human complement factor H in moss, a protein from the immune system that may be used to treat specific kidney diseases.

A Factor H hereditary deficiency is an orphan disease which has major implications. The researchers now hope to gain an insight from their experiments with mice, as to whether the human glycoprotein produced in moss is suitable for use in medication.

The biologists in Freiburg are specialists in moss research and helped to establish Physcomitrella as a model organism for basic biology, biotechnology, and synthetic biology on a worldwide scale. “I am excited about this collaboration between the University Hospital, the University and Greenovation”, says says Professor Ralf Reski, Head of the Department of Plant Biotechnology at the University of Freiburg. “Such public-private co-operations are at the heart of the bioeconomy and will help in developing new medication and generating job opportunities.” # www.uni-freiburg.de

Life Sciences plus 01 I 2015

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