The Humanion Arkive Year Delta 2018-19
September 24: 2018-September 23:2019
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First Published: September 24: 2015





























Molecular Biology

Molecular Biology Arkive

Cell Division: Image: The Institute of Cancer Research, London


New £01.5 Million Wellcome Trust Award for a Joint Study to Identify Disease-Causing Mechanisms in the Cycle Cycle

Image: University of Liverpool 


|| August 07: 2017: University of Liverpool News || ά.  Biochemists at the Universities of Liverpool and Dundee have been awarded a Wellcome Trust Collaborative Award in Science of more than £01.5 million to study potential disease-causing mechanisms in the cell cycle. The research team is composed of Professor Sonia Rocha at the University of Liverpool’s Institute of Integrative Biology and Professor Angus Lamond, Professor Jason Swedlow and Professor Stewart Fleming at the University of Dundee will investigate the interplay between oxygen sensing proteins and cell division.

Oxygen is essential for life in multi-cellular organisms and animals have evolved mechanisms to cope with decreased oxygen concentration, a condition known as hypoxia. In humans, sensing and responding to changes in oxygen involves a class of proteins, called, prolyl-4 hydroxylase domain:PHDs. The new study follows on from earlier work by the research team, which showed that these oxygen-sensing PHD proteins, also, target proteins required for the key process of cell division. Professor Sonia Rocha, Head of the Department of Biochemistry, said, “Our previous finding was unexpected and suggests that sensing oxygen is crucial for regulating cell division.

This project will build on our discovery by studying the molecular mechanisms, that connect oxygen-sensing enzymes and their targets to regulate cell cycle progression.” The research team will test the hypothesis that the sensing of oxygen levels regulates cell division in human tissues and investigate how dysregulation of these mechanisms, may, contribute to human disease, with a major focus on diseases of the kidney, where PHD enzymes are known to have important roles.

Wellcome Trust Collaborative Awards promote the development of new ideas and speed the pace of discovery. They are given to groups of researchers to work together on the most important scientific problems, that can only be solved through collaborative efforts.

Professor Rocha said, “We’re delighted to have received this award, which also, marks the start of a formal partnership between Liverpool and Dundee, two universities with recognised strengths in basic and clinical science.”

Professor Angus Lamond said, “This collaborative project provides an exciting opportunity to apply the world leading expertise we have developed in protein analysis at the University of Dundee to discover clinically important mechanisms that control cell divisions in human tissues.”

For more information about Wellcome Trust Collaborative Awards in Science visit.

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The Immunotype: New Tool Demonstrates the Individuality of Human Immune System


Petter Brodin. Foto: Frida Hansson


|| July 13: 2017: Karolinska Institutet News || ά. Immune system function varies significantly between individuals and up to now, there has been no effective means of measuring and describing these differences. Now, researchers at Karolinska Institutet have shown that white blood cell composition is unique in individuals and that the composition of these cells, may, predict immune system response to various forms of stimulation. The study, which is published in Proceedings of the National Academy of Sciences, paves the way for more individualised treatment of diseases, involving the immune system, e.g, autoimmune disorders, allergies and various forms of cancer.

The human immune system comprises with a complex network of different white blood cells, which co-ordinate their efforts in order to combat different external and internal threats. This network varies widely between different individuals but the differences have been difficult to measure and understand. Together with colleagues at the Massachusetts Institute of Technology:MIT and Stanford University in the USA, researchers at Karolinska Institutet and the Science for Life Laboratory:Sci-Life-Lab, have developed a tool for measuring the unique composition of white blood cells in individuals. Researchers have, further, found that the test, may, predict how individuals will respond to a given treatment, e.g, individual response to an influenza vaccine.

“By measuring all populations of white blood cells in the blood at the same time, we can describe the composition of an individual’s immune system and show that this is unique for the individual. We call this measure, the individual’s 'immunotype'. We have, also, found that this immunotype makes the complex immune system more understandable and predictable.” says Dr Petter Brodin, Physician and Researcher at Sci-Life-Lab and the Department of Medicine, Solna, at Karolinska Institutet.

A human immunotype is not constant but varies over time in response to external factors. In previous studies, Dr Petter Brodin and his research colleagues have shown that in humans individual differences in immune defence, can be, attributed primarily, to the many different environmental factors unique to each individual, e.g, diet, infections, vaccines and microflora.

In the study in question, the researchers analysed blood samples from approximately 1,500 healthy individuals and tested in vitro how their white blood cells respond to different stimuli. They have, also, vaccinated individuals against influenza and studied which antibody protection the individuals developed thereafter. ''It transpired that all different types of stimulation could be predicted based on the individual’s immunotype, which was surprising.'' according to Petter Brodin.

"Our technique, can be, scaled up, and my hope is that, eventually, it will be used clinically to predict those individuals, who may, benefit from a particular immunological treatment or a certain vaccine. The technique, may also, contribute to more individualised drugs to treat autoimmune disease and allergies, as well as immunotherapy to treat cancer, which can be, adapted based on the individual’s immune response.” says Dr Brodin.

The study was financed by the National Institute for Health:NIH, the Ragon Institute of MGH, MIT and Harvard, the National Science Foundation, the European Research Council, the Swedish Research Council, the Swedish Society for Medical Research, the Swedish Cancer Society and Karolinska Institutet. ω.

The Paper: Continuous Immunotypes describe human immune variation and predict various responses: Kevin J. Kaczorowski, Karthik Shekhar, Dieudonné Nkulikiyimfura, Cornelia L. Dekker, Holden Maecker, Mark M. Davis, Arup K. Chakraborty, Petter Brodin. Proceedings of the National Academy of Sciences online July 10: 2017.

For more information, contact: Petter Brodin, physician, researcher: Department of Medicine, Solna, Karolinska Institutet: Science for Life Laboratory: Phone: +46:0:8 524 813 96: Email: Petter.Brodin at

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Research Could Give Insight Into the Genetic Basis of the Human Muscle Disease Myopathy

Image: University of Exeter

|| July 06: 2017: University of Exeter News || ά. Pioneering research using the tropical zebrafish could provide new insights into the genetic basis of myopathy, a type of human muscle disease. An international research team, led by Professor Philip Ingham FRS, inaugural Director of the University of Exeter’s Living Systems Institute has taken the first steps in determining the central role a specific gene mutation in a poorly characterised human myopathy. Myopathies are diseases, that prevent muscle fibres from functioning properly, causing muscular weakness. At present, there is no single treatment for the disease, as it can develop via a number of different pathways.

One particular type is nemaline myopathy, which primarily affects skeletal muscles and can lead to sufferers experiencing severe feeding and swallowing difficulties as well as limited locomotor activity. Mutations in a specific gene, called MY018B, have recently been found to be present in people exhibiting symptoms of this disease, but the role these mutations play in muscle fibre integrity has until now been unclear. In this new research, the Ingham Team, based in Singapore and Exeter, has used high-resolution genetic analysis to create a zebrafish model of MYO18B malfunction; this research takes advantage of the remarkable similarity between the genomes of zebrafish and humans, which have more than 70 per cent of their genes in common.

The Singapore:Exeter team found that the MYO18B gene is active specifically in the ‘fast-twitch’ skeletal muscles of the zebrafish, typically used for powerful bursts of movement. Crucially, by studying fish in which the MYO18B gene is disrupted, they were able to show that it plays an essential role in the assembly of the bundles of actin and myosin filaments that give muscle fibres their contractile properties.

The team believe this new research offers a vital new step towards understanding the cause of myopathy in humans, which in turn could give rise to new, tailored treatments in the future. The leading research is published in the scientific journal, Genetics. Professor Ingham, said, “The identification of a MYO18B mutation in zebrafish provides the first direct evidence for its role in human myopathy and gives us a model in which to study the molecular basis of MYO18B function in muscle fibre integrity.”

A pioneer in the genetic analysis of development using fruit flies and zebrafish as model systems, Professor Ingham is internationally renowned for his contributions to several influential discoveries in the field of developmental biology over the last century. This is the latest research by Professor Ingham that has revealed important links between the processes that underpin normal embryonic development and disease.

His co-discovery of the ‘Sonic Hedgehog’ gene, recognised as one of 24 centennial milestones in the field of developmental biology by Nature, in 2004, led directly to the establishment of a biotechnology company that helped develop the first drug to target non-melanoma skin cancer. The research comes at the University of Exeter holds the official opening of the Living Systems Institute with an Opening Symposium event, taking place now, July 05-06.

Two Nobel Laureates, Sir Paul Nurse FRS and Christiane Nüsslein-Volhard ForMemRS, who separately won the Nobel Prize for Physiology or Medicine, are to deliver keynote speeches as part of the opening event. The high-profile event, held at the University’s Streatham Campus marks the official opening of the LSI, a £52 million inter-disciplinary research facility designed to bring new, crucial insights into the causes and preventions of some of the most serious diseases facing humanity. ω.

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Breaking Up is Hard to Do: New Insights Into Cell Signalling

Immunofluorescence microscopy reveals the different protein profiles of immature stem cells, coloured pink and mature stem cells, coloured
green. Image: Sarita Panula 


|| June 28: 2017: University of Liverpool News || ά. Scientists at the Universities of Liverpool and Washington present important new insights into how cells communicate with each other. The research has been published on June 23 in the journal Science. Cell signalling refers to the mechanisms cells use to communicate with each other. In humans, signalling normally regulates cell growth and repair, and therefore, contributes to diverse basic processes, that control tissue physiology and brain function. However, abnormal cell signalling contributes to many diseases, including diabetes, cancer and neurodegeneration.

For this reason, the proteins controlling disease signalling are important targets for many types of clinically-approved drugs. In the new study, the researchers focused on understanding how proteins assemble into higher order signalling complexes, which control aspects of cell communication and cell fate, such as, the decision to live or die, using the ‘textbook’ cyclic AMP:cAMP signalling pathway. In the late 1960s, cAMP was shown to activate an enzyme complex, termed as, Protein Kinase A:PKA, which can exist in both ‘active’ and ‘inactive’ forms, depending upon the type of complex assembled.

It had long been thought that cAMP levels were sensed in cells by a release of the active kinase component from the larger PKA complex, rather like ice cubes breaking apart after being added to a drink. Using contemporary scientific strategies, including electron microscopy, mass spectrometry, chemical genetics and real-time imaging, the researchers found that instead of being broken apart by physiological levels of cAMP, the signalling complexes remain intact, directly delivering the appropriate message into the correct part of the cell interior.

The study was carried out by Dr Dominic Byrne and Dr Matthias Vonderach in the laboratories of Dr Patrick Eyers and Professor Claire Eyers in the Department of Biochemistry at the University’s Institute of Integrative Biology, in collaboration with Dr Donelson Smith and Professor John Scott and colleagues at the University of Washington.

Dr Patrick Eyers explained, ''We believe the finding that PKA protein complexes respond to the second messenger cAMP in a different way than we had assumed for nearly half a century, might bring about other changes in how we understand cell communication, especially, the type of signalling we study, that involves protein modification, phosphorylation by protein kinases.

“It might, also, prove important for a better biochemical understanding of how medicines affect PKA signalling complexes, allowing us to develop drugs with fewer side-effects.”

The next challenge for the team will be to try to explain how the larger PKA signalling complex functions in cells and how it is regulated by various factors. The research received funding support from the UK Biotechnology and Biological Sciences Research Council, North West Cancer Research and the Howard Hughes Medical Institute.

The paper ‘Local protein kinase A action proceeds through intact holoenzymes’ is published in Science: DOI: 10.1126/science.aaj1669

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The Biology of Uterine Fluid: How It Informs the Foetus of the Mother's World


|| June 25: 2017: Chinese Academy of Sciences News || ά. A developing foetus bathes in a mixture of cellular secretions and proteins, unique to its mother's uterus. Before fertilisation, the pH of uterine fluid helps create a conducive environment for sperm migration, and afterward, its volume supports the embryo as it implants onto the wall of the uterus. Recent evidence suggests that uterine fluid, may, play another key role in embryonic development: communicating the mother's outside conditions to the foetus, so that the latter can prepare accordingly. A review of this research appears on June 22 in Trends in Molecular Medicine.

Studies in livestock, rodents and humans have shown that information from a mother's environment, e.g, food availability, stress and pollutant exposure, can leave epigenetic tags on the DNA of her foetus, potentially, influencing the progression and long-term health of the developing embryo. Scientists have hypothesised that blood flow via the placenta, might, constitute one way the body communicates the mother's condition to the foetus; yet, there is evidence that the foetus can react to changes, such as, those stemming from the mother's diet long before the establishment of the placenta.

"This suggests the involvement of uterine fluid as the communication medium to transfer information between the maternal environment and the floating embryo." says Senior Author En-Kui Duan, a Reproductive Biologist at the Institute of Zoology, Chinese Academy of Sciences. "The pre-implantation period is a critical time for programming offspring health, and thus, expecting mothers should keep a good diet and good mood and stay away from harmful chemicals during this critical window."

While there is much to be learned about how mother-foetus communication takes place, the theory is that information in extracellular vesicles, molecular packages, that move from cell to cell, within uterine fluid and tissue deliver their cargo, including microRNAs and amino acids, to the foetus. These molecules, may be, tagging foetal cell DNA in ways, that alter which genes are being expressed, and thus, can contribute to 'programming' how the embryo and:or placenta develop.

Consequently, researchers are interested in learning which specific maternal environmental exposures and:or behaviours could change the composition of molecules transported via the uterine fluid to the foetus.

For example, mouse studies have shown that a low-protein maternal diet can reduce the level of certain amino acids in uterine fluid and affect gene expression of nutrition-transport-related genes. While these changes might prevent malnutrition in the developing embryo, once grown, the mouse offspring are more predisposed to heart disease when compared to animals on a regular diet.

Hongmei Wang, one of the author of this paper, speculates that uterine fluid could, someday be used, to analyse or even manipulate what signals are being received by a foetus. "For now, uterine fluid collection is not a standard biomarker; yet, many studies have revealed its potential role for non-invasive analysis, and we also, see great potential in it." she says.

"One, it can be screened by using ultrasound recording coupled with computational:biomechanical analysis; and two, uterine fluid can, also be, collected during an endometrial examination." This work is supported by the National Basic Research Program of China, the Ministry of Science and Technology of China, the National Natural Science Foundation of China, the National Key Research and Development Program of China, and the Youth Innovation Promotion Association, CAS. ω.

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TET1 Protein Helps Prevent Congenital Defects and Late-Onset Diseases

Image: University of Leuven


|| June 25: 2017: University of Leuven News || ά. In the earliest stages of embryonic development, a protein known as TET1, may be, the factor, that tips the balance toward health or disease. The first evidence for this vital role of TET1 is presented in Nature Genetics by researchers from KU Leuven. They found that TET1 was necessary to prevent congenital defects, such as, spina bifida, as well as, mental retardation and cancer later in life. Every mammal starts off as a cluster of cells with the same genetic material.

As the embryo develops, this DNA is used to generate the cell-specific building blocks for lungs, the brain and every other tissue and organ in the body. To determine which genetic information is needed for a specific cell, and when, chemical marks or methyl groups are added to the DNA at specific positions. Erasing a mark often switches on a specific message, whereas adding a mark usually switches it off. This determines how proteins interpret the genetic information.

In 2009, a team of researchers at Harvard University demonstrated that the TET protein family erased marks from the DNA and was, thus, essential for the proper development of the embryo. The precise role of the three family members, however, remained a mystery.

Professor Kian Koh from the KU Leuven Department of Development and Regeneration, who was a Co-lead Author of the Harvard study, has now been able to shed light on the role of the first family member, TET1. He found that the protein played a vital role in the embryonic stage that preceded the development of individual organs.

“TET1 is the only TET protein found in detectable amounts at this stage.” Professor Koh explains. “This suggests that it has a unique function. To find out which one, we created mice, that lack TET1. The protein prevents the incorrect marking of DNA.

We found that the loss of TET1, may, lead to severe defects, that cause the brain or spinal cord to develop outside the body. The causes of such defects, including spina bifida, are very complex, of course, but our findings suggest that TET1 plays a pivotal role in preventing them.”

But incorrect marking of the DNA, may also, cause late-onset diseases. “This is because TET1 is necessary to control the speed of embryonic development. If the timing for the start of a specific stage is off, the foetus, may, die. And if it survives, the marks on the DNA, may, still be improperly erased, possibly, leading to mental retardation and cancer later in life.”

These findings open up new avenues of research into the origin and prevention of both congenital disorders and various late-onset diseases. The research was supported by Research Foundation Flanders, Flanders Odysseus grant, the KU Leuven Stem Cell Programme funding, the Ministry of the Flemish Community and the Marie Curie Career Integration Grant. ω.

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New Trial to Fight Cancer Caused by Asbestos

Image: Queen's University Belfast


|| May 21: 2017: University of Southampton News  || ά. Patients with a hard to treat type of cancer are being given new hope in a new clinical trial. Researchers at the University of Southampton and the University of Leicester are trialling a drug, that could boost the body’s immune system to fight off mesothelioma, which can be caused by asbestos. The trial will be one of many to be conducted at the University of Southampton’s Centre for Cancer Immunology, which will be the UK’s first and only centre dedicated to cancer immunology research.

Mesothelioma rates are rising. Since the late 1970s, mesothelioma incidence rates have increased almost six-fold, 497 per cent increase, in Great Britain. There were around 2,700 new cases of mesothelioma in the UK in 2013, more than seven cases diagnosed every day. Current treatment methods include chemotherapy, radiotherapy or surgery and are mainly aimed at keeping the cancer under control. The phase III randomised controlled trial, which is funded by Cancer Research UK and supported by Bristol Myers Squibb, will test whether nivolumab, a drug already used to successfully treat advanced melanoma and advanced kidney cancer, can be used to target mesothelioma.

It works by finding and blocking a protein called PD-1 on the surface of certain immune cells called T-cells. Blocking PD-1 activates the T-cells to find and kill cancer cells. The trial has been launched ahead of International Clinical Trials Day, which is marked on May 20 each year.

Professor Gareth Griffiths, the study’s Co-Chief Investigator from the Southampton Clinical Trials Unit at the University of Southampton, said, “The UK has one of the world’s highest incidences of mesothelioma and currently there aren’t many ways to treat it. Boosting the immune system by releasing killer T-cells that have previously been blocked could offer us a new way to treat more patients with this devastating disease.”

The trial, which is being run in collaboration with the clinical lead Professor Dean Fennell at the University of Leicester, plans to recruit 336 patients, who have relapsed mesothelioma, across 20 UK-wide sites including Southampton and Leicester.

Professor Fennell said, “Preliminary studies targeting PD-1 in mesothelioma have shown promising activity. CONFIRM aims to definitively assess the true benefit of nivolumab for patients with relapsed mesothelioma in a setting where there is an unmet need. Critically, we aim to understand why patients respond or not to this drug and identify biomarkers to ensure that we can personalise therapy to maximize the benefit for patients.”

One person, who has already benefited from using the immune system to fight mesothelioma is Ms Mavis Nye, who was diagnosed with the disease in 2009. After various courses of treatments which failed, she joined a phase 1 immunotherapy trial to test the drug, Keytruda, on how well it blocked the PD-1 protein and enabled the body to fight off a number of cancers, including mesothelioma. After the first two years, scans revealed the tumours had decreased by 81 per cent, with three disappearing completely. Mavis is now cancer-free and spends her time raising awareness about the importance of clinical trials.

She said, “I was just an ordinary woman, whose husband worked at the dockyards in Chatham. We didn’t know what the effects of the asbestos on his clothes might be. Cancer is a terrible and devastating disease, that turns everything on its head. I am so thankful that the trial I took part in worked. But it didn’t work for every participant. We need more trials to help improve treatments and survival rates for cancer and this new trial is a big step in the right direction.”

Dr Catherine Pickworth, Cancer Research UK’s science information officer, said, “Immunotherapy treatments work by turning the power of our immune system against cancer. They are already being used routinely to treat advanced skin and kidney cancers, and are showing promise for other types of cancer too. This clinical trial will find out whether an immunotherapy drug could benefit people with mesothelioma, which is hard for doctors to treat successfully. We urgently need trials like this to help improve survival for patients with this aggressive type of cancer.”

The construction of the Centre for Cancer Immunology is expected to be completed by September and aims to be in full operation in summer 2018. It will bring world-leading cancer scientists together under one roof and enable interdisciplinary teams to expand clinical trials and develop lifesaving drugs.

The Centre, which is based at Southampton General Hospital site, is being funded by a £25 million fundraising campaign by the University of Southampton.

Professor Tim Elliott, Director of the Centre for Cancer Immunology, said, “The University has made major advances in tumour immunology and immunotherapy over the past 40 years and we enjoy a strong reputation for our ‘bench to bedside’ approach. The new Centre will go a long way in helping many more people with cancer become free of the disease, and we hope this new trial to fight a particularly sinister type of cancer will be the first of many successful trials.”

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Researcher Gets Funding for Research Into Growing Tumour-Like Cells to Better Understand Mouth Cancer

Image: Plymouth University


|| April 15: 2017: Plymouth University News || ά. A dental student from Plymouth University Peninsula Schools of Medicine and Dentistry has received an award of £1,000 from the Peninsula Medical Foundation, a charity, which fundraises for the School, to pursue a research project to produce a three-D model to grow tumour-like cells to better understand a common type of mouth cancer, potentially opening the door to treatments and therapies.

The project is part of 'the Inspire Scheme', which is led by the Academy of Medical Sciences and is funded by the Wellcome Trust. It aims to encourage medical and dental undergraduates to pursue scientific research. The award has been made to first-year dental student Mr Michael Daldry. Mr Daldry will work with Dr Vehid Salih and Ms Sam Gould from the School. Mr Daldry will use three-D artificial oral muscosa cells, developed by Ms Gould and Professor Salih to emulate oral cancer in laboratory conditions.

In 2014, 11,449 new cases of oral cancer were diagnosed in the UK and there were 2,386 deaths caused by the condition. Survival rates for head and neck cancers range from 19-59% and 91% are preventable.

By mimicking tumour cell creation, development and growth in squamous cell carcinoma, the most common type of mouth cancer accounting for 90% of cases, it is hoped that Mr Daldry's project will create a replicable and reliable system to test a number of factors including the impact of connective tissue on tumour development, how oral cancer moves and how tumour cells communicate with each other.

Whereas in the past such research might be limited to the number of animal cells, that could be acquired and used, this system allows for almost limitless availability of three-D cell models.

The creation of such a sustainable method may well lead to the development of new treatments and therapies. Mr Daldry said, “I’ve a keen interest in maxillofacial and oral surgery and shadowing consultants in preparation for interviews I realised many were active in research. As the concept of evidence-based practice continues to advance, I feel it is important to have fundamental cell biological research knowledge and understanding to provide the best possible care for our patients.

This is why I am involved with this exciting tissue engineering research and my thanks go to PUPSMD and the Peninsula Medical Foundation for this opportunity.”

Dr Vehid Salih, Associate Professor in Oral and Dental Health Research at PUPSMD, commented, “We were impressed by Michael’s application and we are looking forward to working with him on this project. Tissue engineering reduces or eliminates the need to use animals in research and provides scientists with more sustainable and flexible call model platforms with which to work. This particular application in relation to the most common form of oral cancer has important potential.”

Mr Denis Wilkins, Chair of the Peninsula Medical Foundation, added, “The Foundation is really pleased to be supporting talented students like Michael, who are helping to further important research and gaining invaluable experience for their future careers in medicine. We wish him well with this exciting venture.” ω.

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Stress: Not Only Important But Also Essential: How Stress Controls the Haemoglobin Levels in Blood

Image: Hebrew University

|| April 06: 2017: Hebrew University News || ά. Our ability to breathe oxygen is critical to our survival. This process is mediated by the haemoglobin in our blood, which carries oxygen. Since air contains less oxygen on high mountains, the body is under pressure to make haemoglobin rapidly, a stressful time. But what role does cellular stress play in the production of haemoglobin? In a paper in the journal Cell Research, published April 04, researchers at the Hebrew University of Jerusalem report the discovery of an entirely new mechanism, through which globin genes are expressed. Discovery of this hitherto unknown property of the haemoglobin genes shows that stress is absolutely needed to allow for the production of haemoglobin.

To produce a globin protein molecule, the DNA of the globin gene is first transcribed into a long RNA molecule, from which internal segments must be excised or spliced out, to generate the RNA template for protein synthesis in the red cell. Now, a team of molecular biologists, led by Professor Raymond Kaempfer in the Hebrew University’s Faculty of Medicine reports that for each of the adult and foetal globin genes, the splicing of its RNA is strictly controlled by an intracellular stress signal. The signal, which has been known for a long time, involves an enzyme present in every cell of the body, called PKR, which remains silent unless it is activated by a specific RNA structure, thought to occur only in RNA made by viruses.

What Kaempfer and collaborators have discovered is that the long RNAs transcribed from the globin genes each contain a short intrinsic RNA element, that is capable of strongly activating PKR. Unless the PKR enzyme is activated in this manner, the long RNA cannot be spliced to form the mature RNA template for globin protein synthesis.

“Surprisingly, we have revealed an entirely new mechanism, through which, hemoglobin gene expression is regulated by stress. An intracellular signal, essential for coping with stress, is absolutely necessary to allow for haemoglobin production. That stress signal is activated by the haemoglobin gene itself. Although, we have long known that this signal strongly inhibits protein synthesis in general, during haemoglobin gene expression it first plays its indispensable, positive role before being turned off promptly to allow for massive haemoglobin formation needed for breathing.” said Professor Raymond Kaempfer, the Dr. Philip M. Marcus Professor of Molecular Biology and Cancer Research at the Hebrew University of Jerusalem.

Once activated, PKR will place a phosphate, a process known as phosphorylation, onto a key initiation factor needed for the synthesis of all proteins, called eIF2-alpha. That in turn, leads to inactivation of eIF2-alpha, resulting in a block in protein synthesis. This process is essential for coping with stress.

Most unexpectedly, they discovered that once activated, PKR must phosphorylate eIF2-alpha and that phosphorylated eIF2-alpha is essential to form the machinery needed to splice globin RNA. In the splicing process, removal of an internal RNA segment causes the mature RNA product to refold such that it no longer will activate PKR, now allowing for unimpeded synthesis on this RNA of the essential globin protein chains at maximal rates, allowing for effective oxygen breathing. In other words, the ability to activate PKR remains transient, serving solely to enable splicing.

Thus, the team has demonstrated a novel, positive role for PKR activation and eIF2-alpha phosphorylation in human globin RNA splicing, in contrast to the long-standing negative role of this intracellular stress response in protein synthesis. The realisation that stress is not only important but also essential, may, have important implications for how we understand haemoglobin expression.

“What this boils down to is that even at the cellular level, stress and the ability to mount a stress response are essential to our survival. We have long known this in relation to other biological processes and now we see that it is at play even for the tiny molecules, that carry oxygen in our blood.” said Professor Kaempfer.

Professor Kaempfer's lab is in the Department of Biochemistry and Molecular Biology at IMRIC, the Institute for Medical Research-Israel Canada, in the Hebrew University's Faculty of Medicine. IMRIC is one of the most innovative biomedical research organizations in Israel and worldwide, bringing together brilliant scientific minds to find solutions to the world's most serious medical problems through a multidisciplinary approach to biomedical research. More information at .

The Paper: PKR activation and eIF2-alpha phosphorylation mediate human globin mRNA splicing at spliceosome assembly. Lena Ilan, Farhat Osman, Lise Sarah Namer, Einav Eliahu, Smadar Cohen-Chalamish, Yitzhak Ben-Asouli, Yona Banai, Raymond Kaempfer. Cell Research, advance online publication on April 04, 2017. doi: doi:10.1038/cr.2017.39

This work was supported by grants from the Israel Science Foundation. ω.

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€01.7 Million Funding for Research in Autoimmune Diseases

Image: University of Dundee

|| April 04: 2017: University of Dundee News || ά. A University of Dundee researcher has been awarded €01.7 million to investigate the role that communication between cells plays in autoimmune diseases. Dr Ignacio Moraga, from the University’s School of Life Sciences, has received a prestigious European Research Council:ERC starting grant to fund his research over the next five years. Dr Moraga and his team are studying immune response action in the hope of designing better medicines for conditions such as lupus, arthritis and other debilitating autoimmune diseases.

“I am delighted to receive this funding from the ERC to allow me to further understand the immune system.” said Dr Moraga. “It will allow my team and I to identify events critical for cellular decisions by learning the exact detail of how cells communicate with each other during an immune response.” The human body is composed of more than thirty trillion cells, all of which receive hundreds of messages alerting them to activity around them. These messages need to be brought together and acted upon to produce appropriate responses. Abnormal immune responses cause the body to attack itself, which is what happens in autoimmune diseases.

This extremely complex interconnected network of cells is controlled by a relatively small number of important signallers. The reason why outcomes can be normal or abnormal remains one of the longest-standing questions in biology.

Dr Moraga wants to understand this process in relation to immune response and will focus his research on cytokines, a group of signallers whose main function is to oversee the correct functioning of the immune system. Most immune disorders can be traced back to errors involving cytokines, making them very attractive drug targets, according to Dr Moraga.

“The way by which cytokines interact with cells can alter the responses seen,” he said. “My studies will provide understanding of these actions which will allow the development of more specific and less toxic cytokine-based therapies than those currently available.” ω.

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Researchers Reveal Structure and Mechanism of a Group-I Cobalt Energy Coupling Factor Transporter

Vesicle at the cell surface over time. The track shows where it moves to and the 'contours' shows where
molecules:mountains are. This shows the biological data combined with the mathematical model.
Image: Heriot Watt University

|| April 02: 2017: Chinese Academy of Sciences News || ά.  Energy-coupling factor:ECF transporters has been identified recently as a new family of ATP-binding cassette:ABC transporters, which comprise a large superfamily of protein complexes that transport a variety of substrates across cell membranes. They are composed of a membrane substrate-binding component EcfS, an ECF module of an integral membrane scaffold component EcfT and two cytoplasmic ATP binding:hydrolysis components EcfA:A’.

Two groups of ECF transporters have been identified with group-I ECF transporters being an EcfS protein associated with a dedicated ECF module and the encoding genes, and group-II ECF transporters which include several EcfS proteins and can bind to a common ECF module. These transporters mainly exist in prokaryote and plants and are responsible for micronutrients, like vitamins and metals, uptake from the environment, ECF. Professor Zhang Peng’s lab at Shanghai Institute of Plant Physiology and Ecology of Chinese Academy of Sciences in the previous studies had showed several structures of the group-II ECF transporters.

They interpreted the mechanisms of substrate binding, ECF module sharing and transport process. However, the molecular mechanism of group-I ECF transporters remains pretty much unknown, partly due to a lack of transporter complex structural information. Using a cobalt ECF transporter-CbiMNQO, researhcers set out to explore the structure and mechanism of group-I ECF transporters. New findings indicated that CbiMNQO constitutes transmembrane subunits CbiM:CbiN, CbiQ and cytoplasm subunits CbiO. The study has been published online in Cell Research.

Researchers set up a transport assay using ICP-MS, inductively coupled plasma mass spectrometry and found that CbiN is essential to the transport activity of CbiMNQO. Yet CbiN has little effect on the ATPase activity of the complex, and CbiM is either required for or greatly stimulates the ATP hydrolysis by the CbiMNQO transporter. This stimulation is independent of the presence or binding of Co2+.

This study analyzed the crystal structure of CbiMQO at a resolution of 2.8Å. By comparing the structures of CbiM without substrate and NikM bound with substrate, L1 loop, connecting transmembrane helix two and three, was found showing major conformational changes and can gate substrate binding and releasing. The structure of CbiO in a closed conformation was further indicated, and was compared with the structure of CbiO in CbiMQO complex which adopts an open conformation, the conformational changes of CbiO upon ATP binding and product release were revealed.

Based on the result that the CbiN subunit is essential for cobalt transport but not required for the complex ATPase activity, one favored postulate from the researchers is that CbiN might interact with CbiM and CbiQ, and is required for coupling TMs movements of CbiQ to CbiM. This is the first structure of group-I ECF transporters, which represents a milestone in the field of ECF-type ABC transporters.

Data collection was technically assisted by the staff members at BL19U:18U of National Centre for Protein Science Shanghai:NCPSS and BL17U of Shanghai Synchrotron Radiation Facility:SSRF. The study was supported by the Ministry of Science and Technology of China, the National Natural Science Foundation of China, the Chinese Academy of Sciences. ω.

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Ageing Leads to Breakdown in Cell Co-ordination

Anna-Gene O’Neal talks with Knox Ownby at Alive Hospice Residence Nashville.
Image: Daniel Dubois:Vanderbilt University


|| April 01: 2017: University of Cambridge News || ά. A team of researchers from across Cambridge has shed light on a long-standing debate about why the immune system weakens with age. Their findings, published today in Science, show that immune cells in older tissues lack co-ordination and exhibit much more variability in gene expression or activity compared with their younger counterparts. As we age, we see a progressive decline of function occur throughout the body but until now it has not been clear why this decline occurs and why it happens at different rates for different parts of the body. To understand this process, scientists must unpick all of the mechanisms of ageing at the molecular level for every tissue.

The immune system is like a symphony orchestra, with many different types and subtypes of cells working together to fight infections. But as the immune system ages, its response to infection weakens for reasons that are not yet clear. One long-standing debate amongst scientists concerns two central hypotheses: either the functional degradation is caused by a loss of cellular performance or it is down to a loss of co-ordination among cells. To resolve the debate, scientists at the University of Cambridge, the European Bioinformatics Institute:EMBL-EBI and the Wellcome Trust Sanger Institute studied many different cell types, analysing ‘average’ gene expression profiles.

To do so, they employed high-resolution single-cell sequencing technology to create new insights into how cell-to-cell variability is linked with ageing. The researchers sequenced the RNA of naïve and memory CD4+ T cells in young and old mice, in both stimulated and unstimulated states. Their findings clearly showed that loss of co-ordination is a key component of the impaired immune performance caused by T-Cell ageing.

“You could think of DNA sequencing as a fruit smoothie.” explains Dr John Marioni, Group Leader at EMBL-EBI and at the University of Cambridge’s Cancer Research UK Cambridge Institute:CRUK-CI. “Traditional sequencing technology is a bit like taking a sip of the smoothie, then trying to guess what the ingredients are. Single-cell genomics now lets us study the ingredients individually, so we get direct insight into the constituent parts. Extrapolating, this means that single-cell sequencing allows researchers to individually look at thousands of genes at any given time.”

Dr Duncan Odom, Group Leader at the CRUK-CI and associate faculty at the Wellcome Trust Sanger Institute, shares a further analogy to explain how immune cells fight infection. “Imagine, the immune system as a ‘cell army’, ready to protect the body from infection. Our research revealed that this army is well co-ordinated in young animals, with all the cells working together and operating like a Greek phalanx to block the infection.”

The researchers say that this tight co-ordination makes the immune system stronger and allows it to fight infection more effectively. The team’s study shows that as the animal gets older, cell co-ordination breaks down. “Although individual cells might still be strong, the lack of co-ordination between them makes their collective effectiveness lower.” Odom adds.

Previous studies have shown that in young animals, immunological activation results in tightly regulated gene expression. This study further shows that activation results in a decrease in cell-to-cell variability. Ageing increased the heterogeneity of gene expression in populations of two mice species, as well as in different types of immune cells. This suggests that increased cell-to-cell transcriptional variability may be a hallmark of ageing across most mammalian tissues

“There is a great deal of interest in how biological ageing happens but not much is known about molecular ageing.” says Dr Celia Pilar Martinez-Jimenez, experimental lead from the CRUK-CI. “This research initiative explored a new facet of cell response to disease, while, also, tackling questions related to ageing.”

Nils Eling, computational lead of the project and PhD student at EMBL-EBI and CRUK-CI adds, “The advantage of analysing gene expression from single cells is to detect how cell populations synchronise their response. It is interesting to see that ageing strongly distorts this response, a phenomenon, which could not be observed before.”

The interdisciplinary study paves the way for a more in-depth exploration of the mechanisms by which different types of cells age. It, also, illustrates the potential of single-cell sequencing to enable a richer understanding of cell development and activity. ω.

The Paper: Martinez-Jimenez, C.P., Eling, N et al Ageing increases cell-to-cell transcriptional variability upon immune stimulation. Science; 30 Mar 2017; DOI: 10.1126/science.aah4115

: This piece, by the University of Cambridge, is licensed under a Creative Commons Attribution 4.0 International License:

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Now, Will You Look at This: New Tools to Study the Origin of Embryonic Stem Cells

Immunofluorescence microscopy reveals the different protein profiles of immature stem cells, coloured pink and mature stem cells, coloured
green. Image: Sarita Panula 

|| March 24: 2017: Karolinska Institutet News || ά. Researchers at Karolinska Institutet have identified cell surface markers specific for the very earliest stem cells in the human embryo. These cells are thought to possess great potential for replacing damaged tissue but until now have been difficult to distinguish from classical embryonic stem cells. The study is published in the prestigious journal Cell Stem Cell. During the first week of fertilisation, the embryo grows from a single cell into a blastocyst, a hollow cluster of a few hundred cells. The blastocyst then attaches itself to the wall of the uterus, implantation and for a limited period from fertilisation to a few days after implantation the embryo contains pluripotent stem cells.

These cells can develop into all the body’s cell types and are therefore of considerable interest to the field of regenerative and reparative medicine. A few years ago, it was discovered that there are two stages for human pluripotent stem cells, corresponding to the pre-implanted and post-implanted embryonic cells. Although the classical stem cells used in regenerative medicine are isolated from the pre-implanted embryo, they have adopted a mature stage that is most likely more similar to a post-implantation embryo. A new type of pluripotent cell that genuinely corresponds to the more immature, pre-implantation stage has been identified and can now be cultivated in the laboratory.

These immature stem cells are of great scientific interest since they are believed to have the potential to build certain cell types that are difficult to obtain from the classical stem cells, and they may also be easier to cultivate and manipulate in the laboratory.

Fredrik Lanner. Image: Ulf Sirborn

Fredrik Lanner’s research team at Karolinska Institutet and their colleagues in Peter Rugg-Gunn’s team at Cambridge’s Babraham Institute in the UK have now developed a tool for separating the two stem cell states. They have screened combinations of antibodies that bind to specific proteins on the surface of the immature and mature stem cells and that can be used for flow cytometry, a common laboratory technique for sorting cells.

“We’ve not had cell surface markers for the different stem cell states before, which has made it hard to study them.” says Fredrik Lanner, Assistant Professor at Karolinska Institutet’s Department of Clinical Science, Intervention and Technology. “We now have a simple tool for identifying and sorting the cells, which benefits future stem cell research and basic research on early embryonic development.”

Mature embryonic stem cells cultivated in the laboratory can, under the right conditions, be backed up in their development to the more immature stem cell type. The researchers tested their technique on such cultivated stem cells of both a mature and immature type, and on donated human embryos left over from IVF treatments. As expected, only the immature stem cell type was identified in such pre-implanted embryos, which indicates that the antibodies are highly specific.

“It is at the point of implantation that the stem cells go through this change and ‘mature’, which is also a highly critical time for the embryo.” says Dr Lanner. “These cells are, therefore, also, of interest to infertility research.”

The study was financed by several bodies, including the Swedish Research Council, the Ragnar Söderberg Foundation, the Swedish Foundation for Strategic Research, the Knut and Alice Wallenberg Foundation, the Centre for Innovative Medicine:CIMED and the Ming Wai Lau Centre for Reparative Medicine.

The Paper: ‘Comprehensive Cell Surface Protein Profiling Identifies Specific Markers of Human Naive and Primed Pluripotent States’, Amanda J. Collier, Sarita P. Panula, John Paul Schell, Peter Chovanec, Alvaro Plaza Reyes, Sophie Petropoulos, Anne E. Corcoran, Rachael Walker, Iyadh Douagi, Fredrik Lanner, Peter J. Rugg-Gunn. Cell Stem Cell, online March 23, 2017, doi: 10.1016/j.stem.2017.02.014.

For more information, ontact: Fredrik Lanner, Assistant Professor: Department of Clinical Science, Intervention and Technology: Karolinska Institutet: Phone: +46 :0:73 024 47 05: email: fredrik.lanner at

Karolinska Institutet is one of the world's leading medical universities. Its vision is to significantly contribute to the improvement of human health. Karolinska Institutet accounts for over 40 per cent of the medical academic research conducted in Sweden and offers the country´s broadest range of education in medicine and health sciences. The Nobel Assembly at Karolinska Institutet selects the Nobel laureates in Physiology or Medicine. ω.

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Therefore There is No Such Thing as Undruggable: Says the Kiss of Death


|| March 18: 2017: University of Dundee News || ά. Scientists at the University of Dundee have reported a major breakthrough in targeting the causes of many diseases, using a 'kiss of death' to destroy proteins, which had previously been regarded as 'undruggable'. Much is known about proteins, such as Ras and Myc, which are known to be culpable in human cancer and Huntingtin, which causes Huntingdon's Disease but as yet, they have proved stubbornly resistant to efforts to find ways of tackling them with drugs. Now researchers, led by Professor Alessio Ciulli in the School of Life Sciences at Dundee have found a way of targeting similar proteins, using a small-molecule approach in an unconventional way to target 'bad' proteins by binding them to neutralising agents to start a process of degradation and thereby, removing them entirely.

“We know of many proteins, which are active in causing diseases but which, we have been unable to block from going 'rogue' or to stop them when they do.” said Professor Ciulli. “The major problem is that we have been unable to find the small molecules, which can successfully bind to these proteins and at the same time, hamper their function. It is a highly complex area, these proteins can often fool regulators within the cell and be extremely difficult to pin down with inhibitors. Research in our lab in the past few years has contributed towards establishing a different approach, one that has been theorised for many years but, which is only now fully realised by this latest work.

Instead of using the small molecule to try and disable the bad protein, we have developed a way of modifying it so that it can be used to attract the neutralising proteins, which then bind to their bad neighbour and act against it, starting a cascade process of degradation.

Crucially, we have, also, found that it is not enough for this neutralising protein to sit close to the bad protein, it has to make direct contact with it, to 'kiss' it. And not just a little peck but a real 'Gone With the Wind' embrace. We call this a ‘kiss of death', as it is the key to ensure the degradation of the bad protein.”

Professor Ciulli and colleagues focused their attention on a bivalent chemical degrading molecule, called, Proteolysis-targeting chimeric molecules:'PROTAC'. They have been able to create the first X-ray crystal structure of a PROTAC bound to both the ‘bad' protein and the ‘neutralising' agent, an E3 ubiquitin ligase, and found that it can successfully be deployed as a 'magnet' to draw the two target proteins together.

“This discovery provides the first ever insights into how PROTACs work and how we can target proteins for degradation in a highly selective manner.” said Professor Ciulli.

“This presents a paradigm shift in how we can ensure selective chemical intervention against proteins which we know are factors in causing disease but which until now have been impossible to successfully target. It points towards the possibility of drugging the 'undruggable'.”

The results of the research are published in the journal Nature Chemical Biology.

The work done by the Dundee team focused on pairing one of the BET bromodomain proteins, BRD4, which is an attractive drug target for cancer, with a selective BRD4 'degrader' called MZ1. They found that MZ1 could pull the two proteins together by folding into itself. Once the two proteins are joined in their 'kiss' the interactions between them lead to degradation of the target.

They showed how similar proteins BRD2 and BRD3 do not kiss the neutralizing protein as well, and guided by this information they were able to design new degraders that spare those proteins completely from the degradation process. This is important because it elucidates how degrading molecules could be designed in future to minimise off-target effects in ways that may not be possible using conventional inhibitors.

The Dundee team working on the project included postdoctoral researchers in Professor Ciulli's laboratory Morgan Gadd, Andrea Testa, Xavier Lucas and Kwok-Ho Chan, and Wengzhan Chan and Dougie Lamont from the Dundee Fingerprints Proteomics Facility.

Professor Ciulli said that there were already promising signs from the pharmaceutical industry of significant investment in this research area.

“We now understand better how to turn inhibitors into degraders. The road to turning degraders into drugs will be long and winding and we cannot get there on our own. It is exciting to see signs of serious commitment from the pharmaceutical industry, which adds to optimism that we will be able to get to a point where we can drug the 'undruggable'.”

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Growing Organs Outside the Body: How Tiny Organoids Help Scientists Study the Big Picture

Image: University of Melbourne

|| March 14: 2017: University of Melbourne Australia News: Kate Stanton Writing || ά. Mirella Dottori  loves looking through the microscope at tiny, embryonic human brains. She describes them as a 'beautiful' network of nerve cells, called neurons, reaching for and passing messages to one another. “They have these long extensions and you can see they're looking for something to attach to.” she says. “It's like a grapevine just spreading out.”  Associate Professor Dottori, a Developmental Neuroscientist and her team of researchers at the University of Melbourne’s Centre for Neural Engineering grow clusters of cells resembling miniature human brains. The Centre is part of an ambitious global effort to use stem cells to grow tiny versions of human organs, called, organoids. These organoids could allow researchers to study how diseases affect human cells. It’s an exciting prospect for scientists used to experimenting with the brains of mice, which might not be accurate reflections of the human brain.

 “We always presume or hope the human brain is similar because that’s the best we have had to work with.” says Associate Professor Dottori. “But now with human stem cells we can really ask that question: how does the human brain form?” In understanding how the brain forms, we can better understand why diseases develop in some humans and not in others. Scientists believe that brain organoids could pave the way for major breakthroughs in the fight against neurological conditions such as autism, epilepsy and schizophrenia. These 'brain fragments in a dish' are made from stem cells derived from human skin tissue. They start out smaller than a crumb, so tiny you need a microscope to see them. After two or three months, however, they can grow to the size of a lentil. They develop in much the same way a human brain would in an embryo.

Organoids were first discovered in 2013 by Austrian researcher Dr Madeline Lancaster, who noticed that the human embryonic stem cells she was studying had started to form layered balls, that looked like a part of the brain. Associate Professor Dottori grows organoids that mimic different regions of the brain so scientists have a better understanding of how the brain develops.

“It's been quite insightful because we can play with the mini-brains.” she says. “We can stimulate them and also add things to see how they respond.” The mature brain in a human body uses external senses, such as touch, sight and smell, to activate the neuronal networks within it to fire. In a dish, however, scientists use a tiny network of electrodes to stimulate the cells. Then they look at the firing patterns of neurons, how nerve cells communicate and transfer information along the network. The next step is to apply their understanding of the brain’s chemical and electrical workings to different neurological diseases. For example, Associate Professor Dottori’s team made brain organoids using stem cells from autistic patients. They can then compare them to organoids from patients without autism.

“We're using stem cells to model autism. To better understand the disease so we can see what regions of the brain are really affected, what are the neurons that should be connecting together that are not.”  Associate Professor Dottori says that she became passionate about the potential for finding a cure to Friedreich’s ataxia, a rare genetic disease that damages the nervous system, after attending a meeting with patients affected by it.

“I saw girls my age in wheelchairs, beautiful girls and very smart and just wanting to live a normal life.” she says. “I thought to myself, ‘that could have been me’.” But there haven’t been good animal models that can mimic the disease, which makes it difficult to test drugs that could cure it. Making organoids from the stem cells of Friedreich’s ataxia patients could help scientists find that cure.

Such research needs an interdisciplinary approach. The Centre for Neural Engineering, led by Professor Stan Skafidas, brings together neurologists, geneticists, electrophysicists and engineers to study the electrical and chemical workings of neurons.  “It's not just about having tissues in a dish,” says Associate Professor Dottori. “We really need the engineering technologies to do something with it.”

She has been working with engineers from the University’s School of Engineering to construct tiny scaffolds that can conduct electricity and provide a 3D structure for the cells to cling to, rather than just growing them flat in the dish.

Creating and watching a lab full of mini-brains at different levels of growth is a full-time job.  “It's like a factory,” she says. “You have to be maintaining the lines, setting up new cells to start becoming neurons, you've got mature neurons, you've got a full spectrum of experiments at different stages constantly happening to keep things moving along.”

But all the work is done in the hope that, eventually, scientists will be able to grow a whole brain in the lab. “There are no boundaries.” says Associate Professor Dottori. “Many years ago people wouldn't have dreamt you could make a stem cell from a person the way we are now. We just had to find a way to do it.” ω.

Find out more about this research

: This work, of the University of Melbourne, is licensed under a Creative Commons Attribution-No Derivatives 3.0 Australia CC BY-ND 3.0 AU This article was first published on Pursuit : ω.

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Fingerprinting the Cell Identities

Image: University of Zambia School of Medicine


|| March 07: 2017: Ludwig-Maximilians-Universität München:LMU News || ά. Every cell has its own individual molecular fingerprint, which is informative for its functions and regulatory states. LMU researchers have now carried out a comprehensive comparison of methodologies that quantify RNAs of single cells. The cell is the fundamental unit of all living organisms. Hence, in order to understand essential biological processes and the perturbations that give rise to disease, one must first dissect the functions of cells and the mechanisms that regulate them. Modern high-throughput protein and nucleic-acid sequencing techniques have become an indispensable component of this endeavor.

In particular, single-cell RNA sequencing:scRNA-seq permits one to determine the levels of RNA molecules, the gene copies, that are expressed in a given cell, and several versions of the methodology have been described in recent years. The spectrum of genes expressed in a given cell amounts to a molecular fingerprint, which yields a detailed picture of its current functional state. “For this reason, the technology has become an extraordinarily valuable tool, not only for basic research but also for the development of new approaches to treat diseases.” says LMU biologist Wolfgang Enard. Enard and his team have now undertaken the first comprehensive comparative analysis of the various RNA sequencing techniques, with regard to their sensitivity, precision and cost efficiency. Their results appear in the leading journal Molecular Cell.

The purpose of scRNA-seq is to identify the relative amounts of the messenger RNA:mRNA molecules present in the cells of interest. mRNAs are the blueprints that specify the structures of all the proteins made in the cell, and represent 'transcribed' copies of the corresponding genetic information encoded in specific segments of the genomic DNA in the cell nucleus. In the cytoplasm surrounding the nucleus, the nucleotide sequences of mRNAs are 'translated' into the amino-acid sequences of proteins by molecular machines called ribosomes.

Thus, a complete catalog of the mRNAs in a cell provides a comprehensive view of the proteins that it produces, and tells one what subset of the thousands of genes in the genome are active and how their activity is regulated. Furthermore, aberrant patterns of gene activity point to disturbances in gene expression and cell function, and reveal the presence of specific pathologies. The scRNA-seq procedure itself can be carried out using commercially available kits, but many researchers prefer to assemble the components required for their preferred formulations themselves.

In order to ascertain which of the methods currently in use is most effective and economical, Enard and his colleagues applied six different methods to mouse embryonal stem cells and compared the spectra of mRNAs detected by each of them. They then used this data to compute how much it costs for each method to reliably detect differently expressed genes between two cell types.

“This comparison revealed that some of the commercial kits are ten times more expensive than the corresponding home-made versions.” Enard says. However, the researchers point out that the choice of the optimal method largely depends on the conditions and demands of the individual experiment. "It does make a difference whether one wants to analyse the activity of hundreds of genes in thousands of individual cells, or thousands of genes in hundreds of cells.” Enard says. “We were able to demonstrate which method is best for a given purpose, and we also obtained data that will be useful for the further development of the technology.”

The new findings are of particular interest in the field of genomics. For example, scRNA-seq is a fundamental prerequisite for the success of the effort to assemble a Human Cell Atlas, one of the most ambitious international projects in genomics since the initial sequencing of the human genome.

It aims to provide no less than a complete inventory of all the cell types and subtypes in the human body at all stages of development from embryo to adult on the basis of their patterns of gene activity. It is estimated that the total number of cells in the human body is on the order of 3.5 × 1013. Scientists expect that such an atlas would revolutionize our knowledge of human biology and our understanding of disease processes. ω.

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Estrogen Explains the Exosome-Carried Messenger Profile in the Circulation Among Post-Menopausal Women

Images: University of Jyväskylä

|| March 05: 2017: University of Jyväskylä Finland News  || ά.A study at the Gerontology Research Centre demonstrated that, in blood circulation, the exosome-carried messenger molecule profile differs between post and pre menopausal women. The differences were associated with circulating estrogen and cholesterol levels as well as body composition and other health indicators. These findings enable using the studied molecules in the evaluation of health status.

''The studied messenger molecules are packed in the exosomes, which are released by the cells into the circulation. Exosomes are spherical nano-scale lipid vesicles. These small packages carry microRNA molecules, which are considered to be messengers between the cells, regulating gene function.'' says Docent Eija Laakkonen. The study was the first to show that specific exosome-packed microRNAs are sensitive to the estrogen levels in the circulation, which is influenced both by age and the use of hormonal therapies.

The results can be exploited in evaluating the effects of hormonal contraceptives and hormone replacement therapies on the overall physiological status of women. When the regulatory mechanisms of the microRNAs are better understood, the microRNA profile can be used for recognising individuals with a high risk for metabolic disorders or even lowering the risk.

''It seems, therefore, that the postmenopausal declining amount of circulating estrogen changes the cargo inside the exosomes. When these exosome packages are delivered to the target tissues, the contents are released to the correct recipient cell. These delivered messages change the function of the cell.'' explains doctoral candidate Reeta Kangas.

''The next step would be to perform functional studies in order to see how estrogen regulates the exosome cargo and how the message is further processed inside the recipient cell.'' concludes Ms Reeta Kangas.

''A study, as significant as this, needs broad collaboration, states the leader of the study.'' Docent Vuokko Kovanen. This study was conducted in collaboration with the Gerontology Research Centre, University of Jyväskylä, the Institute of Biomedicine, University of Turku and the Turku Clinical Sequencing Laboratory. In addition, researchers from the University of Oulu and The Finnish Institute for Molecular Medicine were involved.

The study has been published in the esteemed Scientific Reports journal by Nature Publishing. The study was funded by the Academy of Finland, the Finnish Cultural Foundation and the Faculty of Sport and Health Sciences, University of Jyväskylä.

Additional information: Doctoral Candidate Reeta Kangas: +358 40 8054573: reeta.m.s.kangas at Docent Eija Laakkonen: +358 40 5326410: eija.k.laakkonen at Docent Vuokko Kovanen: +358 40 5491486:
vuokko.kovanen at ω.

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New Algorithm Identifies Gene Transfers Between Different Bacterial Species

Mosaic pneumococcal population structure caused by horizontal gene transfer is shown on the left for a subset of genes.
Matrix on the right shows a genome-wide summary of the relationships between the bacteria, ranging from
blue, distant, to yellow, closely related. Kuva: Pekka Marttinen.

|| March 03: 2017: Aalto University News  || ά. When mammals breed, the genome of the offspring is a combination of the parents' genomes. Bacteria, by contrast, reproduce through cell division. In theory, this means that the genomes of the offspring are copies of the parent genome. However, the process is not quite as straightforward as this due to horizontal gene transfer, through which, bacteria can transfer fragments of their genome to each other. As a result of this phenomenon, the genome of an individual bacterium can be a combination of genes from several different donors. Some of the genome fragments may even originate from completely different species.

In a recent study combining machine learning and bioinformatics, a new computational method was developed for modelling gene transfers between different lineages of a bacterial population or even between entirely different bacterial species. The method was used to analyse a collection of 616 whole-genomes of a recombinogenic pathogen Streptococcus pneumoniae. In the study, several individual genes, in which, gene transfers were considered particularly common were identified. These genes also included genes causing resistance to antibiotics.

''In the case of antibiotic-resistance genes, the number of gene transfers may be related to how useful these genes are to bacteria and to the resulting selection pressure.'' says Academy Research Fellow Pekka Marttinen from the Aalto University Department of Computer Science.

''The study will not provide a direct solution to antibiotic resistance because this would require a profound understanding of how the resistance occurs and spreads. Nevertheless, knowing the extent to which gene transfer occurs between different species and lineages can help in improving this understanding.'' he says.

The study was able to show that gene transfer occurs both within species and between several different species. The large number of transfers identified during the study was a surprise to the researchers. ''Previous studies have shown that gene transfers are common in individual genes, but our team was the first to use a computational method to show the extent of gene transfer across the entire genome.'' Marttinen says.

''The method also makes it possible to effectively examine ancestral gene transfers for the first time, which is important in examining transfers between different species.''

Molecular Biology and Evolution published the results in February. More information: Academy researcher Pekka Marttinen: Aalto University, Department of Computer Science: tel. +358 44 303 0349: pekka.marttinen

Paper: Rafal Mostowy, Nicholas J. Croucher, Cheryl P. Andam, Jukka Corander, William P. Hanage and Pekka Marttinen: Efficient inference of recent and ancestral recombination within bacterial populations. Molecular Biology and Evolution 2017. DOI: 10.1093/molbev/msx066. ω.

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Seipin: The Story of the Anchor Working for the Attachment of Lipid Droplets to the Endoplasmic Reticulum

A confocal microscopy image of a living human cell in which seipin, green, connects lipid droplets, magenta, to the endoplasmic reticulum, blue.
Image: Veijo Salo:Ikonen Lab


|| February 28: 2017: University of Helsinki News: Päivi Lehtinen Writing || ά.  Yes, our cells contain organelles called lipid droplets. Lipid droplets are connected to a protein called, seipin and disturbances in their relationship may have harmful consequences. A few years ago, the research group led by Academy Professor Elina Ikonen took on a new field of research when Veijo Salo, then a medical student, began his thesis project in the group by studying lipid droplets and a protein seipin. Lipid droplets are cell organelles that act as reservoirs for excess lipids and release lipids when cells are in need of energy. Fat or adipose cells are filled with these droplets but they can also be found in other eukaryotic cells.

Lipid droplets are generated in the largest endomembrane of the cell, the endoplasmic reticulum but the mechanisms underlying their formation are not well understood. “What we do know is that, in addition to lipid metabolism, they are connected to many other intracellular processes, such as autophagy, protein degradation and virus assemby.” says Salo. “Perturbations in lipid droplet morphology and function are related to many common diseases, such as diabetes, Alzheimer’s disease and fatty liver.” Salo's research is focused on the membrane protein seipin, which seems to have a particularly, important role in the formation and functioning of lipid droplets.

The story of seipin begins in the 1950s when two doctors, one from Norway and the other from Brazil, took an interest in muscular and otherwise seemingly healthy patients who were, however, suffering from a serious metabolic disorder: their bodies produced no fat tissue at all. In 2001, French researchers identified the gene mutation underlying the disease. The gene was named seipin after the Norwegian physician who described the disease. “Mutations in other genes also cause a similar disease but the clinical features of the disease caused by seipin deficiency are the most severe." Salo points out.

Some years after the mutation was identified, it was found that seipin plays an essential role in the ability of yeast cells to store lipids. Scientists were excited: if that works in yeast cells, why not in animal cells as well? Researchers studying seipin started to use animal models, such as mice and fruit flies. In 2011, researchers realised, to research in fruit fly, that seipin has a cell-specific effect on the formation of lipid droplets.

“Seipin research was going full speed ahead when we came along.” says Ikonen. “We thought that the protein still had a lot of interesting unexplored aspects and wanted to investigate it in more detail in human cells." The effort paid off. In November 2016, the EMBO Journal published the article 'Seipin regulates ER–lipid droplet contacts and cargo delivery' by Ikonen’s group.

“We still don’t know what seipin exactly does but we have made significant progress in finding out. An important molecular-level mechanism where seipin is involved was described in the article published by EMBO.''

High resolution microscopy is key to seipin research, much like in many other membrane studies. The best resolution is obtained with electron microscopy in which the group co-operates closely with Eija Jokitalo's group at the Institute of Biotechnology, University of Helsinki. At Ikonen’s laboratory, living cells can be imaged with high precision.

“We have high-quality equipment, not the best in the world but very good nonetheless and Veijo has become very adept at using these devices and methods." enthuses Ikonen.

“The critical factor was the ability to examine what lipid droplets go through right after their formation in cells with seipin deficiency." explains Salo. “In order to do this, we needed cells where the gene that encodes seipin was disrupted. Another important factor is the growth environment of cells; we grew them in a completely lipid-free environment. We acutely added lipids into this environment and observed the results. This way, we were able to observe the earliest moments when the problems began.”

What happened next in seipin deficient cells? Lipid droplets formed inside the cell but they looked peculiar. “The droplets were abnormally small and their mobility was much higher than usual. Furthermore, they did not grow in size normally, even when lipids were added to the cell.” says Salo.

The results suggest that seipin regulates the contacts between the endoplasmic reticulum and lipid droplets; the protein may serve as an anchor attaching the droplets to the reticulum, thus facilitating lipid and protein cargo transfer between these two organelles. To make a long story short, you could say that seipin keeps the droplets connected to the reticulum in order for the cells to effectively store lipids.” Salo sums up.

Seipin is an endoplasmic reticulum:ER network membrane protein, whose mutations cause three rare genetic diseases in humans: i: A recessively inherited, severe form of congenital generalised lipodystrophy:BSCL2. Patients develop no fat tissue at all, which causes, among other things, a severe metabolic syndrome; ii: A dominantly inherited motor neuron disease, a type of hereditary spastic paraplegia:HSP and iii: fatal and early onset neurodegenerative disease:Celia’s encephalopathy. The disease is identified by a progressive developmental disorder and short life expectancy, six-eight years.

The research focus at the laboratory of Academy Professor Elina Ikonen on the Meilahti Campus is on membranes. “We study biomembranes, the layers that encircle cells and help to separate compartments within cells.” says Professor Ikonen. Ikonen is the Director of the Centre of Excellence in Biomembrane Research, which investigates the interactions of the main components of cells, proteins and fat, or lipids.

Along with Ikonen’s group, the Centre of Excellence consists of a research group led by Professor Pekka Lappalainen at the Institute of Biotechnology on the Viikki Campus and the biological physics research group on the Kumpula Campus, led by Professor Ilpo Vattulainen.

“So far, very little is known about lipid-protein interactions. We do know, however, that the functional capacity of proteins is largely dependent on their membrane environment. Lipid bilayer membranes both enable and inhibit interactions between proteins." says Ikonen. The research group, led by Ikonen, studies lipids and proteins that are, in one way or another, connected to membranes.

“Among other things, we are studying lipid storage diseases and the connection between cholesterol metabolism and cardiovascular diseases and metabolic syndrome.” The group, led by Lappalainen, is studying the actin cytoskeleton of cells and its impact on cell shape, motility and adhesion. The group led by Vattulainen, in turn, predicts the way biomembranes interact with proteins through nanoscale modelling.

“Our research is being conducted at the intersection of several disciplines, which is very productive and, in addition, interesting to us as researchers. Our scope ranges from the clinical symptoms of disease to molecular interactions at the nanoscale. That is pretty breathtaking. ” exults Ikonen. “This type of research is impossible without the right kind of tools.” Academy Professor Elina Ikonen points out. ω.

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Stem Cells Collected From Fat May Have Use in the Prevention and Treatment of Aging-Related Diseases

Images: University of Pennsylvania

|| February 26: 2017: University of Pennsylvania: USA News || ά. Adult stem cells collected directly from human fat are more stable than other cells, such as fibroblasts from the skin and have the potential for use in anti-aging treatments, according to researchers from the Perelman School of Medicine at the University of Pennsylvania. They made the discovery after developing a new model to study chronological aging of these cells. They published their findings this month in the journal Stem Cells. Chronological aging shows the natural life cycle of the cells, as opposed to cells that have been unnaturally replicated multiple times or otherwise manipulated in a lab.

In order to preserve the cells in their natural state, Penn researchers developed a system to collect and store them without manipulating them, making them available for this study. They found stem cells collected directly from human fat, called adipose-derived stem cells:ASCs, can make more proteins than originally thought. This gives them the ability to replicate and maintain their stability, a finding that held true in cells collected from patients of all ages. “Our study shows these cells are very robust, even when they are collected from older patients.” said Ivona Percec, MD, Director of Basic Science Research in the Centre for Human Appearance and the study’s Lead Author. “It also shows these cells can be potentially used safely in the future, because they require minimal manipulation and maintenance.”

Stem cells are currently used in a variety of anti-aging treatments and are commonly collected from a variety of tissues. But Percec’s team specifically found ASCs to be more stable than other cells, a finding that can potentially open the door to new therapies for the prevention and treatment of aging-related diseases. “Unlike other adult human stem cells, the rate, at which these ASCs multiply, stays consistent with age.” Percec said. “That means these cells could be far more stable and helpful as we continue to study natural aging.”

ASCs are not currently approved for direct use by the Food and Drug Administration, so more research is needed. Percec said that the next step for her team is to study how chromatin is regulated in ASCs. Essentially, they want to know how tightly the DNA is wound around proteins inside these cells and how this affects aging. The more open the chromatin is, the more the traits affected by the genes inside will be expressed. Percec says that she hopes to find out how ASCs can maintain an open profile with aging.

Funding for this study was provided by the National Institutes of Health.

About Pennsylvania Medicine: Pennsylvania Medicine is one of the world's leading academic medical centres, dedicated to the related missions of medical education, biomedical research, and excellence in patient care. It consists of the Raymond and Ruth Perelman School of Medicine at the University of Pennsylvania, founded in 1765 as the nation's first medical school and the University of Pennsylvania Health System, which together form a $05.3 billion enterprise. The Perelman School of Medicine has been ranked among the top five medical schools in the United States for the past 18 years, according to U.S. News and World Report's survey of research-oriented medical schools. The School is consistently among the nation's top recipients of funding from the National Institutes of Health, with $373 million awarded in the 2015 fiscal year.

The University of Pennsylvania Health System's patient care facilities include: The Hospital of the University of Pennsylvania and Penn Presbyterian Medical Centre, which are recognised as one of the nation's top 'Honour Roll' hospitals by U.S. News and World Report, Chester County Hospital; Lancaster General Health; Penn Wissahickon Hospice and Pennsylvania Hospital, the nation's first hospital, founded in 1751. Additional affiliated inpatient care facilities and services throughout the Philadelphia region include Chestnut Hill Hospital and Good Shepherd Penn Partners, a partnership between Good Shepherd Rehabilitation Network and Penn Medicine. Pennsylvania Medicine is committed to improving lives and health through a variety of community-based programmes and activities. In fiscal year 2015, Pennsylvania Medicine provided $253.3 million to benefit the community it seeks to and does serve.

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Mathematics Predicts the Molecular Biology of Cells

Vesicle at the cell surface over time. The track shows where it moves to and the 'contours' shows where
molecules:mountains are. This shows the biological data combined with the mathematical model.
Image: Heriot Watt University

|| February 23: 2017: Heriot Watt University News || ά. The Heriot Watt University has challenged a long-held theory about the way cells behave on the nanoscale, which could revolutionise future research into diabetes and neurological treatments. Biologists have long agreed that cells that secrete hormones, like insulin or neurotransmitters, like serotonin, package their cargo in vesicles, bubble-like intra-cellular structure and move them in a very regulated way, following the same paths to similar places in the cells, akin to a railroad. But you build a model of the railroad, that you copy from the diagram, that was copied from the human mind, that conceived the concept, before you build one in reality so that a comparative study is possible. And here, is mathematical modelling with the right size and shape to offer assistance.

The integration of advanced microscopy, with cell biology and mathematical modelling, could be applied to many other problems in biomedicine and will accelerate discovery in the years to come. Professor Rory Duncan, Institute of Biological Chemistry, Biophysics and Bioengineering hopes. Although the world’s most powerful microscopes couldn’t see these specific tracks, biologists were convinced they were there because of the observed behaviour of the vesicles. Clusters of nano-sized molecules inside cells were believed to overlap with the vesicles but this theory has now been challenged using sophisticated mathematical modelling.

To test the theory, Professor Rory Duncan, Head of the Institute for Biological Chemistry, Biophysics and Bioengineering worked with Professor Gabriel Lord from the School of Mathematical and Computer Sciences. Using new ‘super-resolution’ microscopy techniques, the team mapped the positions of hundreds of thousands of molecules within the cells on a nanoscale, as small to humans as Jupiter is large to us.

These molecular data, known as ‘big-data’ due to the large amount of information generated, proved ideal for mathematical analysis. Professor Lord explains, “One of Heriot-Watt’s strengths is cross-disciplinary working. Together with our colleagues in Biology, we approached how these cells behave in a radically new way, asking what if the vesicles don't follow paths to special molecular depots inside cells but instead, avoid the cluster of molecules, like a boulder does when following a valley between mountains?

“Working with the biology team and combining microscopy information from diverse experiments into a ‘mathematical model,’ we were able to run multiple experiments on a computer that aren’t possible in the real world.”

Professor Rory Duncan continued, “In contrast to what biology theorised, vesicles actually avoid areas in the cell previously thought to attract them, following ‘valleys’ in between groups of the specific molecules known to drive secretion. The net result to the observer is the same, vesicles re-use similar routes and move to nearly identical places in the cell but the mechanism is the opposite of previous thinking and the physical tracks do not exist.

“Our new approach allows us to run experiments on the computer and our resulting model predicts how the vesicles and molecules behave in cells, particularly, if they are disrupted or mutated, as happens in disease states. This predictive ability is powerful because it tells biologists which molecules to target in future studies and lays the way for larger and more thorough modelling of complex biological processes.

These findings have wide-reaching possibilities for studying cellular dynamics. For example, when something goes wrong with the transport of neurotransmitters in these vesicles, it leads to a variety of neurological disorders. We don’t yet know what goes wrong but now we are starting to understand how cells behave at a molecular level, science may be able to make breakthroughs for conditions like epilepsy or diabetes.

The integration of cutting-edge microscopy, with cell biology and mathematical modelling, could be applied to many other problems in biomedicine and will accelerate discovery in the years to come.”

The work was funded through the Next Generation Optical Microscopy Initiative, led by the Medical Research Council:MRC and with co-funding from the Biotechnology and Biological Sciences Research Council:BBSRC and the Engineering and Physical Sciences Research Council:EPSRC. Funding was also received from Wellcome. The research was published in the journal, Current Biology. ω.

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Autoimmunity: What It Is: How Does It Work: What Does It Do

Image: Victoria University of Wellington

|| February 20: 2017: Victoria University of Wellington New Zealand News || ά. What happens when your immune system turns against you? Researchers at Victoria are devising ways to treat auto-immune disorders using repurposed medications. What happens when your body's defences attack you? Sometimes, your immune system doesn’t recognise your healthy cells as part of you and attacks them as though they’re foreign organisms. When the body is at war with itself, it’s known as autoimmunity. Multiple Sclerosis, often called MS, is an autoimmune disease that attacks the central nervous system and interrupts the flow of information in the brain and between the brain and body.

Understanding why autoimmunity diseases occur is incredibly challenging. Some people may have a genetic predisposition to MS, which can then be triggered by environmental factors. For instance, MS is more prevalent in people born some distance from the equator. Researchers at Victoria University of Wellington are working to piece together the autoimmunity puzzle. By repurposing medications designed to treat other illnesses, we’re targetting the neuroinflammation caused by MS. Well-resourced research is vital. It helps in the fight against MS and other autoimmune diseases, so that one day we can find a cure.

When your body declares war on itself: Professor Anne Camille La Flamme  describes herself as a ‘card-carrying immunologist’. “I’m deeply interested in the immune system and particularly, immune regulation.” she says. “Your immune system is like your own personal army that defends you from invading organisms. But sometimes, that army turns on you and your immune system starts to attack your body’s perfectly healthy cells. So a lot of my research focuses on what causes this auto-immunity.”

She is overseeing a project at the Malaghan Institute of Medical Research, based at Victoria University, which is focused on finding a treatment for the progressive form of Multiple Sclerosis. MS is an auto-immune disease that affects thousands of New Zealanders, the debilitating condition interferes with mobility, vision and cognition and in the progressive form, which affects around 50 percent of people with MS, paralysis can develop.

Professor La Flamme says that there are currently more questions about the condition than there are answers, but there is hope. “What’s heartening is that we are closer to understanding the genetic and environmental factors associated with MS than we were even just ten years ago.”

Repurposing medication: One of the projects Professor La Flamme and her colleagues have been working on is repurposing drugs. “It’s about taking medicines that are already approved for one use and applying them to another disease, in this case, progressive MS. We are currently testing two anti-psychotic medications, clozapine and risperidone, which are used to treat schizophrenia, autism and other neurological disorders and investigating their effect on people with progressive MS, for which there is currently no treatment.

MS Society

“There are cellular pathways that may be involved in a number of disease processes, which aren’t specific to a particular disease.” she says. “For example, the anti-psychotics we are investigating are atypical, meaning, they target a range of different cellular receptors other than those that are known to be involved in psychosis. So drugs have additional functions, one of those seems to be resolving neuro-inflammation which helps with schizophrenia. But, because neuro-inflammation also happens in MS, we’re hoping to target it with these medicines.”

Professor La Flamme says that using existing medications that are already on the market could potentially save time getting a treatment to people with progressive MS. “The fact that these medicines are being repurposed means we already know a lot about them, they are already approved for use, and they’ve been used by millions of people for a number of decades.”

Professor Anne Camille La Flamme: Immunology and Cell Biology School of Biological Sciences: My primary research interest is in the immune regulation of disease. In particular, my research focuses on the pivotal role of one specific immune cell, the macrophage, in the regulation of pro-inflammatory diseases such as multiple sclerosis. These studies investigate the factors that regulate macrophage activation to induce or suppress disease. Recent work, in collaboration with other New Zealand and international researchers, is aimed at identifying new therapeutic targets and drugs to treat multiple sclerosis.

Additionally, I am investigating several aspects of schistosomiasis, a parasitic worm infection. This disease affects over 200 million people worldwide, and one of the applied goals of this research is to identify and develop biomarkers that predict the development of severe disease in patients. Finally, in partnership with other researchers in the Allan Wilson Centre, I am involved in elucidating the immune responses of tuatara. ω.

Further on Cell and Immunology

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Healthy Gut: Time to Assemble the Nineins

Image: University of East Anglia

|| February 19: 2017: University of East Anglia News || ά. Scientists have uncovered key processes in the healthy development of cells which line the human gut, furthering their understanding about the development of cancer. A University of East Anglia:UEA study, published in the journal Open Biology, shows that a protein called ninein is essential for normal tissue development in the gut. The research aimed to unravel some of the poorly-understood mechanisms involved in rearranging the internal ‘skeleton’, cytoskeleton, in cells that are undergoing a shape change during normal development.

Using ‘mini-guts’ created in the lab, they studied the tubular filaments which are part of the cytoskeleton called microtubules and their dramatic rearrangements during the formation of certain types of cells found, for example, in the gut. Dr Mette Mogensen from UEA’s School of Biology said, “Formation of columnar epithelial cells like those lining the gut involves reorganisation of the microtubules, as well as the assembly of new Microtubule Organising Centres:MTOCs, which anchor one end of the microtubules to cell surfaces.

We found that this process can only happen correctly when the protein ninein is present. We also found that the microtubule-associated protein CLIP-170 is needed for the relocation of ninein to the new MTOCs.

The correct organisation of microtubules in columnar gut cells leads to the formation of a transcellular array. As well as influencing cell shape, microtubules form tracks for the transport of vesicles and molecules within cells, which enables nutrient uptake. Loss of this transcellular microtubule array leads to loss of tissue architecture, function and ultimately cancer, so formation of these new MTOCs is critical.”

Researchers used ‘mini-guts’ during the study, generated from stem cells that are isolated from gut tissue and grown in a special medium. They formed structures in culture that mimic normal gut including columnar cells with transcellular microtubule arrays and new MTOCs.

The research was funded by the BBSRC, Anatomical Society and BigC Appeal.

‘Ninein is essential for apico-basal microtubule formation and CLIP-170 facilitates its redeployment to non-centrosomal microtubule organizing centres’ is published in the journal Open Biology:

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A Fish-Tale of Airinemes

An adult zebrafish: Image: Dae Seok Eom:David Parichy:University of Washington

|| February 17: 2017: University of Washington News: James Urton Writing || ά. Scientists at the University of Washington have discovered that a common type of cell in the vertebrate immune system plays a unique role in communication between other cells. It turns out that these cells, called macrophages, can transmit messages between non-immune cells. Their paper, published online February 16 in the journal Science, describes how pigment cells in a species of fish have co-opted macrophages to deliver messages important for pigment patterning in skin. This is the first reported instance of macrophages relaying messages over a long distance between non-immune cells.

But since the macrophages are common to all vertebrates, the researchers believe their discovery is no quirk of aquatic life. Macrophages may be common interlocutors for long-distance messages among cells. “If pigment cells have figured out how to use macrophages for signalling, it stands to reason that others have as well.” said Senior Author and University of Washington Biology Professor David Parichy. “This could occur in a variety of cells and animals.” Parichy and Lead Author Dae Seok Eom, a University of Washington Postdoctoral Researcher, discovered this new role for macrophages while studying zebrafish.

They had wanted to understand how the zebrafish gets its telltale stripes of silver-yellow and black. Each color, black, yellow and silver, arises from a different type of pigment cell. When zebrafish are juveniles, these pigment cells migrate to the right spot to create the stripes. “As they migrate, communication among these three populations of pigment cells is critical to forming the stripes we see in adult zebrafish.” said Parichy.

Eom and Parichy used laboratory genetic tools to make zebrafish pigment cells glow fluorescent colours, making these cells easier to track using a microscope. In the process, they discovered that xanthoblasts, the precursors to yellow pigment cells, produced unique, elaborate projections during the peak time for pigment pattern formation.

“Xanthoblasts sent these thin projections out in circuitous, almost random directions.” said Parichy. “The projections would eventually encounter another pigment cell, the black melanocyte and stop.” Eom discovered that these projections, which they named 'airinemes' for mathematician and astronomer Sir George Airy, who described the optical limits to view small objects, as well as the Greek messenger goddess Iris, contained tiny, membrane-bound packages of proteins that provide molecular signals to melanocytes, the black pigment cells.

The researchers showed that when an airineme from a xanthoblast encountered a melanocyte, the signal proteins from the airineme would cause the black pigment cell to migrate into the stripe. But they didn’t understand how airinemes found melanocytes or why they took such a seemingly random route, until Eom made a critical observation.

“I saw a macrophage interacting with an airineme and then another and then another.” said Eom. “In one experiment, I counted 178 airinemes coming from xanthoblasts and 94 percent of them were obviously associated with a macrophage.”

Macrophages are constantly on the move. In fish, people and everything in between, they wander the tissues of the body, 'crawling' along like amoebae. Along the way, they sample their environment, picking up and ingesting debris. Their scavenged prizes are often harmless cellular detritus. But if they ingest a bit of a pathogen or receive signals that a cell nearby is under assault from an invader, macrophages can alert other cells of the immune system.

Armed with this knowledge, Eom tested whether macrophages were truly facilitating the dialogue between yellow and black pigment cells. Using genetic tools, he created zebrafish without macrophages and saw that xanthoblasts produced far fewer airinemes. And under these conditions, melanocytes did not migrate properly to form stripes.

Under the microscope, Eom captured images and movies of how macrophages behaved when they randomly encountered an airineme. A macrophage would seemingly 'engulf' one of the round, globular protein packages on the airineme and drag it along, stretching the airineme out. “Now we know why airinemes seem to take such a meandering, random route.” said Eom. “They are being dragged by macrophages that are themselves moving along randomly.”

But when that same macrophage encountered a melanocyte, the macrophage appeared to 'hand off' the airineme to the melanocyte and wander away, presumably, delivering the message, via the airineme, to the melanocyte.

Eom showed that airineme membranes contain a type of lipid that is often an 'eat me' signal for macrophages, which may explain why the macrophages attach to and drag along these projections. He and Parichy plan to investigate why macrophages do not digest the airinemes and how the airineme is 'handed off' specifically to a melanocyte.

But given the macrophage’s tendency to wander and pick up objects, Parichy believes this is unlikely to be the sole instance of macrophage co-option by cells outside of the immune system. “It’s very plausible that what we’ve seen here occurs in other contexts where macrophages play important roles, from tissue development and regeneration to cancer.” said Parichy. “We can easily see how macrophages might facilitate signalling between cells in a variety of situations.”

The research was funded by the National Institutes of Health. For more information, contact Parichy at dparichy at or 206-734-7331 and Eom at dseom at or 512-350-9454.

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The Cell That Makes Mistakes That Causes a Major Lung Cancer

Colonies of lung cells grown in the laboratory from a single lung stem cell from a cancer patient. Image: Clare Weeden, Walter and Eliza Hall Institute.

|| February 12: 2017: University of Melbourne Australia News: Andrew Trounson
Writing || ά. For four years straight medical researcher Clare Weeden would go on alert whenever lung surgery was underway anywhere across Melbourne. No matter the time, she would have to be ready in her lab to receive samples of fresh tissue as part of a project to isolate and research the stem cells that repair our lungs as they constantly breathe in contaminants from air pollution to cigarette smoke. She didn’t know it at the time, but she was hot on the trail of the lung’s basal stem cells that now appear to be the likely culprits that trigger a major lung cancer closely tied to smoking, squamous cell carcinoma. It is the second most common form of lung cancer.

Basal stems cells are very quick at repairing DNA damage caused by inhaled chemicals such as those from cigarette smoke, but they are prone to making mistakes. It means that the more repair work they have to do, the greater the chance of a cancer-causing mutation. “What we have found is a genetic fingerprint in squamous cell carcinoma that has been left from basal stem cells in the lung whose repair work has gone awry and led to the cancer.” says Ms Weeden, from the Walter and Eliza Hall Institute of Medical Research and a PhD candidate at the University of Melbourne. “It isn’t definitive but the evidence is that lung basal stem cells are the likely cells of origin.”

The unmasking of basal stem cells, published in the US-based Public Library of Science: Biology, is the culmination of years of painstaking laboratory work and data-crunching that has now provided a crucial new target for developing drugs that may be able to turn off the progress of the cancer. Ms Weeden was sometimes up until to 03:00 at the Institute, isolating and processing cells from the freshly operated-on lung tissue, especially when there was a flurry of samples in one day. It is a complex process that took up to six hours for each of the eventual 140 samples.

To extract and isolate the cells she first had to mince the tissue into tiny pieces with scalpels before adding enzymes and other media. She then put the material through micro strainers and a centrifuge, after which the cells could then be stained with antibodies and passed through a laser cell-sorting machine. She would then have to get the stem cells to grow into colonies to prove they were indeed stem cells and that her isolation process was working.

Lung stem cells cultured in the laboratory. The green, blue and purple colours emerging from behind the orbs are a
protein expressed by lung basal stem cells. Image: Clare Weeden, Walter and Eliza Hall Institute of Medical Research.

But one day she came across a sample that she could barely get to grow at all. Intrigued, she contacted the Victorian Cancer Biobank for basic information on the donor. It was likely that the donor was a smoker or ex-smoker since most people having lung surgery have a history of smoking. But this patient had never smoked. Sensing a possible link she went back to the Biobank to get information on all the previous tissue donors and over that weekend plotted out a chart.

The correlation was stark. Samples from those that had never smoked had low basal cell growth and the more heavily a patient had smoked, the higher the growth rate. “It completely grabbed my curiosity.” she says. “I remember on Monday morning going straight into my supervisor’s office, Dr Marie-Liesse Asselin-Labat and putting the chart down in front of her. We both realised we were onto something significant. The question was what?”

By using the same process that Weeden had developed to accurately isolate lung stem cells, she and Dr Asselin-Libat set to examining how the basal stem cells worked. They discovered that basal stem cells were very efficient at repairing damaged DNA but that the process the cells use, called non-homologous repair, is prone to making errors that can lead to cancer-causing mutations. In non-homologous repair the break in a damaged DNA chain is simply closed over rather than copied. They also found evidence of the accumulation of mutations in the basal stem cells of the smokers.

“While we need more experimentation, this gave us a model of what may be happening.” says Weeden. “Our lungs are constantly being exposed to what we inhale. When we breathe in something like cigarette smoke that causes lung damage, these basal cells receive a signal to grow and repair the damage.

But they have to first repair their own DNA damage and the process they use is very quick. The advantage is that it helps the cells to survive, but the disadvantage is that they are prone to making errors that can lead to cancer.” To test that model they turned to Institute bioinfomaticians Professor Gordon Smyth and Dr Yunshun:Andy: Chen who used statistics and computer science to extract a genetic 'signature' for lung basal stem cells. They then compared that signature with the genetics of various lung cancers.

They discovered that this same signature was highly correlated with lung squamous cell carcinoma, the second most common form of lung cancer and the most closely linked to smoking, some 96 per cent of people with lung squamous cell carcinoma are either smokers or ex-smokers. It was clear evidence that basal stem cells are the likely culprits in how the cancer is triggered.

By identifying a cell of origin Weeden that says we now have a drug target to aim at that has the potential to stop the progress of the cancer. Previous Institute research in 2009 lead by Professor Jane Visvader and Professor Geoff Lindeman had similarly identified a likely cell of origin for inherited breast cancer and last year that same team identified an existing drug, denosumab, that in laboratory models could switch off the problematic cell growth and curtail the cancer. Clinical trials are now underway.

“In the breast cancer research they similarly used correlations to identify a cell of origin like we have and now further work has solidified that.” says Weeden. Does this mean that at some point in the future smokers could breathe easier by taking a drug that could stop the cancer being triggered? No. Weeden points out that if someone took such a drug and continued to smoke the damage could be even worse than the cancer.

“Basal stem cells have a job to do in the lung, they repair any damage. If a person was treated with a drug that turned off basal cells and continued to smoke, I would imagine that other lung problems may develop due to the inability of the stem cell to repair the lung airways from cigarette smoke-induced damage.” says Weeden. She points out that smoking also causes other lung cancers that don’t arise from basal stem cells.

She says the biggest beneficiaries of any such drug could be ex-smokers. “This is particularly relevant as lung squamous cell carcinoma can occur in ex-smokers who have quit perhaps 20 or 30 years ago. But the best way to reduce the risk of lung cancer is to simply quit smoking because no matter how long you’ve smoked for, the risk of lung cancer is reduced when you quit.”

Ms Clare Weeden: PhD Student, Asselin-Labat Laboratory, Stem Cells and Cancer Division, Walter and Eliza Hall Institute of Medical Research; Department of Medical Biology, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne.

: This work, of University of Melbourne, is licensed under a Creative Commons Attribution-No Derivatives 3.0 Australia: CC BY-ND 3.0 AU, so you can republish our articles for free, online or in print. All republished articles must be attributed in the following way and contain links to both the site and original article: “This article was first published on Pursuit. Read the original article:

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Have You Seen Obesity: Look Inside the Mitochondrion

||February 08: 2017: University of Helsinki News: Elisa Lautala Writing || ά. Professor Kirsi Pietiläinen has located the impact of obesity in the mitochondria of fat tissue. Mitochondrial dysfunction likely results in excess weight that is difficult to lose. Kirsi Pietiläinen, a recently appointed Professor at the Faculty of Medicine, has studied obesity since the early 90s. Professor Jaakko Kaprio’s extensive twin study data is central to her research. After studying the genetics and epidemiology of obesity as well as conducting extensive surveys, she is now studying the metabolism of fat tissue through identical twins, in which, one of the twins is obese. As identical twins share a genome, the discrepancy in their weight must be caused by living environment or lifestyle.

"This has become something of a Pandora’s box, or a treasure trove that has led our research group into many unexpected areas we had no idea of when we started our research." said Professor Pietiläinen. The study revealed that the some of the obese twins had disorders in the mitochondria of their fat tissue cells, which are responsible for the metabolism of the cells. Pietiläinen’s group is currently investigating whether weight loss can rectify the dysfunction. "I’m almost willing to bet my doctoral hat on the disorder being a consequence of obesity, which triggers a vicious circle. The body begins to slow down metabolic activity at a very early stage, and it is very difficult to get rid of the accumulated fat. In addition, such people are quick to regain the weight they have lost.”

The twins in Pietiläinen’s study were young, between 25 and 30 years of age, their obesity was not severe, and they had no prominent mutations in their mitochondria or hereditary diseases. In her follow-up studies, Pietiläinen has found that obesity can nearly always be traced back to the mitochondria, but the severity of the dysfunction varies. Among healthy obese people, with no preliminary stages of diabetes or vascular diabetes, the dysfunctions are milder or non-existent.

However, the reason for the variation in the severity of the mitochondrial dysfunction is unknown. "At the moment we’re trying to find out how we could prevent mitochondrial disorder and treat existing disorders." In this, Pietiläinen’s research group is working together with docent Eija Pirinen and Academy Professor Anu Wartiovaara who study mitochondrial diseases.

Earlier studies have revealed that certain B vitamins are effective against some mitochondrial diseases, and the same vitamins are now being administered to the twins in the current research. Preliminary results can be expected later this year, when the research subjects are studied after a five-month course of the vitamin. Pietiläinen wants to next examine the muscle metabolism of the twins in the study, with the goal of finding out whether the metabolic changes associated with obesity are different in the muscles than they are in fat tissue.

A study on the metabolism of patients before and after bariatric surgery is also underway. The intention is to discover whether the changes effected by the surgery influence the behaviour of the mitochondria in morbidly obese patients.  "In my research career, one thing has led to the next, and one answer has always opened the door to new questions." she says. 

Professor Kirsi Pietiläinen is the first person to complete the tenure track and receive a permanent tenured professorship at the University of Helsinki. In the three-level tenure track, launched in 2010, a fixed-term assistant professorship of three to five years leads to an associate professorship of three to five years and culminates in a permanent professorship, if the criteria for granting tenure are met.

The intention of the system is to increase the predictability, competitiveness and attractiveness of academic careers. The University strives to find the most talented and motivated people for the tenure track. Currently there are 54 tenure track assistant and associate professors working in the University of Helsinki. 18 of them have been recruited outside Finland and 19 are women. Professor Pietiläinen heads a 15-person Obesity Research Unit at the Faculty of Medicine, teaches future doctors and nutritional scientists and serves as the senior physician at the Obesity Centre of Jorvi Hospital.

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Breathing Molecule That Regulates CO2 in Blood Discovered: It's Vital to Treating Respiratory Conditions

||February 02: 2017: University of Warwick News || ά. A vital molecule which regulates breathing has been discovered by University of Warwick researchers. Connexin26 detects CO2 levels in body and activates body’s breathing mechanisms and has been specially tuned by evolution for this purpose. Discovery could lead to more targeted treatments of respiratory problems and congenital deafness. Professor Nicholas Dale at the School of Life Sciences has exploited evolutionary principles to identify Connexin26:Cx26 as a key molecule that reacts to CO2 in our bodies and activates breathing.

Cx26 molecules detect levels of CO2 in the blood-stream, and when levels reach a certain point, they tell our bodies to excrete the CO2 and take in oxygen, the vital life-preserving process that allows us to breathe and creates blood flow to the brain. Without this essential molecular function, harmful levels of CO2 would remain in the bloodstream, making breathing difficult or impossible. Mutations in Cx26 are directly connected to a number of serious conditions, ranging from congenital deafness, to respiratory conditions, and serious syndromes that affect skin, vision and hearing. As Cx26 is vital to breathing well, people carrying these mutations may be at risk of sleep apnoea.

Identifying these mutations and working out how to restore the molecule to its normal function could lead to effective, targeted, personalised treatments to mitigate these risks and improve quality of life. Different animals have varying levels of sensitivity to CO2.

Professor Dale’s group exploited this idea to see whether the properties of Cx26 matched the physiological requirements of: birds, which fly at high-altitude and can tolerate low levels of CO2; humans and rats which are broadly similar at an intermediate level and mole rats, which live exclusively underground and tolerate very high levels of CO2.

The researchers found that the CO2 binding properties matched the sensitivities of these different animals. Evolutionary natural selection has thus modified the CO2-binding properties of Cx26, showing that this molecule is a universally important sensor of CO2 in warm blooded animals.

Professor Dale comments on the significance of the research, “Important molecules with universal physiological functions are shaped by evolution. We have exploited this simple fact to show that the CO2-binding characteristics of Cx26 are important in our bodies, too. This is likely to open up new ways to identify and treat people at risk of sleep apnoeas.”

The research, ‘Evolutionary adaptation of the sensitivity of Connexin26 hemichannels to CO2’, is published in the Proceedings of the Royal Society B. February 01, 2017

Warwick School of Life Sciences is an international centre of excellence with more than 80% of its research rated as 'world leading or internationally excellent' in the REF 2014 assessment and 94% overall student satisfaction in the National Student Survey:NSS.

Images: University of Warwick

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Metastasis: How Does It Happen: Epithelial to Mesenchymal Transition

Examples of developed prostate cancer cell line spheres with differing levels of EMT. Image: NTU

|| January 26: 2017: Nottingham Trent University News || ά. Scientists have taken a major leap forward in being able to fully understand how prostate cancer cells acquire their ability to spread from the initial tumour to other areas of the body. The Nottingham Trent University study is significant because metastasis, when cells begin to invade other sites such as the bone or brain, is responsible for the vast majority of prostate cancer-related deaths.

For the first time, a team at the university has been able to generate a panel of prostate cancer cells in the laboratory which spontaneously undergo a process thought to be involved in the spread of disease. The work is expected to provide vital insight into the biology and spread of aggressive prostate cancers and help to improve the management, treatment and survival of patients with therapy-resistant disease. Cells can acquire the invasive and migratory properties needed for metastasis by reactivating a programme called ‘epithelial-to-mesenchymal transition’:EMT.

This is when epithelial cells which don’t move and are tightly joined, undergo a change to become mesenchymal, where they lose their tight cell connections, change shape and gain the ability to migrate and invade surrounding tissues and blood vessels. Although important, the process via which this happens in the body is poorly understood. A main limitation of prostate cancer research has been the lack of available cells which come from a primary tumour in the prostate and can be grown in the laboratory.

In the past, the most commonly-studied prostate cancer cells have been those which are generated from disease which has already spread from the prostate to other sites, such as the bone or brain. But now scientists will be able to study the cells before this process occurs, getting a vital glimpse into cancer progression and metastasis at the earliest possible stage.

The researchers, based in the university’s John van Geest Cancer Research Centre, were able to develop new and unique sub-populations of cells from a primary prostate cancer tumour cell line – and noticed that some of the cells spontaneously took on the features of those which can move to other tissues.

“Prostate cancer is the most common male cancer in Europe and 90% of cancer-related deaths are due to disease which is resistant to therapy and which has spread to other parts of the body.” said Dr David Boocock, a scientist in Nottingham Trent University’s John van Geest Cancer Research Centre.

He said, “Cancer cells acquire the capacity to move from the primary tumour to other sites by activating biological processes which allow them to survive the journey and establish themselves in their new ‘home’.

“It is clear that understanding these processes is crucial if we are to reduce the number of prostate cancer-related deaths.” Professor Graham Pockley, the Director of the university’s John van Geest Cancer Research Centre, added, “This work provides a novel and important platform for future studies that will help us to predict prostate cancer metastasis and better understand cancer progression.

As such, it could also be crucial in providing valuable insight into potential new therapies and approaches for the treatment and management of prostate cancer.” The work is reported in Nature Publishing Group journal Scientific Reports.

John van Geest Cancer Research Centre: Nottingham Trent University’s John van Geest Cancer Research Centre is a unique purpose-built scientific facility. Its aim is to save lives and speed recovery by improving the early diagnosis and treatment of cancer. The Centre focuses on two key approaches to the treatment of patients with cancer:  improving the diagnosis and management of cancer; developing effective vaccines and immunotherapies that will significantly improve the quality of life and survival of cancer sufferers. The Queen's Anniversary Prize for Higher and Further Education was awarded to Nottingham Trent University in November 2015.  It is the highest national honour for a UK university and recognises the institution’s world-class research. Pioneering projects to improve weapons and explosives detection in luggage, enable safer production of powdered infant formula, and combat food fraud, led to the prestigious award. ω.

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Connections: Communications: Linkages: Interactions: Between Oganelles Working Together

Image: Darryl Leja, NHGRI

|| January 22: 2017: University of Exeter News || ά. Scientists have made a breakthrough in understanding how different compartments or organelles of human cells interact. Organelles are the functional units of a cell. Like organs in a body, they perform specialised functions. To allow survival of the cell, organelles have to interact and cooperate. How this is mediated and regulated in the cell is an important and challenging question in cell biology.

Researchers at the University of Exeter have now discovered how two cell organelles, called peroxisomes and the endoplasmic reticulum:ER, associate with each other at the molecular level and work together. This co-operation is crucial for the production of specific lipids, which are essential for the function of nerve cells and can protect cells from oxidative damage. Loss of peroxisome function leads to a range of severe or fatal disorders associated with developmental and neurological defects.

“Close contacts between peroxisomes and the ER were observed more than 50 years ago in ultrastructural studies, but the molecular mechanism remained a mystery.” said lead author Dr Michael Schrader, of the University of Exeter. “This is the first molecular tether identified in humans, which mediates the contact between these two important cell compartments.”

The study showed that a protein at the peroxisomes called ACBD5 directly interacts with a protein at the ER, called VAPB. This interaction links both organelles together and allows transfer of lipids between them. When the interaction between VAPB and ACBD5 is lost, the ER and peroxisomes can no longer interact and this lipid transfer appears to be prevented.

The researchers are working with experts from the Academic Medical Centre in Amsterdam, where a patient with an ACBD5 deficiency has been identified and linked to a peroxisomal defect. This patient has severe damage to the brain and retinas, affecting eyesight, and Dr Schrader said Exeter’s research and similar studies were essential if such conditions are to be diagnosed and ultimately treated.

“If we understand organelle interaction better we might also be able to use this knowledge to protect cells from certain stress conditions which are linked to age-related conditions like neurodegeneration, blindness and diabetes.” he said. People with severe peroxisomal disorders, also known as Zellweger Spectrum Disorders, often die as children or young adults, and a new charity called Zellweger UK exists to raise awareness and to support families and sufferers.

The University of Exeter’s research, funded by BBSRC, was carried out in cooperation with the University of Heidelberg:Mannheim. The paper, entitled, 'ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER' is published in the Journal of Cell Biology.

The authors were Joseph Costello, Ines Castro, Christian Hacker, Tina A Schrader, Jeremy Metz, Dagmar Zeuschner, Afsoon S Azadi, Luis F Godinho, Victor Costina, Peter Findeisen, Andreas Manner, Markus Islinger and Michael Schrader.

The journal will publish it alongside another paper, from researchers at the Hospital for Sick Children in Toronto, which independently came to similar findings, and will highlight the new discoveries in a Spotlight article.

Zellweger UK: Zellweger Spectrum Disorder, otherwise known as GPD or Generalised Peroxisomal Disorder, is a very rare genetic condition belonging to a group of diseases called Peroxisomal Biogensis Disorder:PBD. Most children don’t live into adulthood and deal with global developmental delays, deaf-blindness:multi-sensory loss and a plethora of other health issues. Life with Zellweger Spectrum Disorder can feel incredibly lonely. Most doctors and paediatricians have never even heard of Peroxisomal Disorders, so specialists experienced in the condition are very few and far between. Zellweger UK is made up of a team of volunteers; parents, family and friends of affected children. Our aim is to offer emotional support and provide financial support to families in the way of small grants towards non state funded equipment. Crucially, we are looking to help fund research into desperately needed, effective treatments, of which currently there are none. We hope, eventually, for a future free of ZSD. There is some exciting research currently being undertaken by dedicated scientists, but this type of research takes time and is costly. Peroxisomal Disorder is rare and therefore receives little funding towards research. Sometimes the needs of a few has to be as important as the needs of many, and our hope is that through research into lesser known conditions exciting discoveries can be made that could benefit many other conditions too. Your support is vital in enabling us to help fund this. Registered Charity Number: 1166389. ω.

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RNA-Based Digital Assay of Circulating Tumour Cells May Improve Diagnosis and Monitoring of Liver Cancer

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|| January 19: 2017: Massachusetts General Hospital News || ά. Use of an advanced form of the commonly used polymerase chain reaction:PCR method to analyze circulating tumour cells:CTCs may greatly increase the ability to diagnose early-stage cancer, increasing the likelihood of successful treatment. In their report published in PNAS, a team from the Massachusetts General Hospital:MGH Cancer Centre describe how combining use of the MGH-developed CTC-iChip with RNA-based digital PCR greatly improved detection of cancer cells in the blood of patients with hepatocellular carcinoma:HCC, the most common type of liver cancer.

“We have developed an assay capable of detecting a single cancer cell within a background of the tens of billions of cells that comprise whole blood.” says Mark Kalinich of the MGH Cancer Centre, Co-Lead Author of the PNAS report. “Our test provides highly specific detection of cancer in patients with HCC, compared with healthy individuals and with those at high risk for developing the disease. These results hold promise for both the early detection of HCC and for the monitoring of treatment over time.”

Liver cancer, including HCC, is the second highest cause of cancer death in the world. HCC is particularly prevalent in developing countries, where its incidence is driven by infection with the hepatitis B virus, which now affects more than 248 million individuals. In developed countries, conditions like hepatitis C infection and alcohol abuse are also increasing the prevalence of HCC. Early diagnosis of the tumor can lead to five-year survival rates of from 50 to 80 percent, but once HCC has spread, survival drops to around 15 percent.

Current blood-based strategies for detecting HCC, such as serum levels of alpha-fetoprotein:AFP, have had poor results. New technologies enabling the isolation and analysis of CTCs have been valuable research tools and can help track treatment response, but the use of microscopy to identify and analyze CTCs required the time-consuming development of specific protocols for particular forms of cancer. While standard PCR is a technique for generating many copies of a specific nucleic acid segment of DNA, digital PCR allows much more precise measurement of the quantity of a given nucleic acid segment in a sample of CTCs.

The research team developed their digital PCR assay by first identifying 10 specific RNA transcripts that were expressed in HCC cells but not in blood components. Using the CTC-iChip developed at the MGH Center for Engineering in Medicine, they assayed blood samples from six groups of individuals: newly diagnosed HCC patients; HCC patients receiving treatment who still had evidence of disease; HCC patients who appeared to be cured after surgical treatment, including liver transplantation; patients at risk for developing HCC because of other chronic liver diseases; patients with other types of cancer, including some with liver metastases; healthy volunteers.

Digital PCR analysis revealed significantly higher levels of the HCC-associated RNA transcripts in blood samples from patients with HCC than from those with other cancers, with chronic liver disease or healthy controls. Use of a CTC score based on the 9 RNA transcripts most significantly associated with HCC generated positive results for more than half of those with untreated HCC but only around 8 percent of healthy controls and 3 percent of those with other liver diseases. Around 28 percent of patients currently being treated had positive scores, and the percentage of positive scores among patients with no evidence of disease after treatment was similar to that of healthy controls.

Follow up with a small group of patients produced evidence suggesting the potential of the CTC score to monitor treatment response. Scores remained high in two patients who had not been treated between blood draws, while the scores of two other patients dropped after surgical tumor removal. Another patient’s score dropped precipitously after treatment with a checkpoint inhibitor and then showed significant further reduction after a radioembolisation procedure that greatly reduced the size of the tumor.

To determine whether CTC scoring could improve the inadequate results of AFP screening, the researchers used both methods to analyze blood samples from 15 newly diagnosed patients. In four of them, the presence of HCC was indicated by CTC score alone; AFP alone detected cancer that the CTC score did not in one patient, and the results of both tests were able to detect HCC in another five. Overall, either CTC score, AFP or both produced positive results in 67 percent of patients, missing the diagnosis in only one third.

While previous studies combining AFP with more specific assays like ultrasound have improved diagnosis of at-risk patients, leading to a 37 percent drop in mortality, the authors note that ultrasound results can be compromised in patients with obesity or cirrhosis and that high-quality ultrasound may not be available in the developing countries where the risk of HCC is highest.

“Although there are major hurdles to global implementation of CTC-based digital PCR to screen for HCC, we believe they are surmountable.” says Kalinich, who is an MD:PhD candidate at Harvard Medical School. “With the blood stabilization techniques currently being developed in Mehmet Toner’s lab at the Center for Engineering in Medicine, blood draws from anywhere in the world could be analyzed at central processing facilities, enabling the high throughput required for global screening efforts.”

He notes that further study is required to confirm the ability of this assay to detect HCC in a large-scale trial, expand the number of HCC-related RNA transcripts to further improve diagnostic accuracy, and determine whether this approach can help detect and monitor treatment for other forms of cancer.

Irun Bhan, MD, of the MGH Cancer Centre is Co-Lead Author of the PNAS report; and Daniel Haber, MD, PhD, director of the MGH Cancer Center and the Isselbacher Professor of Oncology at Harvard Medical School, and Shyamala Maheswaran, PhD, MGH Cancer Center, associate professor of Surgery at HMS, are co-senior authors. Support for this study includes National Institutes of Health grants and grants from the Howard Hughes Medical Institute, the National Foundation for Cancer Research, the Department of Defense, the Prostate Cancer Foundation and the National Science Foundation. The MGH has filed a patent application for the work described in this study.

Massachusetts General Hospital: Founded in 1811, is the original and largest teaching hospital of Harvard Medical School. The MGH Research Institute conducts the largest hospital-based research program in the nation, with an annual research budget of more than $800 million and major research centers in HIV:AIDS, cardiovascular research, cancer, computational and integrative biology, cutaneous biology, human genetics, medical imaging, neurodegenerative disorders, regenerative medicine, reproductive biology, systems biology, photomedicine and transplantation biology. The MGH topped the 2015 Nature Index list of health care organisations publishing in leading scientific journals and earned the prestigious 2015 Foster G. McGaw Prize for Excellence in Community Service. In August 2016 the MGH was once again named to the Honor Roll in the U.S. News & World Report list of "America’s Best Hospitals." ω.

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Molecular Volume Control May Help Combat Tumours

Professor Stephen Keyse: Image: University of Dundee

|| January 08: 2017: University of Dundee News || ά. A ‘molecular volume control’ may one day be used to manipulate enzyme activity in order to control the development and treatment of cancer, according to research at the Universities of Dundee and Bath. The researchers have uncovered new functions of an enzyme called Dual-specificity phosphatase 5, DUSP5, which will help scientists to better understand how tumours develop.

DUSP5 is known to switch off the activity of another enzyme, called ERK, which controls cell growth in a number of cancers, including colon, lung and melanoma. This would suggest that DUSP5 is a tumour suppressor, but studies have also shown that increased DUSP5 activity is observed in several human cancers. Using cell-based models, the Dundee-Bath team have shown that the loss of DUSP5 can completely stop cancer cell formation by driving ERK activation to such high levels that it engages a natural protective mechanism within cells that makes them shut down.

An analogy can be made to listening to the radio and being unable to hear comfortably when the volume is either too quiet or too loud. In a similar manner, it appears that increasing or decreasing DUSP5 levels in cells can push ERK activity into ranges that are either too low or too high to cause cancer growth.

The University of Dundee’s Professor Stephen Keyse said, “When the link between DUSP5 and ERK was established it was logically thought that it acted to suppress tumour growth, but levels of DUSP5 are observed to be increased in many human tumours, which doesn’t make intuitive sense.

What we are now seeing is that DUSP5 does indeed inhibit ERK activity, but sometimes this allows cancer cells to persist and grow by preventing them from engaging natural tumour suppressive responses. This suggests that targeting DUSP5 and thus increasing the level of ERK signalling beyond a tolerable level may offer a new route for combatting the progression of some tumours.”

The paper, published in Proceedings of the National Academy of Sciences, highlights the need to better understand how normal and mutant genes interact in tumour cells to predict how they might develop. The next stage for the team is to look at human cancer cells to determine whether DUSP5 has an effect on them.

Dr Jim Caunt, from the University of Bath, said, “These results confounded our initial predictions and show just how important it is to understand how different types of mutation cooperate to influence cancer cell behaviour. An exciting prospect raised by the study is that monitoring DUSP5 could help us predict which types of drugs could be most effective in cancer treatment.”

The research is funded by the Medical Research Council:MRC. ω.

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Scientists Find Out How Cancer Cells Can Shrug off Physical Constraints on Growth and Spread

An image showing 384 experiments from the study, including around 20,000 single cells. Each square is an image from one experiment;
in each case researchers have depleted:knocked down a single gene. Within each experiment, the blue is DNA, the green is YAP,
the yellow is paxillin, the red is actin. Image: Dr Chris Bakal:the ICR

|| January 06: 2017: The Institute of Cancer Research London News || ά. Scientists have discovered how cancer cells are able to break free of the physical restraints imposed by their surroundings in order to grow and spread around the body. The research could point to new ways to treat or prevent the spread of cancer cells, which is the biggest cause of cancer death. Scientists at The Institute of Cancer Research, London, found that cancer cells that spread around the body have a broken switch which continually activates a key molecule called YAP. YAP acts as a ‘mechano-sensor’, allowing cells to ‘feel’ the matrix around them, which they can grasp onto to move around tissues in the body.

The new study is published in the journal Cell Systems and was funded by Cancer Research UK and the ICR itself. Normally cell movement is suppressed by contact with other cells, but YAP can help to overcome these physical restraints by turning on various genes that are usually switched off. In most cells, YAP’s activity is carefully regulated but the researchers found that cancer cells that spread are able to produce YAP all the time, helping them to overcome the physical barriers to movement. The ICR team systematically switched off 950 different genes in cancer cell lines grown in the laboratory to work out which ones influenced YAP signalling. They found that it was partially controlled by a molecule called beta-PIX.

Beta-PIX boosts YAP activity as the cell binds to the extracellular matrix while moving through tissue. When the researchers forced the cells to remain stuck to the matrix, as if the cells had licked an icy pole, YAP activity was even higher. But when beta-PIX molecules were depleted, YAP activity was greatly reduced. To find out how YAP activity was controlled in cancer cells, the team looked at triple-negative breast cancer cells in the lab that were either derived from a primary tumour or from a site of distant spread.

When the researchers disabled the beta-PIX pathway in cancer cells from the primary tumour, YAP failed to activate, as would be expected. But when they did the same to the metastatic cells, YAP did activate. This suggests invasive cancer cells have broken the pathway that links beta-PIX to YAP, allowing them to sustain high levels of YAP even when not bound to the surrounding matrix.

Study leader Dr Chris Bakal, Leader of the Dynamical Cell Systems Team at the ICR, said, “Our research shows how cancer cells that have become invasive are able overcome the normal constraints on cell movement. Cancer cells that have spread around the body have a switch which is jammed on, allowing them to produce a molecule called YAP all the time. This allows them to keep growing and spreading throughout the body, ignoring the physical controls that would normally stop this happening.

Understanding more about the physical processes which constrain and control the growth and movement of cells can open up exciting new avenues for cancer treatment, which may have been missed until now.”

Dr Emma Smith, Science Information Manager at Cancer Research UK, said, “When cancer spreads it’s a lot more difficult to treat. This research identifies the signals that can go wrong in cancer cells, helping them to break free from the tumour.

Understanding more about how cancer spreads could be a crucial first step towards new treatments, but further work is needed to find out if blocking these signals can stop cancer spreading in people.” ω.

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Invoke The Yamanaka and See How Magic Unfolds: Say The Salk Institute Researchers

Now, tell us, which of you, did which part, of the Yamanaka! Ergin Beyret, Mo Li, Aida Platero-Luengo, Pradeep Reddy, Paloma Martinez-Redondo,
Eric Vazquez, Tomoaki Hishida, Toshikazu Araoka, Concepcion Rodriguez Esteban, Fumiyuki Hatanaka, Alejandro Ocampo, Juan Carlos Izpisua Belmonte
Image: The Salk Institute

|| December 16: 2016: The Salk Institute News: La Jolla: San Diego: California: US || ά. Graying hair, crow’s feet, an injury that’s taking longer to heal than when we were 20, faced with the unmistakable signs of aging, most of us have had a least one fantasy of turning back time. Now, scientists at the Salk Institute have found that intermittent expression of genes normally associated with an embryonic state can reverse the hallmarks of old age. This approach, which not only prompted human skin cells in a dish to look and behave young again, also resulted in the rejuvenation of mice with a premature aging disease, countering signs of aging and increasing the animals’ lifespan by 30 percent. The early-stage work provides insight both into the cellular drivers of aging and possible therapeutic approaches for improving human health and longevity.

Induction of reprogramming improved muscle regeneration in aged mice. Left. Impaired muscle repair in aged mice; rright
improved muscle regeneration in aged mice subjected to reprogramming.

“Our study shows that aging may not have to proceed in one single direction.” says Juan Carlos Izpisua Belmonte, a Professor in Salk’s Gene Expression Laboratory and senior author of the paper appearing in the December 15, 2016, issue of Cell. “It has plasticity and, with careful modulation, aging might be reversed.” As people in modern societies live longer, their risk of developing age-related diseases goes up. In fact, data shows that the biggest risk factor for heart disease, cancer and neurodegenerative disorders is simply age. One clue to halting or reversing aging lies in the study of cellular reprogramming, a process in which the expression of four genes known as the Yamanaka factors allows scientists to convert any cell into induced pluripotent stem cells or iPSCs. Like embryonic stem calls, iPSCs are capable of dividing indefinitely and becoming any cell type present in our body.

The Salk Institute researchers discover that partial cellular reprogramming reversed cellular signs of aging such as accumulation of
DNA damage. Left. Progeria mouse fibroblast cells; right, progeria mouse fibroblast cells rejuvenated by partial reprogramming.

“What we and other stem-cell labs have observed is that when you induce cellular reprogramming, cells look younger.” says Alejandro Ocampo, a research associate and first author of the paper. “The next question was whether we could induce this rejuvenation process in a live animal.” While cellular rejuvenation certainly sounds desirable, a process that works for laboratory cells is not necessarily a good idea for an entire organism. For one thing, although rapid cell division is critical in growing embryos, in adults such growth is one of the hallmarks of cancer. For another, having large numbers of cells revert back to embryonic status in an adult could result in organ failure, ultimately leading to death. For these reasons, the Salk team wondered whether they could avoid cancer and improve aging characteristics by inducing the Yamanaka factors for a short period of time.

To find out, the team turned to a rare genetic disease called progeria. Both mice and humans with progeria show many signs of aging including DNA damage, organ dysfunction and dramatically shortened lifespan. Moreover, the chemical marks on DNA responsible for the regulation of genes and protection of our genome, known as epigenetic marks, are prematurely dysregulated in progeria mice and humans. Importantly, epigenetic marks are modified during cellular reprogramming.

Using skin cells from mice with progeria, the team induced the Yamanaka factors for a short duration. When they examined the cells using standard laboratory methods, the cells showed reversal of multiple aging hallmarks without losing their skin-cell identity. “In other studies scientists have completely reprogrammed cells all the way back to a stem-cell-like state.” says co-first author Pradeep Reddy, also a Salk research associate. “But we show, for the first time, that by expressing these factors for a short duration you can maintain the cell’s identity while reversing age-associated hallmarks.”

Encouraged by this result, the team used the same short reprogramming method during cyclic periods in live mice with progeria. The results were striking: Compared to untreated mice, the reprogrammed mice looked younger; their cardiovascular and other organ function improved and, most surprising of all, they lived 30 percent longer, yet did not develop cancer. On a cellular level, the animals showed the recovery of molecular aging hallmarks that are affected not only in progeria, but also in normal aging.

“This work shows that epigenetic changes are at least partially driving aging.” says co-first author Paloma Martinez-Redondo, another Salk research associate. “It gives us exciting insights into which pathways could be targeted to delay cellular aging.” Lastly, the Salk scientists turned their efforts to normal, aged mice. In these animals, the cyclic induction of the Yamanaka factors led to improvement in the regeneration capacity of pancreas and muscle. In this case, injured pancreas and muscle healed faster in aged mice that were reprogrammed, indicating a clear improvement in the quality of life by cellular reprogramming.

“Obviously, mice are not humans and we know it will be much more complex to rejuvenate a person.” says Izpisua Belmonte. “But this study shows that aging is a very dynamic and plastic process, and therefore, will be more amenable to therapeutic interventions than what we previously thought.” The Salk researchers believe that induction of epigenetic changes via chemicals or small molecules may be the most promising approach to achieve rejuvenation in humans. However, they caution that, due to the complexity of aging, these therapies may take up to 10 years to reach clinical trials.

Other authors included: Aida Platero-Luengo, Fumiyuki Hatanaka, Tomoaki Hishida, Mo Li, David Lam, Masakazu Kurita, Ergin Beyret, Toshikazu Araoka, Eric Vazquez-Ferrer, David Donoso, Jose Luis Roman, Jinna Xu and Concepcion Rodriguez of the Salk Institute; Estrella Nuñez Delicado of Universidad Católica San Antonio de Murcia; Gabriel Núñez of the University of Michigan Medical School; Josep Maria Campistol of Hosplital Clinic of Barcelona and Isabel Guillén and Pedro Guillén of Fundación Dr. Pedro Guillén.

The work and the researchers involved were supported in part by a National Institutes of Health Ruth L. Kirschstein National Research Service Award Individual Postdoctoral Fellowship, the Muscular Dystrophy Association, Fundación Alfonso Martin Escudero, the Hewitt Foundation, the Uehara Memorial Foundation, the Nomis Foundation, a JSPS Postdoctoral Fellowship for Research Abroad, the University of California, San Diego, the G. Harold and Leila Y. Mathers Charitable Foundation, The Leona M. and Harry B. Helmsley Charitable Trust (2012-PG-MED002), The Glenn Foundation, Universidad Católica San Antonio de Murcia (UCAM) and Fundación Dr. Pedro Guillén. ω.

Images: The Salk Institute

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Larger Brain Size Linked to Longer Life in Deer

Image: Salla, Lapland, Finland

|| December 15: 2016: University of Cambridge News || ά. The size of a female animals' brain may determine whether they live longer and have more healthy offspring, according to new research led by the University of Cambridge. The study, published in the Royal Society Open Science journal, shows that female red deer with larger brains live longer and have more surviving offspring than those with smaller brains. Brain size is heritable and is passed down through the generations. This is the first extensive study of individual differences in brain size in wild mammals and draws on data comparing seven generations of deer.

Across species of mammals, brain size varies widely. This is thought to be a consequence of specific differences in the benefits and costs of a larger brain. Mammals with larger brains may, for example, have greater cognitive abilities that enable them to adapt better to environmental changes or they may have longer lifespans. But there may also be disadvantages: for instance, larger brains require more energy, so individuals that possess them may show reduced fertility.

The researchers, based at the University of Cambridge's Zoology Department and Edinburgh University's Institute of Evolutionary Biology, wanted to test if they could find more direct genetic or non-genetic evidence of the costs and benefits of large brain size by comparing the longevity and survival of individuals of the same species with different sized brains.

Using the skulls of 1,314 wild red deer whose life histories and breeding success had been monitored in the course of a long-term study on the Isle of Rum, they found that females with larger endocranial volumes lived longer and produced more surviving offspring in the course of their lives.

Lead author Dr Corina Logan, a Gates Cambridge Scholar and Leverhulme Early Career Research Fellow in Cambridge's Department of Zoology, says, ''The reasons for the association between brain size and longevity are not known, but other studies have suggested that larger brains are a consequence of the longer-lived species having longer developmental periods in which the brain can grow.

These hypotheses were generated from cross-species correlations; however, testing such hypotheses requires investigations at the within-species level, which is what we did."

Dr Logan adds: "We found that some of the cross-species predictions about brain size held for female red deer, and that none of the predictions were supported in male red deer. This indicates that each sex likely experiences its own set of trade-offs with regard to brain size.”

The study also showed that females' relative endocranial volume is smaller than that of males, despite evidence of selection for larger brains in females.

"We think this is likely due to sex differences in the costs and benefits related to larger brains," adds Dr Logan. "We don’t know what kinds of trade-offs each sex might encounter, but we assume there must be variables that constrain brain size that are sex specific, which is why we see selection in females, but not males."

Professor Tim Clutton-Brock, who set up the Rum Red Deer study with Fiona Guinness in 1972 and initiated the work on brain size, points out that the reason that this kind of study has not been conducted before is that it requires long term records of a large number of individuals across multiple generations and data of this kind are still rare in wild animals.

Reference: C.J. Logan, R. Stanley, A.M. Thompson, T.H. Clutton-Brock. Endocranial volume is heritable and is associated with longevity and fitness in a wild mammal. Royal Society Open Science; 14 Dec 2016; 10.1098/rsos.160622

:The text in this piece is by University of Cambridge and is licensed under a Creative Commons Attribution 4.0 International License:  ω.

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Mother Nature's Precision Bio-Genetic Engineering at Work: Scientists Discover Safety Catch Within All Dividing Cells

The above image is a composite made of images by the ICR and the inset one by Professor Chris Bakal


|| December 12: 2016: The Institute of Cancer Research London News || ά. Researchers have made a major discovery about how cells control when to divide, representing a step forward in scientists’ understanding of one of the most fundamental processes of life. Their study has revealed a ‘safety catch’ within cells that prevents them from dividing until DNA is allocated equally to the two daughter cells. It could lead to new treatments that prevent cancer cells from dividing, or kill them by forcing them to divide prematurely.

A research team from The Institute of Cancer Research, London, The University of Cambridge and University College Dublin set out to reveal the role of a key part of the cellular machinery which helps to govern cell division. The study is published on Thursday, December 08 in the journal Molecular Cell and was funded by Cancer Research UK, Wellcome, Science Foundation Ireland and the European Union. The researchers examined the role of a molecule called BubR1 in mitosis, the process during which a cell copies its chromosomes and pulls them apart into two separate cells.

BubR1 forms part of a molecular machine which prevents cells from dividing until they are ready by stopping the two sets of chromosomes from being pulled apart. The team focused in on a small part of BubR1 which has been conserved across evolution in all the kingdoms of life except bacteria, pointing to a fundamental function.

They removed normal BubR1 from cells, replacing it with an altered form which was misshapen in the crucial area. They then timed how long cells with mutant BubR1 could be held up in mitosis using time-lapse photography on high-powered microscopes. The researchers found that cells with mutant BubR1 were unable to delay in mitosis as normal, meaning chromosomes were divided unevenly between daughter cells.

They concluded that the crucial part of BubR1 they were studying, which they called the ABBA sequence, acts as a safety catch, preventing the progression of mitosis until the chromosomes are properly positioned to be pulled apart. Cancer cells rely on this safety catch much more than normal cells because they often have more chromosomes to get into place and so need more time in mitosis.

It might be possible to treat cancer by rushing cancer cells into premature division and potentially killing them by causing fatal errors within them. Some drugs that force cancer cells into premature division are already undergoing clinical trials. The new study shows that new classes of small-molecule drugs that switch off BubR1’s safety catch could also be developed in the future.

Professor Jon Pines, Head of Cancer Biology at the ICR, said, “Our study has found a ‘safety catch’ in the cell division machinery, which prevents cells from dividing before they have confirmed that their chromosomes have been successfully aligned in the cell.

In the future it might be possible to disable this safety catch in cancer cells with drugs, which would force cells into dividing before they are ready, and potentially kill them by introducing major errors into the division process.”

Professor Paul Workman, Chief Executive of the ICR, said, “Unravelling the complexity of cell division is fundamental to understanding cancer. We know cells rely on safety mechanisms to prevent them from dividing before they are ready, and these may be particularly crucial in cancer cells, with their complex, unstable genomes and extra chromosomes.

If we could find a way to inactivate this safety catch, we might be able to kill cancer cells specifically by forcing them through division prematurely.”

Dr Áine McCarthy, Cancer Research UK’s Senior Science Communication Officer, said, “By looking at how cells divide, these scientists have identified a potential new way to destroy cancer cells, which could in the future lead to the development of new anti-cancer drugs. Early-stage studies like this highlight the importance of carrying out fundamental research which increases our understanding of how cells work, without this, no new treatments can be developed.”

Enlighten Universana The Humanion Beacon Organisations: The Institute of Cancer Research London

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Human Cells with a Built-in Circuit Help Prevent Tumour Growth

Professor Ali Tavassoli: Image: University of Southampton

|| November 27: 2016: University of Southampton News || ά.  Researchers at the University of Southampton have engineered cells with a ‘built-in genetic circuit’ that produces a molecule that inhibits the ability of tumours to survive and grow in their low oxygen environment. The genetic circuit produces the machinery necessary for the production of a compound that inhibits a protein which has a significant and critical role in the growth and survival of cancer cells. This results in the cancer cells being unable to survive in the low oxygen, low nutrient tumour micro-environment.

As tumours develop and grow, they rapidly outstrip the supply of oxygen delivered by existing blood vessels. This results in cancer cells needing to adapt to low oxygen environment. To enable them to survive, adapt and grow in the low-oxygen or ‘hypoxic’ environments, tumours contain increased levels of a protein called Hypoxia-inducible factor 1:HIF-1. HIF-1 senses reduced oxygen levels and triggers many changes in cellular function, including a changed metabolism and sending signals for the formation of new blood vessels. It is thought that tumours primarily hijack the function of this protein:HIF-1 to survival and grow.

Professor Ali Tavassoli, who led the study with colleague Dr. Ishna Mistry, explains: “In an effort to better understand the role of HIF-1 in cancer, and to demonstrate the potential for inhibiting this protein in cancer therapy, we engineered a human cell line with an additional genetic circuit that produces the HIF-1 inhibiting molecule when placed in a hypoxic environment.

“We’ve been able to show that the engineered cells produce the HIF-1 inhibitor, and this molecule goes on to inhibit HIF-1 function in cells, limiting the ability of these cells to survive and grow in a nutrient-limited environment as expected.

“In a wider sense, we have given these engineered cells the ability to fight back, to stop a key protein from functioning in cancer cells. This opens up the possibility for the production and use of sentinel circuits, which produce other bioactive compounds in response to environmental or cellular changes, to target a range of diseases including cancer.”

The genetic circuit is incorporated onto the chromosome of a human cell line, which encodes the protein machinery required for the production of their cyclic peptide HIF-1 inhibitor. The production of the HIF-1 inhibitor occurs in response to hypoxia in these cells. The research team demonstrated that even when produced directly in cells, this molecule still prevents the HIF-1 signalling and the associated adaptation to hypoxia in these cells.

The next step for the researchers is to demonstrate the viability of this approach to the production and delivery of an anticancer molecule in a whole tumour model system.

Professor Tavassoli adds: “The main application for this work is that it eliminates the need for the synthesis of our inhibitor, so that biologists conducting research into HIF function can easily access our molecule and hopefully discover more about the role of HIF-1 in cancer. This will also let us understand whether inhibiting HIF-1 function alone is enough to block cancer growth in relevant models.

Another interesting aspect to the work is that it demonstrates the possibility of adding new machinery to human cells to enable them to make therapeutic agents in response to disease signals.”

The study, which was funded by Cancer Research UK and the Engineering and Physical Sciences Research Council, is published in the journal ACS Synthetic Biology.

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Looking Into The Epigenesis: And This is What The Humanion Means When It Says: Symphonic Work

Detail of Epigenome: Image: haha works

|| November 18: 2016: University of Cambridge News || ά. University of Cambridge researchers have played a leading role in several studies released today looking at how variation in and potentially heritable changes to our DNA, known as epigenetic modifications, affect blood and immune cells, and how this can lead to disease. The studies are part of BLUEPRINT, a large-scale research project bringing together 42 leading European universities, research institutes and industry entrepreneurs, with close to €30 million of funding from the EU. BLUEPRINT scientists have this week released a collection of 26 publications, part of a package of 41 publications being released by the International Human Epigenome Consortium.

One of the great mysteries in biology is how the many different cell types that make up our bodies are derived from a single stem cell and how information encoded in different parts of our genome are made available to be used by different cell types. Scientists have learned a lot from studying the human genome, but have only partially unveiled the processes underlying cell determination. The identity of each cell type is largely defined by an instructive layer of molecular annotations on top of the genome, the epigenome, which acts as a blueprint unique to each cell type and developmental stage. Unlike the genome, the epigenome changes as cells develop and in response to changes in the environment. Defects in the proteins that read, write and erase the epigenetic information are involved in many diseases.

The comprehensive analysis of the epigenomes of healthy and abnormal cells will facilitate new ways to diagnose and treat various diseases, and ultimately lead to improved health outcomes. “This huge release of research papers will help transform our understanding of blood-related and autoimmune diseases.” says Professor Willem H Ouwehand from the Department of Haematology at the University of Cambridge, one of the Principal Investigators of BLUEPRINT. “BLUEPRINT shows the power of collaboration among scientists across Europe in making a difference to our knowledge of how epigenetic changes impact on our health.”

Among the papers led by Cambridge researchers, Professor Nicole Soranzo and Dr Adam Butterworth have co-led a study analysing the effect of genetic variants in our DNA sequence on our blood cells. Using a genome-wide association analysis, the team identified more than 2,700 variants that affect blood cells, including hundreds of rare genetic variants that have far larger effects on the formation of blood cells than the common ones. Interestingly, they found genetic links between the effects of these variants and autoimmune diseases, schizophrenia and coronary heart disease, thereby providing new insights into the causes of these diseases.

A second study led by Professor Soranzo looked at the contribution of genetic and epigenetic factors to different immune cell characteristics in the largest cohort of this kind created with blood donors from the NHS Blood and Transplant centre in Cambridge.

Dr Mattia Frontini and Dr Chris Wallace, together with scientists at the Babraham Institute, have jointly led a third study mapping the regions of the genome that interact with genes in 17 different blood cell types. By creating an atlas of links between genes and the remote regions that regulate them in each cell type, they have been able to uncover thousands of genes affected by DNA modifications, pointing to their roles in diseases such as rheumatoid arthritis and other types of autoimmune disease.

Dr Frontini has also co-led a study with BLUEPRINT colleagues from the University of Vienna that has developed a reference map of how epigenetic changes to DNA can programme haematopoietic stem cells, a particular type of ‘master cell’, to develop into the different types of blood and immune cells.

Professor Jeremy Pearson, Associate Medical Director at the British Heart Foundation, which helped fund the research, said: “Our genes are critical to our health and there’s still a wealth of information hidden in our genetic code. By taking advantage of a large scale international collaboration, involving the combined expertise of dozens of research groups, these unprecedented studies have uncovered potentially crucial knowledge for the development of new life saving treatments for heart disease and many other deadly conditions.

Collaborations like this, which rely on funding from the public through charities and governments across the globe, are vital for analysing and understanding the secrets of our genetics. Research of this kind is helping us to beat disease and improve millions of lives.”

Departmental Affiliations
Professor Nicole Soranzo: Department of Haematology
Dr Adam Butterworth: Medical Research Council:MRC:British Heart Foundation:BHF:Cardiovascular Epidemiology Unit
Dr Mattia Frontini: Department of Haematology, and Senior Research Fellow for the BHF Cambridge Centre for Research Excellence
Dr Chris Wallace: Department of Medicine and MRC Biostatistics Unit

References: Astle, WJ et al. The allelic landscape of human blood cell trait variation. Cell; 17 Nov 2016; DOI: 10.1016/j.cell.2016.10.042
Chen, L et al. Genetic drivers of epigenetic and transcriptional variation in human immune cells. Cell; 17 Nov 2016; DOI: 10.1371/journal.pbio.0000051
Javierre et al. Lineage-specific genome architecture links enhancers and non-coding disease variants to target gene promoters. Cell; 17 Nov 2016; DOI: 10.1016/j.cell.2016.09.037
Farlik et al. Cell Stem Cell; 17 Nov 2016; DOI: 10.1016/j.stem.2016.10.019

:The text in this piece is by University of Cambridge and is licensed under a Creative Commons Attribution 4.0 International License:  ω.

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Stem Cells Provide Sound in Vitro Models for Deafness: Juntendo University Research

Schematic of the culture of connexin 26 expressing gap junction plaque forming cells
and the recapitulation of disease symptoms in cells cultured from connexion
deficient mice. Cx26, connexin 26; Cx30, connexin 30; GJ, gap junction. Courtesy Stem Cell Reports


|| November 11: 2016: Juntendo University: Japan || ά. A collaboration, including researchers from Juntendo University, demonstrate differentiation from stem cells into specialised cells thought to be the most important therapeutic target for the treatment of hereditary deafness. One in a thousand children suffers deafness or hearing loss, and hearing is the most common sense to be affected by congenital disease. Deafness at birth is often caused by mutations in a specific gene known as Gap Junction Beta 2:GJB2, which codes for the protein connexin 26. In some populations mutations of this gene are responsible for as many as half the instances of congenital hearing loss.

 Now, Kazusaku Kamiya and the co-authors of his recent report demonstrate a means of producing supplies of these cells on demand for use in therapeutic studies. “Human cochlear cells are not readily accessible for biopsy or direct drug administration because of anatomical limitations.” state the researchers in their report. “Therefore, ES:iPS, embryo stem:induced pluripotent stem, cells are an important tool for studying the molecular mechanisms underlying inner-ear pathology as well as for generating cells for replacement therapies.”

We are Juntendo University Japan: Image:JUP

To culture the cells the researchers followed standard protocol for the first seven days at which point specific proteins were added to increase mRNA expression of connexins. On day 07-11 the cells were transferred to a flat 2D culture with inner-ear cells that are especially resistant to enzymes that break down proteins. They successfully cultured induced pluripotent stem cells that differentiated into gap junction plaque cells expressing connexin 26.

The researchers were also able to demonstrate that their stem-cell-derived gap junction cells were functionally and structurally characteristic of developing cochlear cells. Importantly the cells differentiated from mice that were deficient in connexin 26 reproduced cellular characteristics of congenital hearing loss. The researchers conclude, “It is expected, then, that these iPS derived cells, which can be obtained from patients, will be particularly useful for drug screening and inner-ear cell therapies targeting GJB2-related hearing loss.”

Stem Cells: Stem cells are a type of cell that can change into another type of more specialised cell through a process described as differentiation. They occur in embryos, embryonic stem cells, and adults as repair cells. Embryonic stem cells can differentiate into several different types of specialised cells to form the range of cells needed in the human body. The ability to differentiate into several different types of cell is described as pluripotency and can be induced in adult cells as well by reprogramming non-reproductive system cells or somatic cells to produce “induced pluripotent stem cells”.

Structure of the Ear: The ear comprises three main parts: outer, middle and inner. The ear canal in outer ear channels sound vibrations to the ear drum in the middle ear. The middle ear contains three bones or ossicles that transfer the vibrations of the ear drum to the cochlea, a fluid filled spiral cavity in the inner ear. The movement of the fluid in the cochlea in response to these vibrations is detected by thousands of hair cells in the cochlea that convert this motion into electrical signals that are then communicated by nerve cells to the brain, which senses them as sound.

Function of Connexin 26 and Gap Junction Plaques: Connexins 26 and 30 form gap junctions that facilitate the movement of ions needed to maintain a balance in conditions, homeostasis, as well as developmental organisation in the cochlea. The researchers were able to demonstrate that their stem-cell-derived gap junction cells were functional for forming gap junction intercellular communication networks typical of the developing cochlea. The cells differentiated from mice that were deficient in connexin 26 demonstrated a disruption in the formation of gap junction plaques.

Reference: Ichiro Fukunaga1, 2, Ayumi Fujimoto1, Kaori Hatakeyama1, Toru Aoki1, Atena Nishikawa1, Tetsuo Noda3, 4, Osamu Minowa3, 4, Nagomi Kurebayashi5, Katsuhisa Ikeda1, Kazusaku

Kamiya1, In vitro models of GJB2-related hearing loss recapitulate Ca2+ transients via a gap junction characteristic of developing cochlea, Stem Cell Reports, Published online November 11, 2016. DOI: 10.1016/j.stemcr.2016.10.005

Department of Otorhinolaryngology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan
Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan
Department of Cell Biology, Japanese Foundation for Cancer Research, Cancer Institute, Tokyo 135-8550, Japan
Team for Advanced Development and Evaluation of Human Disease Models, RIKEN BioResource Center, Tsukuba 305-0074, Japan
Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan

Corresponding author e-mail: kkamiya at

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Differences in Wiring of “Exhausted” and Effective T-Cells Indicate Possible Gene-Editing Targets

Image: Dana Farber Boston Children's Cancer and Blood Disorders Centre

|| October 31: 2016: Dana Farber Boston Children's Cancer and Blood Disorders Centre News || ά. In people with chronic infections or cancer, disease-fighting T-cells tend to behave like an overworked militia, wheezing, ill-prepared, tentative, in a state of “exhaustion” that allows disease to persist. In a paper posted online today by the journal Science, researchers at Dana-Farber:Boston Children’s Cancer and Blood Disorders Centre report that, in mice with chronic viral infection, exhausted T-cells are controlled by a fundamentally different set of molecular circuits than T-cells effectively battling infections or cancer, a finding that suggests a way to increase the staying power of CAR T-cells, a promising form of immunotherapy for cancer.

An accompanying study led by researchers at the University of Pennsylvania and co-authored by Dana-Farber scientists reports that these differences in circuitry remain largely unchanged by a type of cancer immunotherapy known as checkpoint inhibition, potentially closing off one avenue of improving this technique. The pair of studies bring renewed focus to the epigenetics of T-cells, the multilayered system of molecular switches, accelerators, and throttles that controls the activity of genes. Scientists have known for years that the pattern of genes is different in exhausted T-cells than in functional T-cells that are fully engaged in fighting disease, but the actual extent of these differences has been uncertain.

One difference that is clear is that exhausted T-cells express the programmed cell death protein-1:PD-1, which commands them not to attack normal, healthy cells, but can also prevent them from striking at cancerous or chronically infected cells. Blocking PD-1 with checkpoint-inhibiting drugs, and thereby restoring the cancer-killing zeal of T-cells, has become one of the most successful new approaches to cancer treatment in nearly a decade. However, it has shown effectiveness in only about a quarter of cases.

“Exhausted T-cells display a variety of functional defects.” says Nicholas Haining, MD, of Dana-Farber:Boston Children’s, senior author of the new paper. “They are paralysed and don’t have the fire-power to destroy cancer or virally-infected cells. For us, the question in this study was, do exhausted cells represent a distinct type of T-cell or are they merely a ‘groggy’ version of functional T-cells?”

With chronically infected mice as their model, the researchers used a new technology called ATAC-seq to map the regulatory regions of the genome,  the sections of DNA involved in switching genes on and off, in the animals’ exhausted and functional CD8+ T cells. CD8+ T-cells are programmed to identify and eliminate cancerous and infected cells.

“We found the landscape of regulatory regions to be fundamentally different in exhausted and functional T-cells.” Haining says. “There were thousands of instances where a regulatory region appeared in exhausted T-cells but not in their functional counterparts, and vice versa. This tells us that the two types of cells use very different wiring diagrams to control their gene activity.”

The researchers then tested whether removing a regulatory stretch of DNA that spurs the production of PD-1 would drive down expression of the protein. Using CRISPR:Cas9 technology, they snipped out that region and indeed, PD-1 expression dropped.

The success of this experiment may offer the key to improving CAR T-cell therapy. CAR T-cells are T-cells that are removed from a patient, genetically engineered to grow a protein “sensor” that targets them to tumour cells, and then re-injected into the patient. Although the retrofitted T-cells have demonstrated effectiveness at tracking down cancer cells, particularly in leukemia, one of the shortcomings of CAR T-cells is that they tend to become exhausted.

The work described in the new study suggests that while T-cells are being engineered to produce the sensor, they could also be re-tooled to delete the genetic wiring that causes them to express excessive levels of PD-1 or other exhaustion genes. The resulting CAR T-cells would not only be better at stalking cancer, but also more aggressive about attacking it.

In the companion paper, researchers explored whether blocking the PD-1 checkpoint rewired exhausted T-cells to make them, from an epigenetic standpoint, more like functional T-cells. Using chronically infected mouse models, as in the first study, the investigators found that while such gain of function does occur briefly, the epigenetic switches from its previous, exhausted state remain largely unchanged.

“This suggests that the benefits achieved by checkpoint blockade result from a transient revving up of exhausted T-cells, not a permanent reshaping of their state.” Haining says.

The findings of the two studies point to the need for a comprehensive atlas of the regulatory regions that are active in exhausted and functional T-cells, he continues. Such a guide would provide targets for rewiring T-cells with genetic engineering or epigenetic drugs to make them more effective cancer killers.

The first authors of the study are Debattama R. Sen of Dana-Farber and James Kaminski of the University of California, Berkeley. Co-authors are R. Anthony Barnitz, PhD, Ulrike Gerdemann, MD, PhD, Kathleen B. Yates, Hsiao-Wei Tsao, PhD, Jernej Godec, PhD, Martin W. LaFleur, Flavian D. Brown, of Dana-Farber; Makoto Kurachi, PhD, and E. John Wherry, PhD, of the University of Pennsylvania; Pierre Tonnerre, PhD, Raymond T. Chung, MD, and Georg M. Lauer, MD, PhD, of Massachusetts General Hospital; Damien C. Tully, PhD, and Todd M. Allen, PhD, of the Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University; and Nicole Frahm, PhD, of the Fred Hutchinson Cancer Research Center.

The research was supported by the National Institutes of Health and the BRAIN Initiative.

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What Determines the Size of Human Cells

Image shows a single human Jurkat cell stained for mitochondria, green, and plasma
membrane, red. Image: University of Dundee

|| October 22: 2016: University of Dundee News || ά. New research from the University of Dundee has discovered that cells of average size are the Olympic athletes of the cellular world, performing better than those which are too big or two small. When cells and tissues are observed under the microscope, the size of the cells is one of the most obvious features. However, while cells are small in general, cell size varies from one cell type to another; a muscle cell, for example, is much bigger than a white blood cell. The research by the team at the School of Life Sciences at the University of Dundee now sheds light on the question of why animal cells are of certain size.

It has been recognised for more than one hundred years that metabolic activity declines with increasing organismal size, a process called metabolic allometry. For example, two smaller dogs will consume more food, energy than one bigger dog of similar mass. In a new study, published in the journal Developmental Cell, the Dundee researchers studied metabolic allometry at the cellular level, focusing on mitochondria, the `power generators’ of the cell which are involved in producing energy and cellular building blocks necessary for growth. Dr Teemu Miettinen, one of the research team, said, “Mitochondria are key organelles setting the overall metabolic activity of cells. What we find is that there is a big difference between the amount of mitochondria and how active they are.

While the mitochondrial amount increases with cell size, as was expected, there is a decline in mitochondrial activity with increasing cell size. This appears to limit cells from growing too large. It appears there is a substantial benefit for cells from growing sufficiently but not too large. The mitochondria in intermediate sized cells are more active, helping cells to perform better.

This provides cells of intermediate size a `fitness advantage’. This fitness advantage is probably best illustrated by comparing cells to athletes. Starved or too fat athletes do not perform optimally in sports. The same applies to cells, where those of average size are the peak, Olympic-level performers.”

The findings suggest that the maintenance of cell size within certain limits may be important for the survival and reproductive potential of cells and organisms. Cells need to be able to actively adjust their size to maintain their optimal cellular function to maximise the success of the whole organism.

Another key implication of this work is that problems in controlling growth and cell size may directly relate to the development of metabolic disease. Cellular ageing, for example, is associated with an increase in cell size and decrease in mitochondrial functionality.

“This work suggests the possibility that loss of cell size control leads to mitochondrial dysfunction, which has been linked to a variety of diseases, including neurodegenerative and metabolic disorders.” said Dr Mikael Bjorklund, another of the research team. “It may be that learning how cells sense their physical dimensions and linking it with their metabolic activity can help us to better understand these conditions.”

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TB Tricks the Body’s Immune System to Allow It to Spread

CT scan showing inflammation and lung destruction in a TB lung

|| October 21: 2016: University of Southampton News || ά. Tuberculosis:TB tricks the immune system into attacking the body’s lung tissue so the bacteria are allowed to spread to other people, new research from the University of Southampton suggests. The concept, published in Trends in Immunology, proposes that current ideas about how TB develops in patients may be incomplete and that, in fact, infection causes autoimmunity, where the immune system reacts incorrectly to its own tissue.

TB kills more people than any other infectious disease, and the causative bacterium, Mycobacterium tuberculosis, is becoming increasingly resistant to antibiotics used to treat the infection. The Southampton research team conducted a review of published studies and found evidence suggesting that an autoimmunity process develops in TB. Professor Paul Elkington, of the University of Southampton, who led the project, said: “We are not disputing that the immune system mainly targets the bacteria to fight it off, but we are suggesting that there is more to the story.

It seems that TB tricks the immune system into damaging our own lung tissue, which, therefore, makes the person highly infectious through coughing and the TB then spreads by aerosol droplets to other individuals.

There is also a group of patients who develop a range of symptoms, such as eye inflammation, joint inflammation and skin rashes, that are not explained by current TB disease concepts. These symptoms are usually associated with diseases like rheumatoid arthritis and Crohn’s disease, which led us to believe autoimmunity plays a key role in the TB disease process.”

Professor Elkington highlights that more research is needed to investigate the hypothesis but if it is proved, the discovery could have major implications for the design of new vaccines and drug treatments.

The Southampton team are now undertaking a programme of work to investigate this new concept. Their approach is to combine the study of cells isolated from TB-infected patients with micro-engineering in 3D in the laboratory to investigate how TB damages the lungs.

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Secondary Breast and Bowel Cancers Found to Grow by Piggy-Backing on Patients’ Blood Vessels

The British Heart Foundation Photography Competition 2016: Dr Simone Rivolo: The Heart with Its Intricate
Artworks of Blood Vessels: Kings College London. Blood vessels embedded within the heart muscle are shown in bright colours. These
vessels play a crucial role in delivering oxygen and nutrients to the heart muscle. The image shows a horizontal slice
of the heart with the embedded blood vessels, which were imaged and reconstructed at high resolution.
The colours show how deep the vessels are embedded within the heart wall, which is related to
the compression these vessels endure throughout each heartbeat.


|| October 19: 2016: The ICR London News || ά. New research into breast and bowel cancers that have spread to the liver shows that some tumours power their growth using pre-existing blood vessels rather than developing new ones. The results help to explain why existing treatments which block the development of new blood vessels have so far failed to have the desired effect of slowing the growth of secondary breast cancer tumours, according to a major new paper published today in the journal Nature Medicine. The researchers believe their findings could also be applicable to a range of cancers that have the potential to spread to the liver and other sites.

Breast Cancer Now-funded scientists at The Institute of Cancer Research, London, looked at whether secondary breast and bowel cancers occurring in the liver used pre-existing blood vessels to grow, known as vessel co-option, or grew new blood vessels, a process called angiogenesis. Previously, scientists thought all cancers needed to establish new blood vessels in order to grow, and several anti-angiogenesis drugs have been developed to block this process. The researchers, working within the Breast Cancer Now Toby Robins Research Centre at the ICR, studied clinical samples of secondary tumours occurring in the liver of breast cancer and bowel cancer patients.

Using 187 samples from 92 bowel cancer patients, who received treatment with the anti-angiogenesis drug Avastin, also known as bevacizumab, before having their liver tumours removed, the researchers found that around 40% of the secondary tumours obtained a blood supply predominantly through vessel co-option, while the remaining tumours relied more on angiogenesis or used a mixture of angiogenesis and vessel co-option. They also showed, for the first time, that the tumours which mainly used vessel co-option responded poorly to combined treatment with Avastin and chemotherapy.

The study also looked at secondary tumours in the liver from a group of 17 patients with breast cancer. Removing secondary breast cancers that have spread to the liver is not common practice and so these tumours have not been extensively studied in terms of how they recruit blood vessels. Notably, the researchers found that 16 out of the 17 tumours examined used vessel co-option to obtain a blood supply. The scientists noted that other studies have shown that secondary breast tumours occurring in the brain, lungs, skin and lymph nodes can also rely on pre-existing vessels for their blood supply.

Researchers also looked at whether it was possible to block the vessel co-option process in secondary tumours occurring in the liver, and combined this approach with an anti-angiogenesis drug, to test the potential of a two-pronged approach to treatment. Using mice, they found that when they ‘switched off’ a gene which helps cancer cells to move towards existing blood vessels and co-opt them, and then treated the tumours with an anti-angiogenesis drug, there was significantly less tumour growth than treating tumours with the anti-angiogenesis drug alone.

Dr Andrew Reynolds, who led the research and is a Team Leader in Tumour Biology at the ICR, said: “It was thought for a long time that cancers that have spread to the liver must generate new blood vessels to thrive but now we’ve shown that they can piggy-back on pre-existing vessels instead. Our work helps to explain why drugs designed to target new blood vessel growth in secondary tumours do not always work as well in patients as was originally hoped for. And we found that this use of pre-existing vessels was especially common in breast cancers that had spread to liver.

We now need to undertake more research to understand exactly how tumours recruit pre-existing blood vessels. Our study has suggested that drug combinations that block both vessel co-option and angiogenesis could potentially offer hope as future treatments, and I look forward to this possibility being explored in follow-up studies.”

Baroness Delyth Morgan, Chief Executive at Breast Cancer Now, said: “This research shows how breast cancers use patients’ pre-existing blood vessels in order to grow after they have spread to different parts of the body. It could lead to the development of new, more effective treatments for the disease. Secondary breast cancer is currently incurable, so research that gives us new hope by helping us understand how secondary tumours develop and thrive is a powerful tool in the battle against this disease.

If we are to reach our goal of ensuring that by 2050, all those who develop breast cancer will live, we need to build a picture of how the disease thrives after it spreads away from the breast, and use this to develop new therapies.''

The research also received NHS funding to the National Institute for Health Research Biomedical Research Centre at The Royal Marsden and the ICR, as well as support from the Liver Disease Biobank, Montreal, De Stichting tegen Kanker, Antwerp. Breast Cancer Now funding includes support from Avon through the ‘Dr Avon’ Clinical Fellowship scheme.

The research was a multidisciplinary collaboration, led by Dr Reynolds from the ICR, and involved an international team of scientists, including Professor David Cunningham from The Royal Marsden Hospital, London, Professor Peter Metrakos from McGill University Health Centre, Montreal and Dr Peter Vermeulen and Professor Luc Dirix from GZA Hospitals St. Augustinus, Antwerp who all provided tumour samples from patients for the study. ω.

Enlighten Universana The Humanion Beacon Organisations: The Institute of Cancer Research London

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A Rapid and Versatile Mechanism That Modifies Proteins is Crucial for the Evolutionary Process

|| October 19: 2016: The ICR London News || ά. Research led by the European Bioinformatics Institute:EMBL-EBI and the University of Washington has shown that the biological diversity needed for evolution can be generated by changes in protein modifications. The findings, published today in Science, provide valuable insights into how different species adapt to different environments and could shed light on how pathogens evolve and become resistant to drugs.

“This study is about understanding how evolution works, which tells you how species adapt to changing environments over many generations,” says Pedro Beltrao, a research group leader at EMBL-EBI. “For example, when you compare humans and chimps, they are obviously different, even though a good part of their genetic makeup is more or less the same. Our task is to figure out how diversity is generated, so that we can see in detail how life evolves. That helps us understand how plants and animals adapt and change, and how cancers or bacteria find their way around drugs.”

A Question of Expression

Research into the drivers of genetic diversity has largely focused on gene expression, which controls how much of a given protein will be made, when, and in what tissue. However, the researchers found that a well-known cellular mechanism, one that controls how proteins acquire new functions, also plays a major role. Proteins are controlled by other proteins by way of ‘post-translational modification':PTM. One type of PTM is phosphorylation: a rapid, versatile protein-regulation mechanism. During evolution PTMs can be acquired via mutations, which allows proteins to gain new functions, turn on or off at different times, and go to different places in the cell.

Previous studies comparing proteins in related species have shown very few mutations, so PTMs have not been considered to be a major factor in generating diversity. In today’s study, the group found that only a few mutations are actually required to change these protein-modification sites. In other words, a small number of changes can have a big impact on how proteins and cells work.

“These mutations were hidden in plain sight, we could see them all along, but didn’t know they could have such significant consequences,” says Beltrao. “We only see it now after many years of developing and refining new experimental methods.”

Change is the Constant

Using experimental and computational methods, the researchers reconstructed the evolutionary history of phosphorylation sites, the modifications that can control proteins, in 18 different single-celled species. They determined how long these control points have existed, when they were acquired and how quickly they have changed across species over millions of years.

The group found that most of the phosphorylation sites had come about relatively recently in evolution, indicating that they are part of what make the species different, and a major contributor to evolutionary diversity.

“If a species needs to adapt to a new setting, it needs to generate a lot of diversity over many generations so that evolution has a pool of options to select from. One way for that to happen is through changes in gene expression, but changes in phosphorylation are equally effective,” explains Beltrao.

Cancer: the Expert Lock Picker

The diversity generated by PTMs is an important consideration in tackling cancer. Some cancer drugs stop tumours by blocking the signalling pathway that allows the tumour to grow, effectively placing a lock on a protein ‘door’. But, through mutations, cancers find ways to create new PTMs and signalling events, effectively manufacturing millions of different keys. Most of the keys will be useless, but one is bound to fit the lock eventually, and the tumour can start growing again.

“Learning more about the role of PTMs in evolution also presents a much more reliable picture of how signalling proteins integrate and relay information inside the cell,” adds Beltrao. “This in turn could present exciting new avenues for therapeutic research.”

Source article: Studer RA, et al., 2016 Evolution of protein phosphorylation across 18 fungal species. Science in press; published online October 14. DOI: 10.1126/science.aaf2144

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New Method to Detect Ageing Cells and Aid Rejuvenation Therapies Developed by Researchers

Image: The University of Manchester

|| October 05: 2016: The University of Manchester News || ά. Scientists have discovered a new way to look for ageing cells across a wide range of biological materials; the new method will boost understanding of cellular development and ageing as well as the causes of diverse diseases. Cellular senescence is a fundamental biological process involved in every day embryonic and adult life, both good, for normal human development,  and, more importantly to researchers, dangerous by triggering disease conditions.

Up to now available senescence detecting biomarkers have very limited and burdensome application. Frustrated by the limitations of commercially available biomarkers, researchers led by The University of Manchester’s Professor Paul Townsend and senior author of the resulting paper, and honorary professor at Manchester, Professor Vassilis Gorgoulis, have developed a universally applicable method to assess senescence across biomedicine, from cancer research to gerontology.

Therefore, a more effective, precise and easy-to-use biomarker would have considerable benefits for research and clinical practice. ''The method we have developed provides unprecedented advantages over any other available senescence detection products – it is straight-forward, sensitive, specific and widely applicable, even by non-experienced users,” said Professor Townsend.

In addition to helping researchers make significant new breakthroughs into the causes of diseases, including cancer, through more effective understanding of senescence in cells, the new process will also aid the impact of emerging cellular rejuvenation therapies. By the better identification,  and subsequently elimination of – senescent cells, tissues can be rejuvenated and the health span extended.”

The research on the new methodology, published as ‘Robust, universal biomarker to detect senescent cells in biological specimens’ in the journal Ageing Cell, has led to two pending UK patents.

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New ‘Ecosystem’ Test Strongly Predicts Ovarian Cancer Survival

A high magnification image of ovarian clear cell carcinoma: Image: Nephron

|| September 24: 2016: Year Beta: Day One: The ICR London News || ά. Assessing the cell ‘ecosystems’ at sites where ovarian cancer has spread round the body strongly predicts the chances of surviving from the disease, a new study reports. A new computerised test using software designed to study plant and animal ecosystems measures diversity among cells in the environment around secondary tumours, or metastases, after cancer’s spread. Scientists at The Institute of Cancer Research, London, with collaborators in China, found a 'staggering' difference in survival rates between women with high and low levels of diversity at these metastatic sites.

The fully automated test could identify those women who have the most life-threatening disease, and who urgently need the most aggressive treatment. The test gives a score for metastasis diversity, known as MetDiv, based on whether a patient's sites of cancer spread have one dominant cell type, low score, or a more diverse cell population containing immune or connective tissue cells, high score. Survival was far poorer among women with high diversity scores than those with low scores. Just 09% of women with diverse metastases survived five years from diagnosis, compared with 42% of those whose metastases were dominated by one cell type. A high diversity score was a stronger predictor of poor survival than any of the clinical factors currently used to try to assess a woman’s prognosis.

The study was published on Monday, September 19, 2016 in the journal Oncotarget and was funded by the ICR, the Wellcome Trust and the National Institute for Health Research Biomedical Research Centre at The Royal Marsden and the ICR. The researchers analysed 192 secondary tumours that had spread to the area around the ovary, peritoneal cavity, lymph nodes or appendix, from 61 women with more than one metastasis treated at Sun Yat-sen University Cancer Centre in China.

Dr Yinyin Yuan, Team Leader in Computational Pathology at the ICR, said: “We used to think of tumours as simply a collection of cancer cells, but we now know that they are often complex ecosystems made up of different types of healthy cell, too. Our study has revealed that diverse cell populations at the sites of cancer spread are a clinically important feature of particularly aggressive ovarian cancers.

We have developed a new test to assess the diversity of metastatic sites, and use it to predict a woman’s chances of surviving their disease. More work is needed to refine our test and move it into the clinic, but in future it could be used to identify women with especially aggressive ovarian cancers, so they can be treated with the best possible therapies available on the NHS or through clinical trials.”

Professor Paul Workman, Chief Executive of the ICR, said: “Ovarian cancer is more likely to spread than many other cancers because there is no barrier between the ovaries and the peritoneal cavity, the fluid-filled space in our torso that houses the body’s organs. It’s, therefore, critical that we understand more about the likely progression of disease among cancers that have spread, and get better at tailoring our treatment for individual women.

Finding ways of treating highly aggressive ovarian cancers is a huge challenge. But by knowing that a woman has an especially lethal form of disease, we can look to explore aggressive combination treatments, and give women choices about the kinds of care they want to receive.”

Enlighten Universana The Humanion Beacon Organisations: The Institute of Cancer Research London

The ICR's campus in Sutton: The ICR London

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Between the Two Auroras Sings Life That We Could Never Fully Grasp and Say: Got It

The Aurora Borealis from a rooftop in Iceland: Image: Marco Ottobelli CC BY-SA 4.0

|| September 19: 2016: 2016: The Institute of Cancer Researc London News: Liz Burtally Writing || ά. Targeting a group of malfunctioning components of cell division named after the Northern Lights may present a new way to treat leukaemia. You wouldn’t usually associate the bright, dancing display of the Aurora Borealis, the Northern Lights, with cells dividing. A protein called Aurora got its name due to its localisation to the poles of the dividing cell, similar to the way the Aurora Borealis is observed at the poles of the earth.

Aurora kinases are essential for cell division and are often overactive or in high levels in cancer cells, which makes them an attractive target for cancer treatment. But, although, Aurora kinase small-molecule inhibitors have been around for a while now, it’s only recently that they have looked close to fulfilling their promise as potential cancer therapeutics. I caught up with Dr Spiros Linardopoulos, Leader of the Drug Target Discovery Team at The Institute of Cancer Research, London, who has recently written a review with Dr Vassilios Bavetsias on the progress of Aurora kinase inhibitors.

What are Aurora Kinases?

In humans, the Aurora kinase family consists of three members: Aurora-A, Aurora-B, and Aurora-C. Together they form an important group of enzymes that regulate many processes in cell division, including helping the dividing cell accurately dispense genetic information into two daughter cells. If Aurora kinase signalling goes wrong, the cell’s chromosomes can be distributed unevenly, which is a dangerous state that can lead to cancer. And sure enough, Aurora kinase genes have been found to be overactive in a broad range of human tumours, including breast and ovarian cancers, gliomas, pancreatic cancers and haematological cancers.

Aurora kinase inhibitors were initially designed to target solid tumours including ovarian, breast, lung and bowel cancer. The clinical trials in these cancers were disappointing, but researchers did notice that the drugs seemed to work well in blood cancers such as leukaemia. Response rates were more encouraging for Aurora kinase inhibitors that additionally targeted other factors that promote cancer. For instance, inhibiting another kinase called FLT3, which is known to drive certain blood cancers, gave promising anti-tumour activity in patients with acute myeloid leukaemia. Researchers are now exploring Aurora kinases that have dual inhibitor activity.

Image of the mitotic spindle in a human cell showing microtubules in green,
chromosomes, DNA, in blue, and kinetochores in red. Image: The ICR London

Double-Pronged Attack

A multidisciplinary drug discovery team led by Dr Linardopoulos has discovered a small-molecule dual inhibitor of Aurora and FLT3 kinases called CCT241736. Although FLT3 kinase has attracted a great interest in recent years as a target for acute myeloid leukaemia treatment, FLT3 inhibitors had limited success when used as single agents, because cancers tended to become resistant.

But in acute myeloid leukaemia cell lines that did not respond to FLT3 selective inhibitors, CCT241736 was able to overcome this resistance. CCT241736 has completed preclinical safety and toxicity studies and is poised to enter phase I clinical trials in patients with acute myeloid leukaemia.

Understanding the Biology of Aurora Kinases

Here at the ICR, one facet of our research strategy is to understand in detail the critical mechanisms used by cells to grow, divide and spread and how these processes go wrong in cancer. As a continuation of the work focusing on Aurora kinases and their role in cancer, Dr Linardopoulos and his team have recently published a paper that sets out to establish what role Aurora-B and another mitotic kinase, namely MPS1, play in regulating the spindle assembly checkpoint, a pause in the cell cycle that allows the cell to check that all the chromosomes are properly attached to the mitotic spindle.

As the cell prepares for division, large protein complexes called kinetochores assemble to connect the chromosomes to the microtubule rope that forms the mitotic spindle. When kinetochores are not correctly attached to the spindle, they activate the spindle assembly checkpoint network, pausing cell division. Cell division can only continue when all kinetochores are stably and properly bound to microtubules, and the chromosomes are safely anchored.

In the Gurden et al. paper it was shown that Aurora-B has a dual role in maintaining the spindle assembly checkpoint signal. Firstly, it enhances the recruitment of MPS1, an additional mitotic checkpoint protein, to the kinetochore, so that there is a rapid establishment of the spindle assembly checkpoint. But mainly, Aurora-B prevents the premature removal of spindle assembly checkpoint proteins, including MPS1, from unattached kinetochores.

Given the cross-regulation between Aurora-B and MPS1, researchers used breast cancer cells to test what happens when Aurora-B and MPS1 inhibitors were given together. Combining the drugs increased their potency, killing breast cancer cells by completely preventing the spindle assembly checkpoint from working. But more importantly, the drug combination sensitised breast cancer cell lines that otherwise did not respond to Aurora-B inhibitors alone. Dr Linardopoulos tells me that combining Aurora-B and MPS1 inhibitors may have great potential in the clinic as a cancer therapy, and his team will be exploring this further.

The Future: Is Always in the Depth of What is Called Past

So are Aurora kinases a new light in the sky for cancer treatments? Aurora kinase inhibitors are certainly emerging as promising targeted therapies for blood cancers, with interesting off-target effects that could be used to tackle the problem of resistance. And our greater understanding of the biology of cancer is starting to open up potential new applications for this intriguing class of cancer treatment.

This piece is by Liz Burtally of The Institute of Cancer Research London: the title has been changed and After 'The Future' is added: Is Always in the Depth of What is Called Past

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Researchers Identify Possible Pathway to Reboot Immune System After Bone-Marrow Transplants

A ViSNE plot of immature T-cells that reside in the thymus, the sole site of T-cell production. Highlighted in red is a population of T-cell progenitors that colonise the thymus and give rise to all T-cells. The entry of these cells into the thymus is important for immune system recover post bone marrow transplantation. Image: University of Birmingham

|| August 27: 2016: University of Birmingham News || ά. New research has shown how a cell surface molecule, Lymphotoxin β receptor, controls entry of T-cells into the thymus; and as such presents an opportunity to understanding why cancer patients who undergo bone-marrow transplant are slow to recover their immune system.

The study, published in the Journal of Immunology, used mouse models to reveal an in vivo mechanism that researchers believe might also represent a novel pathway for immunotherapeutic targeting to support patients following transplantation. The thymus, which sits in front of the heart and behind the sternum, imports T-cell precursors from the bone marrow and supports their development into mature T-cells that fight off dangerous diseases.

T-cells are often the last cells to recover in cancer patients receiving bone marrow transplants. Though the cancer is cured, patients are often left with an impaired immune system that can take years to recover. The Birmingham team, supported by US-based collaborators at The Sanford Burnham Medical Research Institute and The Trudeau Institute, found that Lymphotoxin β receptor was required to allow the entry of T-cell progenitors to the thymus both in a healthy state, and during immune recovery following bone-marrow transplantation.

Significantly, the team also found that antibody-mediated stimulation of Lymphotoxin β receptor in murine models enhanced initial thymus recovery and boosted the number of transplant derived T-cells. Professor Graham Anderson, from the University of Birmingham, explained, “The thymus is often something of an ignored organ, but it plays a crucial role in maintaining an effective immune system.”

Post-transplantation, T-cell progenitors derived from the bone marrow transplant can struggle to enter the thymus, as if the doorway to the thymus is closed. Identifying molecular regulators that can ‘prop open’ the door and allow these cells to enter and mature, could well be a means to help reboot the immune system.”

Beth Lucas, also at the University of Birmingham, added, “This is just one piece of the puzzle. It may be that there are adverse effects to opening the door to the thymus, but identifying a pathway that regulates this process is a significant step.”

Following these positive findings the team aim to move towards in-vitro samples of human thymus to examine the role that Lymphotoxin b receptor might play in regulation of thymus function in man.

The research was funded by the Medical Research Council:MRC and Cancer Research UK:CRUK, together with support from the Biotechnology and Biological Sciences Research Council:BBSRC and Arthritis Research UK:ARUK. ω.

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New Research Suggests That Diabetes Could Be Due to Failure of Beta Cell 'Hubs'

Image: WHO

|| July 23: 2016: University of Birmingham News || ά. The significant role of beta cell ‘hubs’ in the pancreas has been demonstrated for the first time, suggesting that diabetes may due to the failure of a privileged few cells, rather than the behaviour of all cells. Researchers used optogenetic and photopharmacological targeting to precisely map the role of the cells required for the secretion of insulin. The team believe that the findings, published in Cell Metabolism, could pave the way for therapies that target the ‘hubs’.

Dr David Hodson, from the University of Birmingham, explained, “It has long been suspected that ‘not all cells are equal’ when it comes to insulin secretion. These findings provide a revised blueprint for how our pancreatic islets function, whereby these hubs dictate the behaviour of other cells in response to glucose.”
According to the NHS, there are currently 3.9 million people living with diabetes in the UK, with 90% of those affected having type 2 diabetes.

Type 2 diabetes occurs when the pancreas fails to produce enough insulin to function properly, meaning that glucose stays in the blood rather than being converted into energy. Beta cells, β cells, make up around 65-80% of the cells in the islets of the pancreas. Their primary function is to store and release insulin and, when functioning correctly, can respond quickly to fluctuations in blood glucose concentrations by secreting some of their stored insulin.

These findings show that just 1-10% of beta cells control islet responses to glucose. Dr Hodson, who is supported by Diabetes UK RD Lawrence and EFSD:Novo Nordisk Rising Star Fellowships, continued, “These specialised beta cells appear to serve as pacemakers for insulin secretion. We found that when their activity was silenced, islets were no longer able to properly respond to glucose. “

Prof Guy Rutter, who co-led the study at Imperial College London, added “This study is interesting as it suggests that failure of a handful of cells may lead to diabetes”. Studies were conducted on islet samples from both murine and human models.

The team note that, though the findings present a significant step forward in understanding the cell mechanisms, the experiments therefore may not be reflected in vivo, where blood flow direction and other molecule dynamics may influence the role of the hubs and insulin secretion.

The research was funded by Diabetes UK and an MRC Project Grant. ω.


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Using Kinesin Inhibitors to Unmask Cancer

Liz Burtally Writing

Stages of cell division showing spindle formation, chromosome segregation and generation of two daughter cells: Image: The Institute of Cancer Research

Breast cancer cells, green, invading through a layer of fibroblasts, red: Image: Luke Henry: The Institute of Cancer Research 2009

|| June 25: 2016: The Institute of Cancer Research News || ά. Kinesins are vital components in the mechanics of cell division, and are emerging as feasible targets for cancer drug development. Liz Burtally finds out more about kinesin inhibitors, and what challenges lie ahead. One of the hallmarks of cancer is the uncontrolled growth of cells that leads to tumours forming. Using chemotherapy that disrupts the machinery needed for cell division is commonly used to tackle this.

Some of these drugs target the mitotic spindle, an essential component of cell division, but can cause nasty side-effects and resistance to treatment often develops. So it’s not surprising that efforts are underway to identify new ways to target the mitotic spindle. Researchers have turned their focus to a group of proteins called kinesins, which play an integral role in maintaining the mitotic spindle, offering an attractive anticancer target.

Professor Ian Collins, Professor of Medicinal Chemistry at The Institute of Cancer Research, London, has recently written a review with Dr Stephanie Myers, an ICR postdoctoral training fellow, on the progress of kinesin inhibitors. Curious about the role of kinesins in cancer, and how they can be targeted, I caught up with Stephanie to find out more.

The mitotic spindle and centrosomes

The mitotic spindle consists of a rope of microtubules organised through structures called centrosomes. Normal cells contain two centrosomes, which migrate to opposite poles of the cell during mitosis (cell division). Chromosomes are lined up at the spindle equator to ensure their correct orientation and segregation, and upon mitosis, each daughter cell receives one centrosome and the correct set of chromosomes.

Supernumerary centrosomes, having more than two centrosomes, have been detected in virtually all human cancers. Having multiple centrosomes creates a myriad of problems for the cell, making the genome unstable and consequently leads to programmed cell death. But this raises the question: how do tumour cells survive with supernumerary centrosomes?

The kinesin HSET is believed to play a key role in the survival of cancer cells with multiple centrosomes. By clustering many centrosomes temporarily into the two poles of the cell, the cancer cell is able to disguise itself as ‘normal’ and escape programmed cell death at the mitotic checkpoint. Because healthy cells with the normal two centrosomes are not reliant on HSET for centrosome clustering, HSET offers a tantalising antimitotic target specific for tumour types with a high incidence of centrosome amplification.

The current landscape

So far, two new HSET small molecule inhibitors, AZ82 and CW069, have been discovered. These inhibitors have been shown to cause centrosome declustering exclusively in cancer cells with amplified centrosomes. Several inhibitors against a kinesin called Eg5 have proceeded further and are in clinical trials, but as a monotherapy, these drugs have been disappointing so far.

Stephanie is working with Professor Collins to develop a new wave of HSET inhibitors. Stephanie tells me that no inhibitor bound crystal structure of HSET is available, so structure-based drug design is a challenge. To add to the hardship, the similarities and differences between the druggable sites on Eg5 and HSET are not yet fully understood.

Fortunately, some clues on how the compounds bind can be gleaned from the structures of other kinesins, and Stephanie remains optimistic in her research. So watch this space for further updates.

Future direction

As is the case with much chemotherapy, the development of resistance to kinesin inhibitors is highly likely, and we have already seen the emergence of resistance to Eg5 inhibitors. Researchers will need to elucidate how resistance to other kinesin inhibitors occurs by looking at the parts of the kinesin that are susceptible to mutation.

Characterisation of where and how inhibitors bind to the kinesins will certainly be essential to the further development of this class of drug. ω.


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Tokyo Institute of Technology Research: Linking Light to Life: New Pathways That Help Plants Adapt to Changing Environments

Researchers: Keisuke Yoshida and Toru Hisabori: Tokyo Institute of Technology

|| June 25: 2016: Tokyo Institute of Technology News || ά. Scientists at Tokyo Tech have identified two redox cascades that co-operatively regulate chloroplast function and contribute to plant survival. A fundamental challenge of the plant life cycle is how to manage fluctuating environmental conditions. To preserve the integrity and efficiency of photosynthesis, chloroplasts have evolved multiple adaptive strategies to changes in light environments.

The thiol-based redox regulation system is important for controlling chloroplast functions in response to light signal. This system has been traditionally considered to be supported only by ferredoxin-thioredoxin reductase:FTR:thioredoxin:Trx: redox cascade. However, emerging genomics and proteomics data indicate that chloroplasts have a complex redox network, not a simple one-directional cascade.

In this advanced study, Yoshida and Hisabori have identified another pathway that works differently but collaboratively with FTR:Trx to regulate chloroplast functions. They show that NADPH-Trx reductase C:NTRC: a unique redox mediator harboring both a NADPH-Trx reductase domain and a Trx domain, plays a distinct role in regulating chloroplast functions and discuss how the co-operative functions of the two redox pathways maintain plant viability.

The scientists employed NTRC-affinity chromatography and biochemical methods to identify molecules targeted by NTRC in spinach chloroplasts. While NTRC and Trx proteins recognised some overlapping targets, their interactive affinities and reducing activities were different. Moreover, NTRC specifically bound Trx-z, one of the Trx proteins, and transferred the reducing power to this unique target.

The deletion of NTRC resulted in pale green leaves, suggesting an important role of NTRC in plant growth. Moreover, further impairment of FTR accumulation was lethal under autotrophic conditions, underscoring the critical importance of the concerted activity of these pathways for plant viability. Since these redox systems can derive signals through different sources:FTR:Trx pathway is exclusively dependent on the light-driven photosynthetic electron transport and NTRC can function even under dark conditions, their integrative functioning significantly contributes to transmitting signals from diverse environmental cues.

These breakthrough findings indicate that co-operative redox regulation by the highly organised network comprising FTR:Trx and NTRC:shown in Figure 1: is essential for chloroplast functional activity and plant survival in varying environmental conditions. Further studies will help elucidate the intricate interplay of these redox networks.

Figure 1. A. Proposed model of the chloroplast redox network. The NTRC and Trx families have distinct target proteins:red arrows and blue arrows, respectively. They have different reducing power transfer efficiencies for common targets:represented with varying thickness of arrows. B. Phenotypic effects of mutating FTR:Trx only, NTRC only, or FTR:Trx and NTRC. Absence of NTRC results in pale green leaves and absence of both FTR:Trx and NTRC results in stunted growth. As published in PNAS.


Authors: Keisuke Yoshida and Toru Hisabori
Title of original paper: Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability
Journal: Proceedings of the National Academy of Sciences of the United States of America
DOI: 10.1073/pnas.1604101113
Affiliations: 1Laboratory for Chemistry and Life Science, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503, Japan
Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo 102-0075, Japan

Research at Tokyo Institute of Technology

About Tokyo Institute of Technology: Tokyo Institute of Technology stands at the forefront of research and higher education as the leading university for science and technology in Japan. Tokyo Tech researchers excel in a variety of fields, such as material science, biology, computer science and physics. Founded in 1881, Tokyo Tech has grown to host 10,000 undergraduate and graduate students who become principled leaders of their fields and some of the most sought-after scientists and engineers at top companies. Embodying the Japanese philosophy of “monotsukuri,” meaning technical ingenuity and innovation, the Tokyo Tech community strives to make significant contributions to society through high-impact research.


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New Research: New Molecules: New Hope for Sufferers of  Cystic Fibrosis

Two anionophores that show high selectivity for carrying chloride ions over protons and hydroxide: Image: University of Southampton

|| June 13: 2016: University of Southampton News || ά. Ion channels are proteins found in a cell’s membrane, which create tiny openings in the membrane that regulate the movement of specific ions. Defective ion channels are the underlying cause of many diseases, notably cystic fibrosis, in which the transport of chloride ions is impaired.

Synthetic transporters that can carry chloride through lipid-bilayer membranes have been developed that could potentially replace the function of faulty channels. However, these transporters may also carry protons or hydroxide ions, which could disrupt pH homeostasis in the human body and lead to undesired toxic effects.

The new study involving an international team of researchers, published in the journal Chem, is the first to show examples of anion transporters with a high selectivity for chloride over protons and hydroxide. The researchers first demonstrated that proton/hydroxide transport is an overlooked side effect of synthetic anion transporters that were previously assumed to just carry anions. To address this problem, the researchers synthesised two new molecules that showed high selectivity for carrying chloride ions over protons and hydroxide. One of these compounds enabled chloride transport in real cells without seriously affecting lysosomal pH.

Lead author and PhD student, Xin Wu from the University of Southampton, said: “These new findings represent a paradigm shift for transporter design and provide important clues on how to develop anion transporters for different biomedical applications. We showed that different classes of anion transporters can have different behaviour in regulating ion gradients, membrane potential and pH gradients in cells. You need to pick the right molecule to have the desired biological effect for treating a certain disease.”

Co-author and Xin’s supervisor Professor Phil Gale, Head of Chemistry at the University of Southampton, said: “We demonstrated the possibility to develop molecules to replace the function of chloride channels without disrupting pH homeostasis. This is a significant step toward real biomedical application of anion transporters in the battle against cystic fibrosis and other diseases caused by faulty ion channels.”

The study involved researchers from the University of Bristol, Universitat de Barcelona, Spain and Xiamen University, China. It was funded by the Engineering and Physical Sciences Research Council:EPSRC, the Spanish government, the EU, and the La Marató de TV3 Foundation.

Read in Biomedicojurisprudence: Professor Anneke Lucassen: Ethics of Genomics: Exploring the Application of Ethics and Law in Clinical Practice


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Let's Talk About Blood and, Oh, Yes, Cancer

Dr Claire Bithell Writing

A white blood cell: Image: Wellcome Images

|| June 13: 2016: The Institute of Cancer Research News || ά. As further developments in immunotherapy-related cancer research are reported in the national media, Dr Claire Bithell reflects on the dramatic recent progress in this field. In a new paper in the journal Nature published this week, researchers have carried out early stage research to turn the body’s own defences against cancer. The study has been covered widely in the media, including The Guardian, The Telegraph and The Independent — and journalists asked The Institute for Cancer Research, London, for comment.

We asked a new team leader at the ICR, what he thought of the research and the field of immunotherapy as a whole. Professor Alan Melcher is Professor of Translational Immunotherapy at the ICR and Honorary Consultant Oncologist at The Royal Marsden NHS Foundation Trust.

He told us: "Immunotherapy for cancer is a rapidly evolving and exciting field. This new study, in mice and a small number of patients, shows that an immune response against the antigens within a cancer can be triggered by a new type of cancer vaccine.

"This vaccine is given into the blood, and comprises very small nanoparticules made up of fat joined to RNA (a type of genetic code for the tumour antigens). These nanoparticles target particular cells in the mice, called dendritic cells, which are key to stimulating an immune response.”
'Great hope'

Although this is exciting news, the potential treatment will take some time to reach the clinic. As Prof Melcher pointed out: "Although the research is very interesting, it is still some way away from being of proven benefit to patients. In particular, there is uncertainty around whether the therapeutic benefit seen in the mice by targeting a small number of antigens will also apply to humans, and the practical challenge of manufacturing nanoparticles for widespread clinical application."

Although many immunotherapies are in an early stage of development, in conjunction with a better understanding of cancer and the mechanisms that drive it, the future looks promising. As Professor Raj Chopra, Head of Cancer Therapeutics described in a previous blog post: “We are in an era of great hope for cancer treatment, with an explosion in knowledge of how cancers develop, evolve and acquire drug resistance, which is feeding through into innovative new treatments like immunotherapy.”

Here at the ICR, there is also a lot going on in the field of immunotherapy.

More to come

For example, at the end of last year a viral immunotherapy known as T-Vec became the first to be approved in Europe. Researchers from the ICR and The Royal Marsden led the UK arm of the major phase III trial for T-VEC, which was the first to definitively show patient benefit for a viral immunotherapy for cancer.

Then, in April this year, the international cancer conference, the American Association for Cancer Research Annual Meeting 2016, was buzzing with news of new data on the immunotherapy drugs known as checkpoint inhibitors.

With new immunotherapy experts joining the ICR recently, such as Professors Chopra and Melcher, we predict there will be lots more of interest in the future. We will keep you posted with developments.


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Tissue Repair and Cancer: How Not to Inflame the Neighbours

Graham Shaw Writing

Colorised scanning electron micrograph of a macrophage (image: NIAID/CC BY 2.0)

|| May 15: 2016: The Institute of Cancer Research News || ά. The human body has remarkable abilities to repair itself after illness and damage, but these same mechanisms could help cancer bounce back from treatments designed to kill cancer cells. Researchers at The Institute of Cancer Research, London, are studying what happens during tissue damage and repair to see if it can provide clues for treating cancer.

Cancer has been described as a wound that doesn’t heal. Despite cancer treatments designed to trigger the machinery that causes cells to die, researchers have known for decades that cancer cells which survive treatment can rapidly repopulate tumours by proliferating at a much higher rate than healthy cells.

When the body suffers an injury, the immune system leaps into action, like the emergency services at the scene of an accident; clearing up dead and dying matter and encouraging cells to divide nearby to replace the damaged tissue.

This capacity to self-repair is truly amazing, but it could also help cancer stem cells to grow out of control or tumours regrow after therapy.

Researcher Dr Pascal Meier and his team at The Institute of Cancer Research, London, are investigating these mechanisms to understand how they might affect treatment and recovery in patients with cancer.

In the journal Current Biology, Dr Meier discusses how dying cells communicate with their healthy neighbours to encourage regrowth after injury.

Using ‘zombie’ fruit fly cells which were kept in a permanent state of dying, researchers saw that dying cells produce a compound called hydrogen peroxide, through the activity of proteins called caspases.

Early warning system

Caspases carry out important inflammatory responses such as apoptosis, or programmed cell death, and ensure that cellular components are degraded in a controlled manner. In the dying cells the production of hydrogen peroxide acts as an early warning of damage for the immune system, triggering a cascade of signalling that starts wound healing.

White blood cells called macrophages are recruited by the cells, which engulf and digest cellular debris, foreign substances, cancer cells or anything else without the right types of proteins on their surfaces.

These macrophages activate a signalling mechanism called JNK that creates a feedback loop producing more and more signals telling the cells to die.

But interestingly, JNK also has an effect on the neighbouring healthy cells, leading them to produce compounds that encourage cell division and tissue repair.

Normally this process works correctly to keep healthy tissue replenished, but in cancer it helps tumours recover after treatments like radiotherapy and chemotherapy, causing cells to spread rapidly.

This dual role in clearing away damaged cells and re-growing tissue to compensate helps cancer to survive therapies designed to destroy it, but it could also point the way to improving treatment in the future.

Rewiring defences

Professor Meier and his team are studying inflammation in breast cancer, to see how these cells communicate with their neighbours when they are dying.

If they can identify the signalling receptors that coordinate inflammation in breast cancer cells, they could prevent this resistance to common cancer treatments and stop tumours regrowing via their healthy neighbours.

They hope that rewiring the macrophages recruited by cancer cells could switch off the signals that encourage cancer cells to proliferate.

They are also investigating a similar phenomenon called cell competition, where stronger cells kill weaker cells, which may play an important role in the development of cancer.

A complex relationship exists between cell death and inflammation — our body’s response to damage and infection — but understanding this relationship could help doctors treat cancer more effectively. ω.


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Cell ‘Skeletons’ Help Keep Cell Division on Track and Cancer in Check


||April 16, 2016|| Scientists at the Francis Crick Institute in London have greatly improved our understanding of how microtubules in the cell cytoskeleton switch between phases of growth and shrinkage.

This has implications for cancer research because correct switching frequency between growth and shrinkage is essential for cell division - and cancer can result when cell division goes awry.

Dr Christian Duellberg of the Crick, in Thomas Surrey's lab, explained: "In analogy to the skeleton, cells have a cytoskeleton. Microtubules form part of this cytoskeleton and are little tubes that span throughout the space inside a cell.

"Unlike our static bones, microtubules change their length constantly and switch between phases of growth and shrinkage. This is important because cells need to be able to drastically change their shape when they divide or move. However, exactly how this switching occurs was unknown and has been a controversial topic in the last decades."

Most scientists agree that there has to be some sort of stabilising 'cap' at the end of microtubules. However, how the size of this cap affects the stability of the microtubules was unknown.

To investigate, the team measured the properties of the cap in space (size) and time (duration of stability). They revealed for the first time a clear link between the cap and microtubule stability - the longer the cap, the more stable the microtubule.

They showed that so-called End Binding (EB) proteins can bind to the cap and make the microtubule unstable, which causes it to switch from growing to shrinking more often. The scientists also showed that these EB proteins, when labelled with dye, can be used to measure the length of the cap by proxy and can therefore be used to track microtubule stability directly and in real time using light microscopy.

Dr Duellberg said: "Because the correct switching frequency between growth and shrinkage of microtubules is essential for cell division, drugs that affect this switching frequency have become powerful tools in chemotherapy as they prevent cells from dividing.

"An example of such a drug is Paclitaxel, which reduces the switching frequency from growth to shrinkage. It is currently used to treat forms of ovarian, breast, lung, pancreatic and other cancers. However, resistance to the drug and side effects are a problem. As our study provides more details into how microtubules switch from growth to shrinkage, this might help to design better drugs that affect microtubule properties more specifically."

As well as offering new insights into cancer, using dye-labelled EB proteins to follow the stability of microtubules in real time will help scientists find out more about diseases caused by altered microtubule stability and incorrect switching between growth and shrinkage. Examples include eye movement disorders such as strabismus, where a patient's eyes don't align correctly. The paper, The size of the EB cap determines instantaneous microtubule stability, is published in the journal eLife.

New insights into how microtubules in the cell cytoskeleton switch between phases of growth and shrinkage have implications for cancer research. Correct switching frequency between growth and shrinkage is essential for cell division - and cancer can result when cell division goes awry. The Francis Crick Institute scientists worked with David Holmes of the London Centre for Nanotechnology. The work was supported by Cancer Research UK, the Francis Crick Institute and the European Research Council.

The Francis Crick Institute is a unique partnership between the Medical Research Council (MRC), Cancer Research UK, the Wellcome Trust, UCL (University College London), Imperial College London and King's College London.


P: 170416


Protein Translocation Channel of Mitochondrial Inner Membrane and Matrix-exposed Import Motor Communicate Via Two-domain Coupling Protein

Rupa Banerjee, Christina Gladkova, Koyeli Mapa, Gregor Witte, Dejana Mokranjac


Rupa Banerjee, Christina Gladkova, Koyeli Mapa, Gregor Witte, Dejana Mokranjac (of Ludwig-Maximilians-Universität, Germany) writes in a recently published research in eLife that the majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.

The function of Tim44 can be rescued by its two domains expressed in trans but not by either of the domains alone. (A) Schematic representation of Tim44 domain structure (numbering according to yeast Tim44 sequence). pre. - presequence (B and C) A haploid yeast deletion strain of TIM44 carrying the wild-type copy of TIM44 on a URA plasmid was transformed with centromeric plasmids carrying indicated constructs of Tim44 under control of endogenous promoter and 3'UTR. Cells were plated on medium containing 5-fluoroorotic acid and incubated at 30°C. The plasmid carrying wild-type Tim44 and an empty plasmid were used as positive and negative controls, respectively. (D) Total cell extracts of wild-type yeast cells transformed with plasmids coding for indicated Tim44 constructs under GPD promoter were analysed by SDS–PAGE and immunoblotting against depicted antibodies. *, ** and *** - protein bands detected with antibodies raised against full-length Tim44.


eLife digest

Human, yeast and other eukaryotic cells contain compartments called mitochondria. These compartments are surrounded by two membranes and are most famous for their essential role in supplying the cell with energy. While mitochondria can make a few of their own proteins, the vast majority of mitochondrial proteins are produced elsewhere in the cell and are subsequently imported into mitochondria. During the import process, most proteins need to cross both mitochondrial membranes.

Many mitochondrial proteins are transported across the inner mitochondrial membrane by a molecular machine called the TIM23 complex. The complex forms a channel in the inner membrane and contains an import motor that drives the movement of mitochondrial proteins across the membrane. However, it is not clear how the channel and import motor are coupled together. There is some evidence that a protein within the TIM23 complex called Tim44 – which is made of two sections called the N-terminal domain and the C-terminal domain – is responsible for this coupling. It has been suggested that mainly the N-terminal domain of Tim44 is required for this role.

Banerjee et al. used biochemical techniques to study the role of Tim44 in yeast. The experiments show that both the N-terminal and C-terminal domains are essential for its role in transporting mitochondrial proteins. The N-terminal domain interacts with the import motor, whereas the C-terminal domain interacts with the channel and the mitochondrial proteins that are being moved.

Banerjee et al. propose a model of how the TIM23 complex works, in which the import of proteins into mitochondria is driven by rearrangements in the two domains of Tim44. A future challenge is to understand the nature of these rearrangements and how they are influenced by other components of the TIM23 complex.


Mitochondria perform a number of essential cellular functions ranging from production of ATP and diverse other metabolic intermediates to initiation of apoptosis. It is thus not very surprising that disturbances in mitochondrial function are associated with a number of human diseases, including neurodegenerative disorders, diabetes, and various forms of cancer (Nunnari and Suomalainen, 2012; Quirós et al., 2015; Youle and van der Bliek, 2012). An essential prerequisite for correctly functioning mitochondria is import of about 1000 different proteins synthesized as precursor proteins in the cytosol. Recent studies revealed that mitochondrial protein import machineries are sensitive indicators of functionality of mitochondria (Harbauer et al., 2014; Nargund et al., 2012; Yano et al., 2014), demonstrating that a deep understanding of mitochondrial protein import pathways and their regulation will be essential for understanding the role mitochondria have under physiological and pathophysiological conditions. Over half of mitochondrial proteins are synthesized with cleavable, N-terminal extensions called presequences. Import of such precursor proteins requires a coordinated action of the TOM complex in the outer membrane and the TIM23 complex in the inner membrane and is driven by membrane potential across the inner membrane and ATP in the matrix (Dolezal et al., 2006; Endo et al., 2011; Koehler, 2004; Mokranjac and Neupert, 2009; Neupert and Herrmann, 2007; Schulz et al., 2015; Stojanovski et al., 2012).

The TIM23 complex mediates translocation of presequence-containing precursor proteins into the matrix as well as their lateral insertion into the inner membrane. The latter process requires the presence of an additional, lateral insertion signal. After initial recognition on the intermembrane space side of the inner membrane by the receptors of the TIM23 complex, Tim50 and Tim23, precursor proteins are transferred to the translocation channel in the inner membrane in a membrane-potential dependent step (Bajaj et al., 2014Lytovchenko et al., 2013; Mokranjac et al., 2009Shiota et al., 2011Tamura et al., 2009). The translocation channel is formed by membrane-integrated segments of Tim23, together with Tim17 and possibly also Mgr2 (Alder et al., 2008; Demishtein-Zohary et al., 2015leva et al., 2014; Malhotra et al., 2013). At the matrix-face of the inner membrane, precursor proteins are captured by the components of the import motor of the TIM23 complex, also referred to as PAM (presequence translocase-associated motor). Its central component is mtHsp70 whose ATP hydrolysis-driven action fuels translocation of precursor proteins into the matrix (De Los Rios et al., 2006Liu et al., 2003; Neupert and Brunner, 2002Schulz and Rehling, 2014). Multiple cycles of mtHsp70 binding to and release from translocating proteins are required for complete translocation across the inner membrane. The ATP hydrolysis-driven cycling of mtHsp70 and thereby its binding to proteins is regulated by the J- and J-like proteins Tim14(Pam18) and Tim16(Pam16) as well as by the nucleotide-exchange factor Mge1 (D'Silva et al., 2003Kozany et al., 2004Mapa et al., 2010Mokranjac et al., 20062003bTruscott et al., 2003). Tim21 and Pam17 are two nonessential components that bind to Tim17-Tim23 core of the TIM23 complex and appear to modulate its activity in a mutually antagonistic manner (Chacinska et al., 2005Popov-Celeketic et al., 2008van der Laan et al., 2005).

The translocation channel and the import motor of the TIM23 complex are thought to be coupled by Tim44, a peripheral inner membrane protein exposed to the matrix (D'Silva et al., 2004; Kozany et al., 2004; Schulz and Rehling, 2014). Like other components of the TIM23 complex, Tim44 is a highly evolutionary conserved protein and is encoded by an essential gene. In mammals, Tim44 has been implicated in diabetes-associated metabolic and cellular abnormalities (Wada and Kanwar, 1998; Wang et al., 2015). A novel therapeutic approach using gene delivery of Tim44 has recently shown promising results in mouse models of diabetic nephropathy (Zhang et al., 2006). In addition, mutations in Tim44 were identified that predispose carriers to oncocytic thyroid carcinoma (Bonora et al., 2006). Understanding the function of Tim44 and its interactions within the TIM23 complex will therefore be essential for understanding how the energy of ATP hydrolysis is converted into unidirectional transport of proteins into mitochondria and may provide clues for therapeutic treatment of human diseases.

Tim44 binds to the Tim17-Tim23 core of the translocation channel (Kozany et al., 2004; Mokranjac et al., 2003b). Tim44 also binds to mtHsp70, recruiting it to the translocation channel. The interaction between Tim44 and mtHsp70 is regulated both by nucleotides bound to mtHsp70 as well as by translocating proteins (D'Silva et al., 2004; Liu et al., 2003; Slutsky-Leiderman et al., 2007). Tim44 is likewise the major site of recruitment of the Tim14-Tim16 subcomplex, recruiting them both to the translocation channel as well as to mtHsp70 (Kozany et al., 2004; Mokranjac et al., 2003b). In this way, Tim44 likely ensures that binding of mtHsp70 to the translocating polypeptides, regulated by the action of Tim14 and Tim16, takes place right at the outlet of the translocation channel in the inner membrane.

Tim44 is composed of two domains, depicted as N- and C-terminal domains (Figure 1A). Recent studies suggested that the N-terminal domain is responsible for the majority of known functions of Tim44. Segments of the N-terminal domain were identified that are important for interaction of Tim44 with Tim16 and with mtHsp70 (Schilke et al., 2012; Schiller et al., 2008). Furthermore, using site-specific crosslinking, residues in the N-terminal domain were crosslinked to the matrix-exposed loop of Tim23 (Ting et al., 2014). However, the C-terminal domain of Tim44 shows higher evolutionary conservation. Still, the only function that has so far been attributed to the C-terminal domain is its role in recruitment of Tim44 to cardiolipin-containing membranes (Weiss et al., 1999). Based on the crystal structure of the C-terminal domain, a surface-exposed hydrophobic cavity was initially suggested to be important for membrane recruitment (Josyula et al., 2006). However, subsequent biochemical studies combined with molecular dynamics simulations, demonstrated that the helices A1 and A2 (residues 235–262 in yeast Tim44), present in the beginning of the C-terminal domain, are important for membrane recruitment (Marom et al., 2009). Deletion of helices A1 and A2 abolished membrane association of the C-terminal domain. Interestingly, attachment of helices A1 and A2 to a soluble protein was sufficient to recruit it to a model membrane (Marom et al., 2009).

We report here that the function of the full-length Tim44 cannot be rescued by its N-terminal domain extended to include membrane-recruitment helices of the C-terminal domain, demonstrating an unexpected essential function of the core of the C-terminal domain. Surprisingly, we observed that the two domains of Tim44, when expressed in trans, can support, although poorly, growth of yeast cells, giving us a tool to dissect the role of the C-terminal domain in vivo. We identify the C-terminal domain of Tim44 as the domain of Tim44 that is in contact with translocating proteins and that directly interacts with Tim17, a component of the translocation channel. Our data suggest that intricate rearrangements of the two domains of Tim44 are required during transfer of translocating precursor proteins from the channel in the inner membrane to the ATP-dependent motor at the matrix face.


The function of Tim44 can be rescued by its two domains expressed in trans

We reasoned that if all important protein–protein interactions of Tim44 are mediated by its N-terminal domain and the only function of the C-terminal domain is to recruit Tim44 to the membrane, then a construct consisting of the N-terminal domain, extended to include the membrane-recruitment helices A1 and A2, should suffice to support the function of the full-length protein. To test this hypothesis, we cloned such a construct in a yeast expression plasmid and transformed it into a Tim44 plasmid shuffle yeast strain. Upon incubation of transformed cells on a medium containing 5-fluoroorotic acid to remove the URA plasmid carrying the wild-type, full-length copy of Tim44, no viable cells were obtained (Figure 1B). A plasmid carrying the full-length copy of Tim44 enabled growth of yeast cells, whereas no viable colonies were obtained when an empty plasmid was used, confirming the specificity of the assay. We conclude that the N-terminal domain of Tim44, even when extended to include the membrane-recruitment helices of the C-terminal domain, is not sufficient to support the function of the full-length protein. Furthermore, this result suggests that the C-terminal domain of Tim44 has a function beyond membrane recruitment that is apparently essential for viability of yeast cells.

We then tested whether the function of Tim44 can be rescued by its two domains expressed in trans. Two plasmids, each encoding one of the two domains of Tim44 and both including A1 and A2 helices, were co-transformed into a Tim44 plasmid shuffle yeast strain and analyzed as above. Surprisingly, we obtained viable colonies when both domains were expressed in the same cell but not when either of the two domains was expressed on its own (Figure 1C). The rescue was dependent on the presence of A1 and A2 helices on both domains (data not shown), as in their absence neither of the domains could even be stably expressed in yeast (Figure 1D).

It is possible that the two domains of Tim44, both carrying A1 and A2 helices, bind to each other with high affinity and therefore are able to re-establish the full-length protein from the individual domains. To test this possibility, we expressed both domains recombinantly, purified them and analyzed, in a pull down experiment, if they interact with each other. The N-terminally His-tagged N-terminal domain efficiently bound to NiNTA-agarose beads under both low- and high-salt conditions (Figure 1—figure supplement 1A). However, we did not observe any copurification of the non-tagged C-terminal domain. We also did not observe any stable interaction of the two domains when digitonin-solubilized mitochondria containing a His-tagged version of the N-terminal domain were used in a NiNTA pull-down experiment (Figure 1—figure supplement 1B). Thus, the two domains of Tim44 appear not to stably interact with each other.

N+C cells are viable, but grow only very poorly even on fermentable medium

We compared growth rate of the yeast strain carrying the wild-type, full-length version of Tim44 (FL) with that of the strain having two Tim44 domains, both containing A1 and A2 helices, expressed in trans, for simplicity reasons named from here on N+C. The N+C strain was viable and grew relatively well on a fermentable carbon source at 24°C and 30°C (Figure 2A). Still, its growth was slower than that of the FL strain at both temperatures. At 37°C, the N+C strain was barely viable. On a nonfermentable carbon source, when fully functional mitochondria are required, N+C did not grow at any of the temperatures tested. Thus, the function of Tim44 can be reconstituted from its two domains separately, although only very poorly.

We isolated mitochondria from FL and N+C strains grown on fermentable medium and compared their mitochondrial protein profiles. Immunostaining with antibodies raised against full-length Tim44 detected no full-length protein in N+C mitochondria but rather two faster migrating bands (Figure 2B). Based on the running behavior of the individual domains seen in Figure 1D, the slower migrating band corresponds to the N domain and the faster migrating one to the C domain. This confirms that, surprisingly, the full-length Tim44 is indeed not absolutely required for viability of yeast cells. The endogenous levels of other components of the TIM23 complex were either not changed at all (Tim17, Tim23, and Tim50), or were slightly upregulated (mtHsp70, Tim14, and Tim16), likely to compensate for only poorly functional Tim44. Levels of components of other essential mitochondrial protein translocases of the outer and inner mitochondrial membranes, Tom40, Tob55, and Tim22, were not altered compared to FL mitochondria. Similarly, we observed no obvious differences in endogenous levels of proteins present in the outer membrane, intermembrane space, inner membrane, and the matrix that we analyzed.

We conclude that Tim44 can be split into its two domains that are sufficient to support the function of the full-length protein, although only poorly.

Protein import into mitochondria is severely impaired in N+C cells

Considering the essential role of Tim44 during translocation of precursor proteins into mitochondria, we tested whether the severe growth defect of the N+C strain is due to compromised mitochondrial protein import. When import of precursor proteins into mitochondria is impaired, a precursor form of matrix-localized protein Mdj1 accumulates in vivo (Waegemann et al., 2015; Wrobel et al., 2015). We indeed observed a very prominent band of the precursor form of Mdj1 in total cell extracts of N+C cells, grown at 24°C and 30°C, that was absent in cells containing full-length Tim44 (Figure 3A). Thus, the efficiency of protein import into mitochondria is reduced in N+C cells.

To analyze protein import in N+C mitochondria in more detail, we performed in vitro protein import into isolated mitochondria (Figure 3B–G,I–J). To this end, various mitochondrial precursor proteins were synthesized in vitro in the presence of [35S]-methionine and incubated with isolated mitochondria. The import efficiencies of all matrix-targeted precursors analyzed, pF1β, pcytb2(1–167)△DHFR, and pSu9(1–69)DHFR, were drastically reduced in N+C mitochondria when compared to wild type. Import of presequence-containing precursor of Oxa1 that contains multiple transmembrane segments was similarly impaired. Likewise, precursor proteins that are laterally inserted into the inner membrane by the TIM23 complex, such as pDLD1 and pcytb2, were imported with reduced efficiency into N+C mitochondria. In agreement with the established role of Tim44 in import of precursors of a number of components of respiratory chain complexes and their assembly factors, we observed a slightly reduced membrane potential in N+C mitochondria as compared to wild type (Figure 3H). However, precursors of ATP/ADP carrier and of Tim23, whose imports into mitochondria are not dependent on the TIM23 complex, were imported with similar efficiencies in both types of mitochondria, demonstrating that observed effects are not due to general dysfunction of mitochondria. We conclude that splitting of Tim44 into two domains in N+C cells severely impairs transport of proteins by the TIM23 complex, suggesting that full-length Tim44 is required for efficient import of presequence-containing precursor proteins into mitochondria.

Both domains of Tim44 assemble into the TIM23 complex

Tim44 is thought to play an important role in connecting the translocation channel and the import motor of the TIM23 complex. We thus reasoned that disassembly of the TIM23 complex in N+C mitochondria might be a reason for its reduced functionality. When wild-type mitochondria are solubilized with digitonin, affinity-purified antibodies to Tim17 and to Tim23 essentially deplete both Tim17 and Tim23 from the mitochondrial lysate and precipitate part of Tim50, Tim44, Tim14, and Tim16 (Figure 4). Similarly, affinity-purified antibodies to Tim16 deplete both Tim16 and Tim14 and precipitate Tim50, Tim17, Tim23, and Tim44 from mitochondrial lysate. We observed essentially the same precipitation pattern when we analyzed digitonin-solubilized N+C mitochondria, demonstrating that the TIM23 complex is properly assembled. Importantly, both N and C domains of Tim44 were recruited to the TIM23 complex.

The TIM23 complex adopts an altered conformation in N+C mitochondria

Since the assembly of the TIM23 complex is not affected in N+C mitochondria, we reasoned that an altered conformational flexibility may be a reason behind its reduced function in N+C cells. Chemical crosslinking is currently the most sensitive assay available to analyze the conformation of the TIM23 complex in intact mitochondria. We thus compared the crosslinking patterns of TIM23 subunits in N+C mitochondria to those in FL. In wild-type mitochondria, Tim16 can be crosslinked to mtHsp70, Tim44, and Tim14 in an ATP-dependent manner (Figure 5A). In N+C mitochondria, the same crosslinks of Tim16 to mtHsp70 and to Tim14 were observed. The crosslink to Tim44 was, as expected, absent in N+C mitochondria and another crosslink to a smaller protein appeared. In addition, a crosslink between two Tim16 molecules became prominent. Interestingly, this crosslink has previously been observed in mutants in which conformation of the TIM23 complex was altered (Popov-Čeleketić et al., 2008). Similarly, we observed prominent changes in crosslinking pattern of the channel component Tim23 (Figure 5B). In addition to the crosslink of Tim23 to Pam17, observed in both FL and N+C mitochondria, a prominent Tim23-dimer crosslink appeared in N+C mitochondria.

To obtain an independent evidence that the conformation of the TIM23 complex is affected in N+C mitochondria, we analyzed the complex by blue native gel electrophoresis. When digitonin-solubilized wild-type mitochondria are separated by BN-PAGE, Tim17, and Tim23 are present in a 90 kDa complex and, to a lesser degree, in higher molecular weight complexes that additionally contain Tim21 and Mgr2 (Chacinska et al., 2005; Ieva et al., 2014). In contrast, with digitonin-solubilized N+C mitochondria, antibodies to Tim17 and Tim23 revealed slightly shifted bands, in particular of the 90 kDa complex (Figure 5C). Since the 90 kDa complex does not contain any other known subunit of the TIM23 complex, this finding further supports the above notion that the conformation of the translocation channel is changed in N+C mitochondria. We observed no obvious difference in the ca. 60 kDa Tim14-Tim16 complex between FL and N+C mitochondria. As expected, full-length Tim44, present in FL mitochondria, was absent in N+C mitochondria (Figure 5C).

Together, these results demonstrate that the conformation of the TIM23 complex is changed in N+C mitochondria. They further show that alterations in the components traditionally assigned to the import motor affect the conformation of the translocation channel in the inner membrane, supporting the notion of an intricate crosstalk within the complex.

Role of the C-terminal domain of Tim44

The data presented so far suggest that full-length Tim44 is required for optimal conformational dynamics of the TIM23 complex. Furthermore, they suggest that the C-terminal domain has an essential function within the TIM23 complex, beyond mere membrane recruitment. So, what is the function of the C-terminal domain of Tim44? We first searched for binding partners of the individual domains. To that end, we recombinantly expressed and purified full-length Tim44 as well as its two domains (Figure 6A). To look for interaction partners of the core domains, both domains now lacked the segment containing A1 and A2 helices. Purified proteins were covalently coupled to the Sepharose beads and were subsequently incubated with mitochondrial lysates. Mitochondria were solubilized with Triton X-100 that, unlike digitonin, dissociates the TIM23 complex into its individual subunits (except for the Tim14-Tim16 subcomplex that remains stable). In this way, direct protein-protein interactions can be analyzed. We observed prominent, specific binding of mtHsp70, Tim16, Tim14 and Tim17, and to a far lesser degree of Tim23 and Tim50, to full-length Tim44 (Figure 6B). None of the proteins bound to empty beads. Also, we observed no binding of two abundant mitochondrial proteins, porin, and F1βß, demonstrating the specificity of observed interactions. mtHsp70, Tim16 and Tim14 also efficiently bound to the N-terminal domain of Tim44, in agreement with previous observations (Schilke et al., 2012; Schiller et al., 2008), and far less efficiently to the C-terminal domain. Since the Tim14-Tim16 subcomplex remains stable in Triton X-100, it is not possible by this method to distinguish which of the two subunits, or maybe even both, directly interacts with the N-terminal domain of Tim44. Binding of Tim17 to the N-terminal domain of Tim44 was drastically lower compared to its binding to the full-length protein. Instead, a strong binding of Tim17 to the C-terminal domain of Tim44 was observed.

We conclude that the N-terminal domain of Tim44 binds to the components of the import motor, whereas the C-terminal domain binds to the translocation channel in the inner membrane, revealing a novel function of the C-terminal domain of Tim44.

We then asked which of the two domains of Tim44 is in contact with translocating proteins. To answer this question, we first affinity-purified antibodies that specifically recognize cores of the individual domains of Tim44 using the above described Sepharose beads. The antibodies, affinity purified using beads with coupled full-length Tim44, recognized full-length Tim44 as well as both of its domains (Figure 6C). In contrast, antibodies that were affinity purified using beads with coupled individual domains recognized only the respective domain and the full-length protein (Figure 6C). This demonstrates that we indeed purified antibodies specific for individual domains of Tim44. Next, we accumulated 35S-labelled precursor protein pcytb2(1–167)△DHFR as a TOM-TIM23-spanning intermediate. Briefly, this precursor protein consists of the first 167 residues of yeast cytochrome b2, with a 19 residue deletion in its lateral insertion signal, fused to the passenger protein dihydrofolate reductase. In the presence of methotrexate, that stabilizes folded DHFR, the b2 part reaches the matrix, whereas the DHFR moiety remains on the mitochondrial surface resulting in an intermediate that spans both TOM and TIM23 complexes. The association of Tim44 and its domains with the arrested precursor protein was analyzed by chemical crosslinking followed by immunoprecipitation with antibodies to full-length Tim44 and its individual domains. In wild-type mitochondria, all three antibodies precipitated a crosslinking adduct of Tim44 to the arrested precursor protein, demonstrating that they are all able to immunoprecipitate the respective antigens (Figure 6D). In contrast, with N+C mitochondria, a faster migrating crosslinking adduct of a Tim44 domain to the arrested precursor protein was immunoprecipitated with the antibodies against the C-terminal domain and against the full-length protein but not with the antibodies against the N-terminal domain. This demonstrates that the C-terminal domain of Tim44 is in close vicinity of the translocating protein.

Mutations identified in human patients can frequently point to functionally important residues in affected proteins. In this respect, Pro308Gln mutation in human Tim44 has recently been linked to oncocytic thyroid carcinoma (Bonora et al., 2006). Since the mutation maps to the C-terminal domain of Tim44, we wanted to analyze functional implications of this mutation and therefore made the corresponding mutation in yeast Tim44 (Pro282Gln). We compared thermal stabilities of wild type and mutant Tim44 proteins by thermal shift assay. The melting temperature of wild-type Tim44 was 54°C, whereas that of the mutant protein was 4°C lower (Figure 6E). This demonstrates that the mutation significantly destabilizes Tim44, providing first clues toward molecular understanding of the associated human disease.


The major question of protein import into mitochondria that has remained unresolved is how translocation of precursor proteins through the channel in the inner membrane is coupled to the ATP-dependent activity of the Hsp70-based import motor at the matrix face of the inner membrane.

Results presented here demonstrate that the two domain structure of Tim44 is essential during this process. We show here that the two domains of Tim44 have different interaction partners within the TIM23 complex. In this way, Tim44 holds the TIM23 complex together. Our data revealed a direct, previously unexpected interaction between the C-terminal domain of Tim44 with the channel component Tim17. This result not only assigned a novel function to the C-terminal domain of Tim44 but also shed new light on Tim17, the component of the TIM23 complex that has been notoriously difficult to analyze. Recent mutational analysis of the matrix exposed loop between transmembrane segments 1 and 2 of Tim17 revealed no interaction site for Tim44 (Ting et al., 2014), suggesting its presence in another segment of the protein. Our data also confirmed the previously observed interactions of the N-terminal domain of Tim44 with the components of the import motor (Schilke et al., 2012; Schiller et al., 2008). We did, however, not observe any direct interaction between Tim23 and the N-terminal domain of Tim44 that has previously been seen by crosslinking in intact mitochondria (Ting et al., 2014). It is possible that this crosslinking requires a specific conformation of Tim23 only adopted when Tim23 is bound to Tim17 in the inner membrane. This notion is supported by our previous observation that the stable binding of Tim44 to the translocation channel requires assembled Tim17-Tim23 core of the TIM23 complex (Mokranjac et al., 2003b). We observed a direct Tim17-Tim44 interaction here probably because of a high local concentration of the C-terminal domain when bound to the beads.

The core of the C-terminal domain is preceded by a segment that contains two amphipathic, membrane-recruitment helices. This central segment connects the two domains of Tim44. Intriguingly, the two currently available crystal structures of the C-terminal domains of yeast and human Tim44s showed different orientations of the two helices relative to the core domains (Handa et al., 2007; Josyula et al., 2006). The conformational change was likely induced upon PEG binding to this region of human Tim44 during crystallization (Handa et al., 2007). It is tempting to speculate that the same conformational change takes place during translocation of proteins in the mitochondria. Such a conformational change would not only reorient the two helices in respect to the core of the C-domain but also change the relative orientation of N- and C-terminal domains. Since the two domains have different interaction partners within the TIM23 complex, such a change could rearrange the entire complex. The importance of this proposed conformational change in Tim44 is supported by the data presented here. The function of the full-length Tim44 could be reconstituted from its individual domains only very poorly. Also, there is obviously a very strong evolutionary pressure to keep the two domains of Tim44 within one polypeptide chain. N+C strain had to be kept at all times on the selective medium - even after only an overnight incubation on a nonselective medium the full-length protein reappeared (our unpublished observation), likely due to a recombination event between two plasmids.

Tim44 can be crosslinked to translocating proteins. Our data revealed that it is the C-terminal domain of Tim44 that interacts with proteins entering the matrix from the translocation channel in the inner membrane. A direct interaction of the same domain with Tim17 would optimally position the C-terminal domain to the outlet of the translocation channel. This raises an interesting possibility that translocating precursor proteins may play an important role in the above postulated conformational changes of Tim44.

A missense mutation Pro308Gln in human Tim44 is associated with familial oncocytic thyroid carcinoma. The corresponding mutation in yeast, Pro282Gln, destabilized the protein but produced no obvious growth phenotype or an in vivo import defect (our unpublished observations), suggesting that the yeast system is more robust. This observation is in agreement with the notion that mutations that would severely affect the function of the TIM23 complex would likely be embryonically lethal in humans. Still, the disease caused by a mutation in the C-terminal domain of human Tim44 speaks for an important role of this domain in the function of the entire TIM23 complex. Furthermore, the mutation maps to the short loop between A3 and A4 helices in the C-terminal domain of Tim44. Based on the crystal structure of Tim44, it was previously suggested that the mutation could affect the conformational flexibility of the A1 and A2 helices (Handa et al., 2007), intriguingly providing further support for the above postulated conformational changes of Tim44.

Based on the previously available data and the results presented here, we put forward the following model to describe how translocation of precursor proteins through the channel in the inner membrane is coupled to their capture by the ATP-dependent import motor at the matrix face of the channel (Figure 7). Tim44 plays a central role in this model. We envisage that two domains of Tim44 are connected by the central segment that contains membrane-recruitment helices, like two cherries on the stalks (Figure 7 insert). This central segment of Tim44 recruits the protein to the cardiolipin-containing membranes. There, through direct protein–protein interactions, the C-terminal domain of Tim44 binds to Tim17 and the N-terminal domain to mtHsp70 and to Tim14-Tim16 subcomplex (1). In this way, Tim44 functions as a central platform that connects the translocation channel in the inner membrane with the import motor at the matrix face. Additional interactions likely stabilize the complex, in particular that between the N-terminal domain of Tim44 and Tim23 (Ting et al., 2014) as well as the one between Tim17 and the IMS-exposed segment of Tim14 (Chacinska et al., 2005). In the resting state, the translocation channel is closed to maintain the permeability barrier of the inner membrane. During translocation of proteins (2), the translocation channel in the inner membrane has to open to allow passage of proteins. Opening of the channel will likely change the conformation of Tim17 that could be further conveyed to the C-terminal domain Tim44. It is tempting to speculate that this conformational change is transduced to the N-terminal domain of Tim44 through the central, membrane-bound segment of Tim44, leading to relative rearrangements of the two domains of Tim44. This change would now allow Tim14-Tim16 complex to stimulate the ATPase activity of mtHsp70 leading to stable binding of the translocating protein to mtHsp70. mtHsp70, with bound polypeptide, will then move into the matrix, opening a binding site on Tim44 for another molecule of mtHsp70 (3). We speculate that the release of mtHsp70 with bound polypeptide from the N-terminal domain of Tim44 will send a signal back to the C-terminal domain of Tim44 and further to the translocation channel. Multiple cycles of mtHsp70 are required to translocate the entire polypeptide chain into the matrix. Once the entire polypeptide has been translocated, the translocation channel will revert to its resting, closed state, bringing also Tim44 back to its resting conformation (1). Thus, the translocation channel in the inner membrane and the mtHsp70 system at the matrix face communicate with each other through rearrangements of the two domains of Tim44 that are stimulated by translocating polypeptide chain.

Material and methods

Yeast strains, plasmids, and growth conditions

Wild-type haploid yeast strain YPH499 was used for all genetic manipulations. A Tim44 plasmid shuffling yeast strain was made by transforming YPH499 cells with a pVT-102U plasmid (URA marker) containing a full-length TIM44 followed by replacement of the chromosomal copy of TIM44 with a HIS3 cassette by homologous recombination. For complementation analyzes, endogenous promoter, mitochondrial presequence (residues 1–42) and the 3’-untranslated region of TIM44 were cloned into centromeric yeast plasmids pRS315 (LEU marker) and pRS314 (TRP marker) and obtained plasmids subsequently used for cloning of various Tim44 constructs. The following constructs were used in the analyzes: Tim44(43–209), Tim44(43–262), Tim44(264–431), and Tim44(210–431). The constructs encompassing the N- and the C-terminal domains of Tim44 were cloned into pRS315 and pRS314 plasmids, respectively. Plasmids carrying the full-length copy of TIM44 were used as positive controls and empty plasmids as negative ones. A Tim44 plasmid shuffling yeast strain was transformed with two plasmids simultaneously and selected on selective glucose medium lacking respective markers. Cells that lost the wild-type copy of Tim44 on the URA plasmid were selected on medium containing 5-fluoroorotic acid at 30°C.

For expression in the wild-type background, the above-described constructs of Tim44, containing endogenous Tim44 presequence, were also cloned into centromeric yeast plasmids p414GPD and p415GPD for expression under the control of the strong GPD promoter. Cells were grown on selective lactate medium containing 0.1% glucose.

FL and N+C cells were grown in selective glucose medium at 30°C, unless otherwise indicated, and mitochondria were isolated from cells in logarithmic growth phase.

Recombinant proteins

DNA sequences coding for various segments of Tim44 were cloned into bacterial expression vector pET-Duet1 introducing a TEV cleavage site between the His6-tag and the protein coding region. The following Tim44 constructs were cloned: Tim44(43–431) (full-length protein lacking the mitochondrial presequence), Tim44(43–209) (referred to as N in Figure 6A), Tim44(43–263), Tim44(211–431), and Tim44(264–431) (referred to as Cc in Figure 6A). Pro282Gln mutation was introduced into the full-length construct using site directed mutagenesis. Proteins were expressed in E. coli BL21(DE3) at 37°C and purified using affinity chromatography on NiNTA-agarose beads (Qiagen, Germany) followed by gel filtration on Superdex 75 column (GE Healthcare, Germany). Unless otherwise indicated, the His6-tags were removed by incubation with the TEV protease. The purified proteins were stored at -80oC in 20 mM HEPES/KOH, 200 mM KCl, 5 mM MgCl2, pH 7.5, until use.

Purified proteins were coupled to CNBr-Sepharose beads (GE Healthcare, Germany) according to manufacturer's instructions and stored at 4°C. The beads were used for purification of domain-specific antibodies from the serum raised in rabbits against recombinantly expressed full-length Tim44. For direct binding analysis, mitochondria isolated from wild-type yeast cells were solubilized with 0.5% Triton X-100 in 20 mM Tris/HCl, pH 8.0, 80 mM KCl, 10% glycerol at 1 mg/mL and incubated with Tim44 constructs coupled to CNBr-Sepharose beads for 30 min at 4oC. After three washing steps, specifically bound proteins were eluted with Laemmli buffer. Samples were analyzed by SDS–PAGE and immunoblotting.

Figure 7. A proposed model of function of the TIM23 complex. See text for details. For simplicity reasons, only essential subunits of the complex are shown.

Thermal shift assay

Thermal stabilities of wild type and P282Q mutant form of Tim44 were analyzed by fluorescence thermal shift assay (Müller et al., 2015). Recombinant proteins (6.2 µM) in 20 mM HEPES/NaOH, 150 mM NaCl, pH 7.1 were mixed with 5x SYPRO Orange and melting curves analyzed in a real-time PCR machine using a gradient from 5°C to 99°C. Three technical replicates of two independent protein purifications were analyzed in parallel. Mutant Tim44 showed significantly decreased thermal stability under all conditions analyzed - in buffers containing different salt concentrations (50, 150, and 450 mM) as well as in different buffers and pHs (HEPES buffer at pH 7.1 and phosphate buffer at pH 8.0).


Previously published procedures were used for protein import into isolated mitochondria, crosslinking, coimmunoprecipitations and arrest of mitochondrial precursor proteins as TOM-TIM23 spanning intermediates followed by crosslinking and immunoprecipitation under denaturing conditions (Mokranjac et al., 2003a; 2003b; Popov-Čeleketić et al., 2008).

The Article can be read in full in eLife DOI

( Published December 29, 2015 Cite as eLife 2015;4:e11897)


P: 080216


Fibroblastic Reticular Cell-derived Lysophosphatidic Acid Regulates Confined Intranodal T-cell Motility

Akira Takeda
Daichi Kobayashi
Keita Aoi
Naoko Sasaki
Yuki Sugiura
Hidemitsu Igarashi
Kazuo Tohya
Asuka Inoue
Erina Hata
Noriyuki Akahoshi
Haruko Hayasaka
Junichi Kikuta
Elke Scandella
Burkhard Ludewig
Satoshi Ishii
Junken Aoki
Makoto Suematsu
Masaru Ishii
Kiyoshi Takeda
Sirpa Jalkanen
Masayuki Miyasaka
Eiji Umemoto

In a recently published research in eLife the above named authors write that lymph nodes (LNs) are highly confined environments with a cell-dense three-dimensional meshwork, in which lymphocyte migration is regulated by intracellular contractile proteins. However, the molecular cues directing intranodal cell migration remain poorly characterized. Here we demonstrate that lysophosphatidic acid (LPA) produced by LN fibroblastic reticular cells (FRCs) acts locally to LPA2 to induce T-cell motility. In vivo, either specific ablation of LPA-producing ectoenzyme autotaxin in FRCs or LPA2 deficiency in T cells markedly decreased intranodal T cell motility, and FRC-derived LPA critically affected the LPA2-dependent T-cell motility. In vitro, LPA activated the small GTPase RhoA in T cells and limited T-cell adhesion to the underlying substrate via LPA2. The LPA-LPA2 axis also enhanced T-cell migration through narrow pores in a three-dimensional environment, in a ROCK-myosin II-dependent manner. These results strongly suggest that FRC-derived LPA serves as a cell-extrinsic factor that optimizes T-cell movement through the densely packed LN reticular network.

eLife digest

Small organs called lymph nodes are found throughout the body and help to filter out harmful particles and cells. Lymph nodes are packed with different types of immune cells, such as the T-cells that play a number of roles in detecting and destroying bacteria, viruses and other disease-causing microbes. Within the lymph node, T-cells crawl along a meshwork made up of cells called fibroblastic reticular cells. The T-cells appear to move in random patterns, but the signals that drive this movement remain ill-defined.

Now, Takeda et al. reveal that a lipid called lysophosphatidic acid (LPA), which is produced by the fibroblastic reticular cells, is responsible for regulating how T-cells move around inside the lymph nodes. T-cells are able to detect LPA via certain receptor proteins on their surface. Takeda et al. engineered mice that were either unable to produce a particular LPA receptor on their T-cells, or that produced less LPA than normal. The T-cells of these mice moved around less than T-cells in normal mice.

Further experiments revealed that LPA signaling also affects the signaling pathway that alters how well the T-cells stick to nearby surfaces. This suggests that LPA helps to optimize T-cell movement to allow the cells to navigate the small spaces found between the fibroblastic reticular cells. In the future, targeting the processes involved in LPA signaling could help to develop new treatments for disorders of the immune system.


Blood-borne naïve lymphocytes migrate along the fibroblastic reticular cell (FRC) network in lymph nodes (LNs) (von Andrian and Mempel, 2003; Miyasaka and Tanaka, 2004; Girard et al., 2012). B cells then migrate into LN follicles, whereas T cells remain in the paracortex and migrate continually along the FRC network (Bajénoff et al., 2006). This intranodal migration provides critical opportunities for T cells to encounter cognate antigen-presenting dendritic cells. Two-photon microscopic analysis has shown that naïve T cells crawl along the FRC network in an apparently random pattern of motion, at an average velocity of 10–15 μm per minute (Miller et al., 2002; Okada and Cyster, 2007; Worbs et al., 2007). FRCs promote intranodal T-cell motility by signaling naïve lymphocytes with CCL21/CCL19 via CCR7, thus activating the small GTPase Rac (Okada and Cyster, 2007; Worbs et al., 2007; Faroudi et al., 2010; Huang et al., 2007), although CCR7 signaling only partially account for the interstitial T cell motility (Okada and Cyster, 2007; Huang et al., 2007).

LPA is a bioactive lysophospholipid produced both extracellularly and intracellularly. Extracellularly produced LPA is involved in such diverse biological functions as vascular remodeling and cell growth, survival, and migration (Choi et al., 2010; Yanagida et al., 2013). Intracellularly produced LPA is an intermediate in the synthesis of triglycerides and glycerophospholipids, and thought to act as a 'housekeeper' inside the cell (Mills and Moolenaar, 2003). Extracellular LPA is predominantly produced by autotaxin (ATX, also referred to as ENPP2 [ectonucleotide pyrophosphatase/phosphodiesterase family member 2]), an ectoenzyme that was originally identified as a tumor-cell motility-enhancing factor (Stracke et al., 1992). ATX is a lysophospholipase D that produces LPA by hydrolyzing lysophosphatidylcholine (LPC) (Okudaira et al., 2010; Moolenaar and Perrakis, 2011). We and others have reported that ATX is strongly expressed in HEV endothelial cells (ECs), and that ATX regulates lymphocyte migration into the LN parenchyma (Kanda et al., 2008; Nakasaki et al., 2008; Umemoto et al., 2011). We also demonstrated that LPA enhances lymphocyte detachment from ECs and promotes lymphocyte transmigration across the HEV basal lamina, at least in part by acting on HEV ECs (Bai et al., 2013). LPA also acts on naïve T cells to induce chemokinesis and cell polarization (Kanda et al., 2008; Katakai et al., 2014; Zhang et al., 2012) and transmigration (Zhang et al., 2012).While a study using pharmacological inhibitors revealed that ATX/LPA promotes intranodal lymphocyte motility in an ex vivo LN explant model (Katakai et al., 2014), the physiological significance of the ATX/LPA axis in interstitial lymphocyte migration remains unknown.

To date, six LPA receptors (LPA1–LPA6) have been identified. LPA receptors couple to multiple G proteins, including Gi, G12/13, Gq, and Gs, and upon ligand binding, these G proteins activate diverse intracellular signaling components including Rho and Rac. Although LPA2 has recently been reported to play a role in intranodal T-cell migration (Knowlden et al., 2014), it remains unclear how LPA2-mediated signaling affects interstitial T-cell motility and whether LPA is the prime activating ligand.

Leukocyte migration in a confined environment is regulated at least partly by cell contraction (Lämmermann et al., 2008). Jacobelli et al. (2010) reported that a contractile protein, myosin IIA, is required for T-cell amoeboid motility in confined environments such as LNs (Jacobelli et al., 2010). Myosin IIA cross-links actin, thus limiting surface adhesion and allowing T cells to exert contractile force. Myosin IIA’s activity is regulated by RhoA and Rho-associated protein kinase (ROCK) signaling. While the cell-extrinsic factor(s) that regulate myosin II’s activity during T-cell migration in a confined environment have been poorly defined, a recent study using zebrafish germ progenitor cells showed that LPA induces cell polarization in a ROCK-myosin II-dependent manner, which enables rapid cell migration in a confined environment (Ruprecht et al., 2015).

In this study, by using mice conditionally deficient for the LPA-generating enzyme ATX in FRCs and those deficient in LPA2, we demonstrated that bioactive LPA species are produced by FRCs in an ATX-dependent manner and that LPA acts locally on LPA2 on T cells. This LPA2-mediated signaling activates the RhoA-ROCK-myosin II pathway and promotes confinement-optimized interstitial T-cell migration. The FRC-derived LPA thus serves as a cell-extrinsic factor that optimizes T-cell movement through the densely packed LN reticular network, to fine-tune T-cell trafficking.


FRCs express the LPA-generating enzyme ATX in a lymphotoxin β receptor (LTβR)-signaling-dependent manner

Although CCR7 ligands are reported to stimulate intranodal T-cell motility (Okada and Cyster, 2007; Worbs et al., 2007; Huang et al., 2007), they are not sufficient to account for effective T-cell migration in LNs. Given that ATX, which generates the motogenic lysophospholipid, LPA, is expressed in HEV ECs to modulate lymphocyte motility (Nakasaki et al., 2008; Bai et al., 2013), we speculated that ATX and its product LPA are also expressed in other stromal cell subsets in the LN parenchyma and may control intranodal lymphocyte migration. Indeed, a recent paper showed that ATX is expressed in CCL21+ CD31- stromal cells in LNs (Katakai et al., 2014). We therefore subdivided the CD45- LN stromal cells into four stromal subsets (Figure 1A). As shown in Figure 1B, Enpp2/Autotaxin was readily detected in GP38+ CD31- FRCs as well as GP38- CD31+ blood endothelial cells (ECs), with negligible expression in lymphatic ECs and double-negative cells. Electron microscopic analysis confirmed that ATX was expressed in the FRCs surrounding collagen fiber bundles (Figure 1C). Interestingly, analyses using Ccl19-Cre x R26-EYFP mice, which constitutively express yellow fluorescent protein (YFP) in FRCs (Chai et al., 2013), revealed that Enpp2 was selectively expressed in LN FRCs but not splenic FRCs (Figure 1D). This Enpp2 expression was apparently dependent on LTβR signaling, because blocking LTβR signaling significantly reduced expression of Enpp2, Cxcl13, and Ccl19, but not Icam1, in FRCs (Figure 1E). This blockade also downregulated transcription of HEV marker genes such as Glycam1 and Ccl21 in BECs (Figure 1E), consistent with a previous report (Browning et al., 2005). These results confirm that similarly to HEV ECs, FRCs constitutively express the LPA-generating enzyme ATX, which is maintained at least in part by LTβR signaling.

Multiple LPA species are produced in the LN parenchyma by FRCs

To verify that LPA is generated in situ by FRC-derived ATX, we crossed Enpp2fl/fl mice and Ccl19-Cre mice to generate Ccl19-Cre Enpp2fl/fl mice that lacked ATX expression specifically in the FRCs. As expected, in the Ccl19-Cre Enpp2fl/fl mice, Enpp2 was completely lost in the FRCs but not in the BECs, whereas Ccl21 and GP38 expression was comparable between these strains (Figure 2A, Figure 2—figure supplement 1). The frequency of FRCs in stromal cells also appeared to be uncompromised by the deficiency of Enpp2 in FRCs (Figure 2—figure supplement 1). We then compared LPA production in the LN of these mice using imaging mass spectrometry (IMS). To this end, we first injected fluorescein-conjugated dextran, which labels lymphatics and the medulla, into the footpad, and LPA (18:0), LPA (18:1), LPA (18:2), and LPA (20:4) were then visualized in LN sections. As shown in Figure 2B, signals corresponding to LPA (18:0) were widely distributed in the LN. The signals were comparable in intensity and frequency in Enpp2fl/fl and Ccl19-Cre Enpp2fl/fl mice; this LPA species appears to be produced mainly within the cell (Aoki, J; unpublished observation) independently of ATX (Yukiura et al., 2011, Nishimasu, et al., 2011). In sharp contrast, signals corresponding to LPA (18:1), LPA (18:2), and LPA (20:4), the major species produced extracellularly by ATX (Yukiura et al., 2011), were predominantly observed in the paracortex both close to and at a distance from HEVs, but only marginally in the medulla. These signals were substantially decreased in the cortex of Ccl19-Cre Enpp2fl/fl as compared with Enpp2fl/fl mice (Figure 2B).

To verify that the cortical LPA signals associated with non-HEV structures were derived from FRCs, we next mapped the LPA signals relative to HEVs in Enpp2fl/fl mice and Ccl19-Cre Enpp2fl/fl mice by measuring the distance between individual signals and the nearest HEV. As shown in Figure 2C, the frequency of LPA (18:1), LPA (18:2), and LPA (20:4) signals within 50 μm of an HEV did not differ significantly between Enpp2fl/fl and Ccl19-Cre Enpp2fl/fl mice. However, the frequency of relatively distant signals (more than 50 μm) decreased substantially when ATX was ablated in FRCs. Hence, the median distance between LPA signals and HEVs was significantly reduced in Ccl19-Cre Enpp2fl/fl compared with Enpp2fl/fl mice, consistent with the idea that distant LPA signals were associated with FRCs. Together with the observations showing robust expression of ATX in FRCs, these findings indicate that the cortical LPA signals not associated with HEVs are mainly produced by FRCs in an ATX-dependent manner.

FRC-derived LPA promotes intranodal T-cell migration

To understand the role of FRC-derived LPA in regulating intranodal T-cell migration, we next examined the CD4+ T-cell interstitial migration in LNs by intravital two-photon microscopy. To this end, CD4+ T cells from WT mice expressing a transgene encoding enhanced GFP (eGFP) were injected intravenously into Enpp2fl/fl and Ccl19-Cre Enpp2fl/fl mice, and the intranodal T-cell migration in popliteal LNs (PLNs) was imaged 15–25 hr later (Video 1). As shown in Figure 3A, B, CD4+ T-cell movement and displacement from the original location in the PLN was substantially restricted in Ccl19-Cre Enpp2fl/fl compared with Enpp2fl/fl mice. The median T-cell velocity was also lower in Ccl19-Cre Enpp2fl/fl than in Enpp2fl/fl mice (Figure 3C, Figure 3—figure supplement 1). Measurement of the mean displacement and the motility coefficient, which represents the volume in which an average cell scans per unit time (Sumen et al., 2004), also indicated that T-cell motility was impaired in the LN parenchyma of Ccl19-Cre Enpp2fl/fl mice (Figure 3D,E), whereas the directionality of the intranodal T-cell movement was comparable in these mouse groups (Figure 3—figure supplement 2), supporting the hypothesis that FRC-derived LPA is required for efficient intranodal T-cell migration.

The Article can be read in full in eLife DOI

( The Authors are from: Osaka University Graduate School of Medicine, Japan; Osaka University, Japan; University of Turku, Finland; Keio University School of Medicine, Japan; JST Precursory Research for Embryonic Science and Technology project, Japan; Akita University, Japan; Kansai University of Health Sciences, Japan; Tohoku University, Japan; Kantonal Hospital St. Gallen, Switzerland; Core Research for Evolutional Science and Technology project, Japan)


P: 040216


Attenuation of AMPK Signaling by ROQUIN Promotes T Follicular Helper Cell Formation

In a recent research published in eLife Roybel R Ramiscal, Ian A Parish, Robert S Lee-Young, Jeffrey J Babon, Julianna Blagih, Alvin Pratama, Jaime Martin, Naomi Hawley, Jean Y Cappello, Pablo F Nieto, Julia I Ellyard, Nadia J Kershaw, Rebecca A Sweet, Christopher C Goodnow, Russell G Jones, Mark A Febbraio, Carola G Vinuesa, Vicki Athanasopoulos writes that T follicular helper cells (Tfh) are critical for the longevity and quality of antibody-mediated protection against infection. Yet few signaling pathways have been identified to be unique solely to Tfh development. ROQUIN is a post-transcriptional repressor of T cells, acting through its ROQ domain to destabilize mRNA targets important for Th1, Th17, and Tfh biology. Here, we report that ROQUIN has a paradoxical function on Tfh differentiation mediated by its RING domain: mice with a T cell-specific deletion of the ROQUIN RING domain have unchanged Th1, Th2, Th17, and Tregs during a T-dependent response but show a profoundly defective antigen-specific Tfh compartment. ROQUIN RING signaling directly antagonized the catalytic α1 subunit of adenosine monophosphate-activated protein kinase (AMPK), a central stress-responsive regulator of cellular metabolism and mTOR signaling, which is known to facilitate T-dependent humoral immunity. We therefore unexpectedly uncover a ROQUIN–AMPK metabolic signaling nexus essential for selectively promoting Tfh responses.

eLife digest

The immune system protects the body from invading microbes like bacteria and viruses. Upon recognizing the presence of these microbes, cells in the immune system are activated to destroy the foreign threat and clear it from the body.

A type of immune cell called T follicular helper cells (or Tfh for short) are formed during an infection and are essential for coordinating other immune cells to produce high-quality antibody proteins that attack the microbes. Without Tfh cells, life-long production of these protective antibodies is severely crippled, which can cause common variable immune deficiency and other serious immunodeficiency diseases. On the other hand, the body must also avoid generating excessive numbers of Tfh cells, which can lead to the production of antibodies that attack healthy cells of the body.

ROQUIN is a protein that inhibits the formation of Tfh cells and other types of active T cells. A region on the protein called the ROQ domain destabilizes particular molecules of ribonucleic acid (RNA) that are required for these specialist T cells to form and work properly. ROQUIN belongs to a large family of enzymes that have a so-called RING domain, which is a feature that enables these enzymes to attach tags onto specific target proteins to modify their activity or stability. However, it was not known whether the RING domain of ROQUIN was active.

Ramiscal et al. now address this question in mice. Unexpectedly, the experiments show that the RING domain is required to promote the formation of Tfh cells, but not other types of active T cells. This domain allows ROQUIN to repress an enzyme called AMPK, which normally blocks cell growth by regulating cell metabolism. The findings suggest that the different roles of the ROQ and RING domains allow ROQUIN to fine-tune the numbers of Tfh cells so that they remain within a safe range. In the future, these findings may aid the development of vaccines that are more efficient at generating protective Tfh cells to prevent infectious diseases.


High-affinity and long-lasting humoral immunity against infection requires controlled cross-talk between limiting CD4+CXCR5highPD1highBCL6high T follicular helper (Tfh) cells and immunoglobulin-maturing germinal center (GC) B cells in secondary lymphoid tissues (King et al., 2008; Victora and Nussenzweig, 2012; Nutt and Tarlinton, 2011; Ramiscal and Vinuesa, 2013). As the GC largely consists of clonally diverse B cells, Tfh cells especially in narrow numbers are best at maintaining a selective pressure for B cell competition, favoring the survival of greater affinity antigen-responsive GC B cell clones (Pratama and Vinuesa, 2014; Victora and Mesin, 2014). Deregulation of Tfh cells can lead to faulty GC selection that may also seed the production of autoantibodies (Weinstein et al., 2012; Vinuesa et al., 2005; Kim et al., 2015; Linterman et al., 2009) and GC-derived malignancies such as follicular lymphoma (Rawal et al., 2013; Klein and Dalla-Favera, 2008). To date, the signals that exclusively govern Tfh cell differentiation over other T cell effector subsets remains poorly characterized.

ROQUIN (also called ROQUIN1; encoded by Rc3h1) acts to post-transcriptionally repress Tfh cells by binding effector T cell transcripts via its winged-helix ROQ domain (Schuetz et al., 2014; Tan et al., 2014; Schlundt et al., 2014) and recruiting proteins of the RNA decapping and deadenylation machinery (Athanasopoulos et al., 2010; Glasmacher et al., 2010; Leppek et al., 2013; Pratama et al., 2013; Yu et al., 2007; Vogel et al., 2013) as well as the endoribonuclease REGNASE-1 (Jeltsch et al., 2014). Some of its RNA targets include the Tfh-polarising Icos (Glasmacher et al., 2010) and Il6 mRNA (Jeltsch et al., 2014) as well as Ox40 (Vogel et al., 2013) and Tnf (Pratama et al., 2013) transcripts. In sanroque mice, an Rc3h1 missense point mutation, encoding for a Met199 to Arg substitution translates into a minor conformational shift in the RNA-binding ROQ domain (Srivastava et al., 2015) of ROQUIN and a loss of function in post-transcriptional repression. This leads to excessive Tfh growth and systemic autoimmunity (Linterman et al., 2009; Vinuesa et al., 2005). Complete ablation of ROQUIN results in unexplained perinatal lethality in C57BL/6 mice and selective deletion of ROQUIN in T cells does not lead to Tfh cell accumulation nor autoimmunity (Bertossi et al., 2011). The latter is at least in part explained by the existence of the closely related family member ROQUIN2 (encoded by Rc3h2), which has overlapping functions with ROQUIN (Pratama et al., 2013; Vogel et al., 2013). The ROQUINM199R mutant protein has been proposed to act as a ‘niche-filling’ variant that has lost its RNA-regulating activity (Pratama et al., 2013) but can still localize to mRNA-regulating cytoplasmic granules to prevent the compensatory activity of ROQUIN2.

ROQUIN contains a conserved amino terminal RING finger with two conforming zinc-chelating sites (Srivastava et al., 2015), despite an atypical aspartate as its eighth zinc ligand synonymous to RBX1 (Kamura et al., 1999). This suggests ROQUIN may function as an E3 ubiquitin ligase (Deshaies and Joazeiro, 2009) but, to date, no such enzymatic activity of the ROQUIN RING domain has been demonstrated in mammals. In vivo attempts to delineate the cellular pathways regulated by ROQUIN are made challenging due to the existence of multiple protein domains in the protein (Figure 1—figure supplement 1a). The Caenorhabditis elegans ROQUIN ortholog, RLE-1, acts through its RING domain to ubiquitinate DAF-16, a pro-longevity forkhead box O (FOXO) transcription factor homolog (Li et al., 2007). We did not find any evidence for molecular binding between ROQUIN and the fruitfly or mammalian FOXO orthologs (Drosophila melanogaster FOXO and Mus musculus FOXO1 or FOXO3a; data not shown) and therefore set out to understand the role of ROQUIN RING signaling in CD4+ T cell development and function by generating mice that selectively lack the ROQUIN RING zinc finger.

We previously demonstrated that ROQUIN RING-deleted T cells in mice 6 days after sheep red blood cell (SRBC) immunization can form normal early Tfh cell responses but fail to promote optimal GC B cell reactions (Pratama et al., 2013). Here, in mice that have developed robust Tfh-dependent GC responses toward SRBC or infected with lymphocytic choriomeningitis virus (LCMV), we identify a novel and unexpected role of the ROQUIN RING domain in selectively promoting mature antigen-specific Tfh cell responses while leaving unaffected the development of other CD4+ effector T cell lineages. ROQUIN directly binds to and limits adenosine monophosphate-activated protein kinase (AMPK), a tumor suppressor and central regulator of T cell glucose uptake and glycolysis (MacIver et al., 2011). Our data indicate that loss of AMPK repression by deletion of the ROQUIN RING domain promotes stress granule persistence. This in turn cripples mTOR activity, otherwise known to play a critical role in driving CD4+ effector T cell expansion (Delgoffe et al., 2009; 2011) and T-dependent antibody responses (Keating et al., 2013; Zhang et al., 2011; Gigoux et al., 2014; De Bruyne et al., 2015).


The ROQUIN RING domain selectively controls Tfh cell formation

To examine the function of the ROQUIN RING domain in vivo, we generated two strains of C57BL/6 mice carrying either a germline deletion (designated ringless; ‘rin’ allele) or a T cell conditional deletion (Tringless; ‘Trin’ allele) of exon 2 in the Rc3h1 gene, which encodes the translation START codon and RING finger domain of the ROQUIN protein (Figure 1—figure supplement 1b, c and Pratama et al., 2013). In these mice, skipping of exon 2 resulted in splicing of exon 1 to exon 3 yielding an alternative in-frame Kozak translation initiation site at Met133 (Figure 1—figure supplement 1d, e). This predicted ROQUIN133-1130 protein product specifically lacks the RING domain (Figure 1—figure supplement 1f). Mice homozygous for the rin allele were perinatally lethal (Figure 1—figure supplement 1g–i), precluding T cell studies in intact animals. In contrast, Tringless mice were viable and showed no severe variations in thymic development and output of CD4 single positive T cells (Figure 1—figure supplement 2a–e). There were also no major changes in Th1 cell differentiation in Tringless mice infected with LCMV (Figure 1a), which predominantly yields LY6Chigh Th1 and LY6Clow Tfh virus-specific effector cells (Hale et al., 2013; Marshall et al., 2011). In Tringless animals immunized with SRBCs, the formation of Th1, Th2, Th17, and regulatory T cells also remained largely unperturbed (Figure 1—figure supplement 2f, g). This was mirrored in vitro with Tringless CD4+ naive T cells activated under Th1, Th2, Th17, or induced Treg (iTreg) polarizing conditions (Figure 1—figure supplement 2h) displaying maximal expression of intracellular TBET, GATA3, RORγT, and FOXP3 comparable to floxed wild-type T cell cultures (Figure 1—figure supplement 2i). Surprisingly in Tringless mice, there was an overall defective Tfh cell primary response to LCMV infection (Figure 1b–d) and to SBRC immunization (Figure 1—figure supplement 3a). ROQUIN RING-deficient T cells were also inefficient in supporting GC formation (Figure 1e, f and Figure 1—figure supplement 3b), which was associated with reduced IL-21 production (Figure 2a), a Tfh signature cytokine vital in supporting GC reactions (Liu and King, 2013).

By stimulating splenocytes ex vivo with GP61-80 peptide to identify virus-responsive IFNγ-producing Th1 cells (Figure 2b) and by examining splenic LYC6high Th1 cells amongst GP66-77+ tetramer stained T cells (Figure 2c), we verified that ROQUIN RING loss did not disrupt protective Th1 responses but caused a severe abrogation of virus-specific Tfh cells during LCMV infection (Figure 2d–f). Virus-specific T cells also showed significantly reduced expression of BCL6 (Figure 2g), an indispensible nuclear factor for Tfh cell terminal differentiation (Liu et al., 2013). Furthermore, we found an increased frequency of FOXP3+ T follicular regulatory (Tfr) cells within the total Tfh pool (Figure 2h) despite these Tfr cells not expressing a GP66-77 virus-specific T cell antigen receptor (TCR; Figure 2i). Nonetheless, as Tfr cells are negative regulators of GC reactions (Ramiscal and Vinuesa, 2013), their abundance may indicate augmented suppression of Tfh cells and long-term B cell responses.

ROQUIN undergoes RING-dependent autoubiquitination and directly limits AMPK activity

We next sought to determine the molecular basis for the ROQUIN RING domain as a determinant in protective Tfh cell responses. Several lines of evidence implicated an involvement of ROQUIN in the negative regulation of AMPK signaling: Rc3h1ringless fetuses displayed skeletal muscle atrophy of the thoracic diaphragm (Figure 1—figure supplement 1j), which is a characteristic phenotype of mice with overactive AMPK (Sanchez et al., 2012) and pointed to perinatal respiratory failure as the cause of the lethality. Also, AMPK over-expression in nematode worms has been shown to extend lifespan (Mair et al., 2011), an observation consistent with the phenotype of worms lacking the ROQUIN ortholog RLE-1 (Li et al., 2007). Since the AMPKα1 catalytic subunit is expressed in T cells and responds to TCR activation (Tamas et al., 2006), we tested the possibility of ROQUIN directly binding to this subunit of AMPK (encoded by Prkaa1). Upon ectopic expression in HEK293T cells, ROQUIN colocalized with AMPKα1 diffusely or in fine cytoplasmic speckles in resting cells and within larger cytoplasmic granules upon induction of oxidative stress (Figure 3a). We also observed colocalization of endogenous AMPKα1 within ROQUIN+ cytoplasmic granules in arsenite-treated primary C57BL/6 mouse embryonic fibroblasts (MEFs) (Figure 3b) with the use of an AMPKα1-specific antibody displaying no cross-reactivity toward the AMPKα2 subunit when ectopically expressed in HEK293T cells (Figure 3—figure supplement 1a). Unlike the AMPKα1 subunit, ectopically expressed AMPK β and γ regulatory subunits did not associate with ROQUIN+ cytoplasmic granules, although AMPKγ2 and AMPKγ3 exhibited generally diffuse cytoplasmic distribution (Figure 3—figure supplement 1b). We next determined if ROQUIN and AMPKα1 interacted by conducting in situ proximity ligation assays (PLAs) on primary C57BL/6 MEFs. Compared to control PLAs accounting for false interactions between endogenous AMPKα1 and non-expressed green fluorescent protein (GFP) detected by optimized anti-GFP immunostaining (Figure 3—figure supplement 1c), we found that endogenously expressed ROQUIN and AMPKα1 proteins localized with very close molecular proximity in both resting and arsenite-stressed cells (Figure 3c, d) at a frequency 15-fold higher or more than weak PLA interactions previously observed between ROQUIN and AGO2 (Srivastava et al., 2015). Moreover, we were able to coimmunoprecipitate ROQUIN and AMPKα1 when over-expressed in HEK293T cells (Figure 3—figure supplement 1d) or expressed endogenously in the mouse T lymphoblast line EL4 cells (Figure 3e). Together with the PLAs, this indicated that ROQUIN bound specifically with the α1 subunit of AMPK and that under physiological conditions, the two proteins could form a stable complex.

( Author are from Australian National University, Australia; Baker IDI Heart and Diabetes Institute, Australia; Walter and Eliza Hall Institute of Medical Research, Australia; McGill University, Canada; Garvan Institute of Medical Research, Australia)

The Article can be read in full in eLife  DOI 



P: 120116



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