January 19 - 25, 2020: Issue 434

Molecular probe maps misfolded proteome state in live cells

January 9, 2020
The folding state of the proteins in live cells often reflect the cell's general health. Australian scientists have developed a molecular probe that senses the state of the proteome -- the entire set of the proteins -- by measuring the polarity of the protein environment. The fluorescence signal of the probe quantifies unfolding and its chameleon-like colour shift maps the cellular regions of enhanced misfolding, says the study published in the journal Angewandte Chemie.

If live cells are stressed, protein-synthesis and folding-correction mechanisms are out of balance. Misfolded proteins remain stuck, enhanced degradation occurs, and inactive proteins and protein debris aggregate to form granules and condensates in the cytoplasm. Such aggregates play an important role in neurodegenerative diseases and cancer. One driving factor for the aggregation of misfolded proteins seems to be the polarity -- the electronic distribution in an environment. Yuning Hong and colleagues at La Trobe University Melbourne and The University of Melbourne, Australia, have designed a two-modal fluorogenic probe to monitor protein aggregation in greater detail.

In one mode, the probe senses misfolded proteins. Correctly folded proteins are often stabilized by bridges made of the amino acid cysteine. These bridges are usually deeply buried, whereas misfolded proteins expose the cysteine residues at the surface. When the probe binds to cysteine exposed by a misfolded protein chain, fluorescence is switched on, explain the authors.

In the other mode, the probe assesses the polarity. Polar environments indicate an unbalanced electronic distribution, which can be measured by the dielectric constant. To measure this parameter, the researchers added an electronic "push-pull" chemical group to the fluorogenic probe. They observed that, in polar solutions with a high dielectric constant, the fluorogenic probe called NTPAN-MI emitted its fluorescence signal with a color shift. This "chameleon-like" color change thus indicates a polarity change.

The authors tested the NTPAN-MI probe on a human cell line, which they stressed by adding drugs that interfered with protein synthesis and folding. The scientists observed normal fluorescence in untreated cells, but bright fluorescence when unfolded or misfolded proteins accumulated in cells treated with toxins or infected by virus. In addition, the color shift signaled the polarity of the environment and thus the proteome state of each cellular compartment. The researchers reported that they visualized the "unfolded protein load" in the nucleus for the first time. Previous methods could only measure unfolded proteins in the cytoplasm.

With its two sensing modes -- measuring unfolding and the polarity of the protein environment -- the NTPAN-MI probe provides a sharper picture of the stress responses of live cells than what can be obtained with only one-modal probes or different methods. The authors point out that their method would allow scientists to obtain more accurate knowledge of the crosstalk of the cellular components in response to stress.

Tze Cin Owyong, Pramod Subedi, Jieru Deng, Elizabeth Hinde, Jason J. Paxman, Jonathan M. White, Weisan Chen, Begoña Heras, Wallace W. H. Wong, Yuning Hong. A Molecular Chameleon for Mapping Subcellular Polarity in an Unfolded Proteome Environment. Angewandte Chemie International Edition, 2020; DOI: 10.1002/anie.201914263

Australian Meteorite contains the oldest material on Earth: 7-billion-year-old stardust

January 13, 2020
Stars have life cycles. They're born when bits of dust and gas floating through space find each other and collapse in on each other and heat up. They burn for millions to billions of years, and then they die. When they die, they pitch the particles that formed in their winds out into space, and those bits of stardust eventually form new stars, along with new planets and moons and meteorites. And in a meteorite that fell fifty years ago in Australia, scientists have now discovered stardust that formed 5 to 7 billion years ago -- the oldest solid material ever found on Earth.

"This is one of the most exciting studies I've worked on," says Philipp Heck, a curator at the Field Museum, associate professor at the University of Chicago, and lead author of a paper describing the findings in the Proceedings of the National Academy of Sciences. "These are the oldest solid materials ever found, and they tell us about how stars formed in our galaxy."

The materials Heck and his colleagues examined are called presolar grains-minerals formed before the Sun was born. "They're solid samples of stars, real stardust," says Heck. These bits of stardust became trapped in meteorites where they remained unchanged for billions of years, making them time capsules of the time before the solar system..

But presolar grains are hard to come by. They're rare, found only in about five percent of meteorites that have fallen to Earth, and they're tiny-a hundred of the biggest ones would fit on the period at the end of this sentence. But the Field Museum has the largest portion of the Murchison meteorite, a treasure trove of presolar grains that fell in Australia in 1969 and that the people of Murchison, Victoria, made available to science. Presolar grains for this study were isolated from the Murchison meteorite for this study about 30 years ago at the University of Chicago.

"It starts with crushing fragments of the meteorite down into a powder ," explains Jennika Greer, a graduate student at the Field Museum and the University of Chicago and co-author of the study. "Once all the pieces are segregated, it's a kind of paste, and it has a pungent characteristic-it smells like rotten peanut butter."

This "rotten-peanut-butter-meteorite paste" was then dissolved with acid, until only the presolar grains remained. "It's like burning down the haystack to find the needle," says Heck.

Once the presolar grains were isolated, the researchers figured out from what types of stars they came and how old they were. "We used exposure age data, which basically measures their exposure to cosmic rays, which are high-energy particles that fly through our galaxy and penetrate solid matter," explains Heck. "Some of these cosmic rays interact with the matter and form new elements. And the longer they get exposed, the more those elements form.

"I compare this with putting out a bucket in a rainstorm. Assuming the rainfall is constant, the amount of water that accumulates in the bucket tells you how long it was exposed," he adds. By measuring how many of these new cosmic-ray produced elements are present in a presolar grain, we can tell how long it was exposed to cosmic rays, which tells us how old it is.

The researchers learned that some of the presolar grains in their sample were the oldest ever discovered-based on how many cosmic rays they'd soaked up, most of the grains had to be 4.6 to 4.9 billion years old, and some grains were even older than 5.5 billion years. For context, our Sun is 4.6 billion years old, and Earth is 4.5 billion.

But the age of the presolar grains wasn't the end of the discovery. Since presolar grains are formed when a star dies, they can tell us about the history of stars. And 7 billion years ago, there was apparently a bumper crop of new stars forming-a sort of astral baby boom.

"We have more young grains that we expected," says Heck. "Our hypothesis is that the majority of those grains, which are 4.9 to 4.6 billion years old, formed in an episode of enhanced star formation. There was a time before the start of the Solar System when more stars formed than normal."

This finding is ammo in a debate between scientists about whether or not new stars form at a steady rate, or if there are highs and lows in the number of new stars over time. "Some people think that the star formation rate of the galaxy is constant," says Heck. "But thanks to these grains, we now have direct evidence for a period of enhanced star formation in our galaxy seven billion years ago with samples from meteorites. This is one of the key findings of our study."

Heck notes that this isn't the only unexpected thing his team found. As almost a side note to the main research questions, in examining the way that the minerals in the grains interacted with cosmic rays, the researchers also learned that presolar grains often float through space stuck together in large clusters, "like granola," says Heck. "No one thought this was possible at that scale."

Heck and his colleagues look forward to all of these discoveries furthering our knowledge of our galaxy. "With this study, we have directly determined the lifetimes of stardust. We hope this will be picked up and studied so that people can use this as input for models of the whole galactic life cycle," he says.

Heck notes that there are lifetimes' worth of questions left to answer about presolar grains and the early Solar System. "I wish we had more people working on it to learn more about our home galaxy, the Milky Way," he says.

"Once learning about this, how do you want to study anything else?" says Greer. "It's awesome, it's the most interesting thing in the world."

"I always wanted to do astronomy with geological samples I can hold in my hand," says Heck. "It's so exciting to look at the history of our galaxy. Stardust is the oldest material to reach Earth, and from it, we can learn about our parent stars, the origin of the carbon in our bodies, the origin of the oxygen we breathe. With stardust, we can trace that material back to the time before the Sun."

"It's the next best thing to being able to take a sample directly from a star," says Greer.


Murchison meteorite at the The National Museum of Natural History (Washington)

This study was contributed to by researchers from the Field Museum, University of Chicago, Lawrence Livermore National Laboratory, Washington University, Harvard Medical School, ETH Zurich, and the Australian National University. Funding was provided by NASA, the TAWANI Foundation, the National Science Foundation, the Department of Energy, the Swiss National Science Foundation, the Brazilian National Council for Scientific and Technological Development and the Field Museum's Science and Scholarship Funding Committee.

Philipp R. Heck, Jennika Greer, Levke Kööp, Reto Trappitsch, Frank Gyngard, Henner Busemann, Colin Maden, Janaína N. Ávila, Andrew M. Davis, Rainer Wieler. Lifetimes of interstellar dust from cosmic ray exposure ages of presolar silicon carbide. Proceedings of the National Academy of Sciences, Jan. 13, 2020; DOI: 10.1073/pnas.1904573117

Novel avian species: 10 new bird taxa in islands of Wallacea

January 10, 2020
Birds are the best known class of animals, and since 1999, only five or six new species have been described each year on average. Recently, a joint research team from the National University of Singapore (NUS) and the Indonesian Institute of Sciences (LIPI) made a quantum leap in the discovery of cryptic avian diversity by uncovering five bird species and five subspecies new to science.

The team, led by Associate Professor Frank Rheindt from the Department of Biological Sciences at NUS Faculty of Science, found the birds in three small island groups off Sulawesi, Indonesia. The islands are situated in Indonesia's Wallacea region, an archipelago at the interface between the Oriental and Australian biogeographical realms, named after Sir Alfred Wallace, who was the most famous historical collector exploring the area.

The results of the study, which were published in the journal Science on January 10th 2020, provide evidence that our understanding of species diversity of complex areas such as Wallacea remains incomplete even for relatively well-known groups such as birds. The findings also suggest that modern exploration to find undescribed species diversity can be targeted to areas of high promise.

Insights from paleo-climatology and history guided discovery of new taxa
Sea depth is an important and long-neglected factor in determining the distinctness of an island's terrestrial communities. The Earth undergoes periods of glacial-interglacial cycles, leading to the formation of land bridges between shallow islands during ice ages, allowing fauna of the different islands to interbreed. Deep sea islands, which have always been isolated, and high elevation islands are more likely to harbour endemism due to absence of land connections even during glacial cycles.

Guided by this knowledge, Assoc Prof Rheindt and his team concentrated their research efforts on the islands of Taliabu and Peleng, which are located off the north-eastern coast of Sulawesi, as bathymetric data indicate the presence of deep sea between these islands and Sulawesi.

The research team also examined the accounts of historic collectors such as Sir Alfred Wallace, and sought to focus on parts of Wallacea that had received the least coverage by historic collectors, as these areas would hold the highest promise of harbouring undescribed avian diversity.

The islands that the team targeted were characterised by such incomplete historic coverage: Taliabu and its neighbours, together forming the Sula group, were only briefly visited by eight historic collecting expeditions, all of which remained in coastal areas and failed to penetrate the highlands of the interior because of poor accessibility; and Peleng and the remaining islands of the Banggai group were visited along their coastline by only three historic collectors who never ventured far uphill into the interior.

New taxa found
Assoc Prof Rheindt and his team undertook extensive fieldwork in the three remote islands for six weeks, from November 2013 to January 2014, and collected 10 new, long-overlooked avian forms.

By integrating genomic and phenotypic research methodologies, the team successfully described five new songbird species and five new subspecies:
  • On Taliabu, they found three new species: the Taliabu Grasshopper-Warbler, the Taliabu Myzomela and the Taliabu Leaf-Warbler; as well as three subspecies: the Taliabu Snowy-browed Flycatcher, Taliabu Island Thrush and Sula Mountain Leaftoiler.
  • On Peleng, two new species -- the Peleng Fantail and the Peleng Leaf-Warbler -- and a new subspecies -- the Banggai Mountain Leaftoiler -- were discovered.
  • On Togian, a new subspecies -- the Togian Jungle-Flycatcher -- was found.

The Taliabu Grasshopper-Warbler is one of the new bird species discovered by the NUS-LIPI team led by Assoc Prof Frank Rheindt, during their expedition in Indonesia’s Wallacea region (Photo: James Eaton/Birdtour Asia)

"Studying the routes and operations of historic collecting expeditions and identifying gaps has been a fruitful approach to pinpoint focal areas in our case. The description of this many bird species from such a geographically limited area is a rarity," shared Assoc Prof Rheindt.

He added, "Going forward, the use of earth-history and bathymetric information could also be applied to other terrestrial organisms and regions beyond the Indonesian Archipelago to identify promising islands that potentially harbour new taxa to be uncovered."

Implications for conservation
During the expedition, the research team found that both Taliabu and Peleng have suffered from rampant forest destruction. There is virtually no primary lowland forest on both islands, and most highland forests have been impacted by some form of logging or forest fires.

"While most of the avifauna we described seems to tolerate some form of habitat degradation and is readily detected in secondary forest and edge, some species or subspecies are doubtless threatened by the immense levels of habitat loss on these islands. As such, urgent, long-lasting conservation action is needed for some of the new forms to survive longer than a couple of decades beyond their date of description," said Assoc Prof Rheindt.

Frank E. Rheindt, Dewi M. Prawiradilaga, Hidayat Ashari, Suparno, Chyi Yin Gwee, Geraldine W. X. Lee, Meng Yue Wu, Nathaniel S. R. Ng. A lost world in Wallacea: Description of a montane archipelagic avifauna. Science, 2020 DOI: 10.1126/science.aax2146

Disclaimer: These articles are not intended to provide medical advice, diagnosis or treatment.  Views expressed here do not necessarily reflect those of Pittwater Online News or its staff.

Most meat eaters support veganism as 'ethical' and good for the environment

January 8, 2020
A recently published study that looked at public opinion on plant-based diets has found widespread support for the ethics and environmental benefits of veganism and vegetarianism among meat eaters, but finds that the barriers are practical matters of taste, price, and convenience.

Analysis published in the journal Sustainability from University of Bath Psychology PhD student Chris Bryant suggests that 73% of meat eaters surveyed considered veganism to be 'ethical', 70% said it was good for the environment and half (50%) considered it healthy. 60% thought veganism was 'acceptable'.

By contrast, over 80% of respondents thought veganism was not easy, 77% thought it 'inconvenient' and over 60% thought it was not enjoyable. Attitudes from respondents towards vegetarianism were significantly more positive on almost all counts.

The study, which involved 1,000 men and women with an average age of 34, was conducted in September 2018. Participants were recruited online through the survey platform Prolific. The work was partially funded by the charity Viva! as part of Chris Bryant's ESRC PhD. His research is focused on shifting preferences away from animal consumption in view of climate change and reducing animal suffering.

Chris Bryant from Bath's Department of Psychology explains: "At a time of year when many people are considering switching to plant-based diets with 'Veganuary', this study shows that most people already agree with the ethics of veganism and are aware of the benefits of vegan diets to the environment.

"That many people agree with the principles of veganism is one thing, but in terms of changing behaviours we need to acknowledge that for many it has been seen as too expensive, inconvenient and a sacrifice in terms of taste.

"Interestingly, in the time since this study was conducted, these things have all changed substantially. Supermarkets, restaurants, and even fast food outlets have developed numerous high quality and affordable vegan options. Having direct replacements for the foods people know and like makes it easier for everybody to consume fewer animal products. If we are to reduce animal product consumption in the UK and around the world, the development of high quality affordable alternatives to animal products is key."

January marks the start of Veganuary, the annual campaign which last year inspired over a million people to try vegan diets for January and beyond.

Earlier this month, the fast food bakery Greggs launched a vegan steak bake. This follows the introduction of Subway's vegan Meatball Marinara sub and even a vegan KFC burger.

Christopher J. Bryant. We Can’t Keep Meating Like This: Attitudes towards Vegetarian and Vegan Diets in the United Kingdom. Sustainability, 2019; 11 (23): 6844 DOI: 10.3390/su11236844

Have your health and eat meat too: Making a Mediterranean diet work Down Under

December 9, 2019: University of South Australia
Barbecued, stir-fried or roasted, there's no doubt that Aussies love their meat. Consuming on average nearly 100 kilograms of meat per person per year, Australians are among the top meat consumers worldwide.

But with statistics showing that most Australians suffer from a poor diet, and red meat production adding to greenhouse-gas emissions, finding a balance between taste preferences, environmental protection, and health benefits is becoming critical.

Now, researchers from the University of South Australia can reveal that Aussies can have their health and eat meat too with a new version of the Mediterranean diet adapted for Australian palates.

Incorporating 2-3 serves (250g) of fresh lean pork each week, the Mediterranean-Pork (Med-Pork) diet delivers cognitive benefits, while also catering to Western tastes, and ensuring much lower greenhouse-gas emissions than beef production.

A typical Mediterranean diet includes extra virgin olive oil, fruits, vegetables, nuts, seeds, legumes, wholegrain breads, pastas and cereals, moderate consumption of fish and red wine, and low consumption of red meat, sweet and processed foods.

This study compared the cognitive effects of people aged 45-80 years and at risk of cardiovascular disease following a Med-Pork or a low-fat diet (often prescribed to negate risk factors for cardiovascular disease), finding that the Med-Pork intervention outperformed the low-fat diet, delivering higher cognitive processing speeds and emotional functioning, both of which are markers of good mental health.

UniSA researcher Dr Alexandra Wade says the new Med-Pork diet will provide multiple benefits for everyday Australians.

"The Mediterranean diet is widely accepted as the world's healthiest diet and is renowned for delivering improved cardiovascular and cognitive health, but in Western cultures, the red meat restrictions of the diet could make it hard for people to stick to," Dr Wade says.

"By adding pork to the Mediterranean diet, we're broadening the appeal of the diet, while also delivering improved cognitive function.

"This bodes well for our ageing population, where age-associated diseases, such as dementia, are on the rise.

"Improving people's processing speed shows the brain is working well. So, in Australia, the Med-Pork diet is an excellent lifestyle intervention where dementia is one of the leading causes of disability and the second leading cause of death.

"Then, when you add the fact that pork production emits only a fraction of the greenhouse gases compared to beef, and the Med-Pork diet is really ticking all boxes -- taste, health and environment."

According to the World Health Organisation (WHO), by 2050, the number of people aged 60 years and older will outnumber children younger than five years old, bringing common health concerns associated with ageing into the fore. Further WHO statistics shows that cardiovascular disease is the number 1 cause of death globally and that dementia is one of the major causes of disability and dependency among older people worldwide.

Dr Wade says the Mediterranean diet with lean pork is an effective adaption of a successful eating plan

"Put simply, a Mediterranean diet encourages healthy eating. It's a food-based eating pattern that, with pork, still delivers significant health benefits," Dr Wade says.

"We're hoping that more people will find this dietary pattern to be more in line with their accustomed eating patterns and therefore more adoptable.

"Making a Mediterranean Diet work 'Down Under' is just one step in a bigger picture for better health."

Alexandra T. Wade, Courtney R. Davis, Kathryn A. Dyer, Jonathan M. Hodgson, Richard J. Woodman, Hannah A. D. Keage, Karen J. Murphy. A Mediterranean Diet with Fresh, Lean Pork Improves Processing Speed and Mood: Cognitive Findings from the MedPork Randomised Controlled Trial. Nutrients, 2019; 11 (7): 1521 DOI: 10.3390/nu11071521

Human immune cells produced in a dish in world first

January 8, 2020: Murdoch Childrens Research Institute
A research team has reproduced and visualised the earliest developmental steps in human immune cell production in the laboratory and are now set to advance our understanding of childhood diseases like leukemia and autoimmune conditions.

One day the advance could lead to a patient's own skin cells being used to produce new cells for cancer immunotherapy or to test autoimmune disease interventions.

The group, led by Professors Ed Stanley and Andrew Elefanty, from the Murdoch Children's Research Institute in Melbourne, Australia, said the work has added definitive evidence about how the body's earliest immune cells are formed.

These lymphocytes are produced by cells which form the embryo's first organs rather than the blood-producing stem cells that sit inside the body's bone marrow.

The research combined two powerful laboratory techniques, genetic engineering and a novel way of growing stem cells, to make the breakthrough, which has been published in the  journal Nature Cell Biology.

First, the team engineered pluripotent stem cells to glow green when a specific protein marker of early immune cells, RAG1, was switched on. RAG1 is responsible for creating the immune response to infections and vaccines.

Next, the team isolated the glowing green RAG1-positive cells and showed that they could also form multiple immune cell types, including cells required for shaping the development of the whole immune system.

"We think these early cells might be important for the correct maturation of the thymus, the organ that acts as a nursery for T-cells" said Professor Stanley.

"These RAG1 cells are like the painters and decorators who set up that nursery, making it a safe and cosy environment for later-born immune cells," he said.

Professor Elefanty said, "Although a clinical application is likely still years away, we can use this new knowledge to test ideas about how diseases like childhood leukaemia and type 1 diabetes develop. Understanding more about the steps these cells go through, and how we can more efficiently nudge them down a desired pathway, is going to be crucial to that process."

Ali Motazedian, Freya F. Bruveris, Santhosh V. Kumar, Jacqueline V. Schiesser, Tyrone Chen, Elizabeth S. Ng, Ann P. Chidgey, Christine A. Wells, Andrew G. Elefanty, Edouard G. Stanley. Multipotent RAG1 progenitors emerge directly from haemogenic endothelium in human pluripotent stem cell-derived haematopoietic organoids. Nature Cell Biology, 2020; 22 (1): 60 DOI: 10.1038/s41556-019-0445-8

Bacterial link in celiac disease

January 8, 2020
Bacterial exposure has been identified as a potential environmental risk factor in developing celiac disease, a hereditary autoimmune-like condition that affects about one in 70 Australians.

It is estimated that half of all Australians are born with one of two genes that cause celiac disease, and approximately one in 40 are likely to develop the condition.

People with celiac disease must follow a lifelong gluten-free diet, as even small amounts of gluten can cause health problems.

While environmental factors are known to trigger celiac Disease in those with the genetic predisposition, exactly how that works has remained unclear.

Scientists from the Monash Biomedicine Discovery Institute (BDI) and ARC Centre of Excellence in Advanced Molecular Imaging, have now provided a molecular foundation for microbial exposure as a potential environmental factor in the development of celiac disease.

The results of the study, done in collaboration with researchers at Leiden University Medical Centre and the Walter and Eliza Hall Institute of Medical Research, have been published in the journal Nature Structural and Molecular Biology.

Co-Lead researcher Dr Hugh Reid, from Monash University, said the team showed, at the molecular level, how receptors isolated from immune T cells from celiac disease patients can recognise protein fragments from certain bacteria that mimic those fragments from gluten.

Exposure to such bacterial proteins may be involved in the generation of aberrant recognition of gluten by these same T cells when susceptible individuals eat cereals containing gluten, he said.

"In celiac disease you get aberrant reactivity to gluten and we have provided a proof-of-principle that there's a link between gluten proteins and proteins that are found in some bacteria," he said.

"That is, it's possible that the immune system reacts to the bacterial proteins in a normal immune response and in so doing develops a reaction to gluten proteins because, to the immune system, they look indistinguishable -- like a mimic."

Dr Reid said the findings could eventually lead to diagnostic or therapeutic approaches to celiac disease.

About celiac disease
Celiac disease is caused by an aberrant reaction of the immune system to gluten, a protein which occurs naturally in grains such as wheat, rye, barley and oats, and therefore is typically found in bread, pastries and cakes. Immune system cells, known as T cells, regard gluten as a foreign substance, and initiate action against it.

In patients with CD, activation of these T cells leads to an inflammatory response in the small intestine causing a wide range of symptoms including diarrhea, bloating and malabsorption of nutrients, to name a few.

People with celiac disease must follow a lifelong gluten-free diet, as even small amounts of gluten can cause health problems. If left untreated, the disease can cause serious issues including malnutrition, osteoporosis, depression and infertility, and there is a small increased risk of certain forms of cancer, such as lymphoma of the small bowel.

Jan Petersen, Laura Ciacchi, Mai T. Tran, Khai Lee Loh, Yvonne Kooy-Winkelaar, Nathan P. Croft, Melinda Y. Hardy, Zhenjun Chen, James McCluskey, Robert P. Anderson, Anthony W. Purcell, Jason A. Tye-Din, Frits Koning, Hugh H. Reid, Jamie Rossjohn. T cell receptor cross-reactivity between gliadin and bacterial peptides in celiac disease. Nature Structural & Molecular Biology, 2019; DOI: 10.1038/s41594-019-0353-4

New auto-inflammatory disease called CRIA syndrome

January 8, 2020
Over the last 20 years, three families have been unknowingly linked to one another by an unknown illness. Researchers at the National Human Genome Research Institute (NHGRI) and other organizations have now identified the cause of the illness, a new disease called CRIA syndrome. The results of their work were published in the journal Nature.

Discovering a new disease
NHGRI scientific director Daniel Kastner, M.D., Ph.D., a pioneer in the field of auto-inflammatory diseases, and his team, had never seen a condition like this one. Symptoms include fevers, swollen lymph nodes, severe abdominal pain, gastrointestinal problems, headaches and, in some cases, abnormally enlarged spleen and liver.

The disorder has characteristics typical of an auto-inflammatory disease, where the immune system appears to be activated without any apparent trigger. Although the condition is not life-threatening, patients have persistent fever and swollen lymph nodes from childhood to old age, as well as other symptoms that can lead to lifelong pain and disability.

When confronted by patients' symptoms, who were first seen at the NIH Clinical Center, researchers looked for infections and cancer as the cause. After those were ruled out, they sought answers in the genome, a person's complete set of DNA. Kastner and his team sequenced gene regions across the genome and discovered only one gene -- RIPK1 -- to be consistently different in all patients.

Researchers identified a specific type of variation in the patients: a single DNA letter at a specific location incorrectly changed. This change can alter the amino acid added to the encoded protein. These are called "missense" mutations.

Remarkably, each of the three families had its own unique missense mutation affecting the very same DNA letter in the RIPK1 gene. Each affected person had one mutant and one normal copy of the gene, while the unaffected family members had two normal copies of the gene.

The researchers also looked at 554 people with sporadic unexplained fever, swollen glands and other symptoms or diseases, and then at over a quarter million people from public sequence databases to see if they encountered the same RIPK1 mutations. When they did not find such mutations elsewhere, it was clear that they were onto something new.

"It was as if lightning had struck three times in the same place," said Kastner, who led the NHGRI team. "This discovery underscores the tremendous power of combining astute clinical observation, state-of-the-art DNA sequencing, and the sharing of sequence data in large publicly-accessible databases. We live in a very special time."

Cause and effect
The RIPK1 gene encodes for the RIPK1 protein, which is involved in the body's response to inflammation and programmed cell death. To make sure that RIPK1 action does not initiate inflammation and cell death in all cells, another protein "cuts" the RIPK1 protein at a specific location in the protein sequence. The research team noticed that all the mutations in CRIA patients occur at the location where RIPK1 usually gets cut, resulting in an uncuttable, seemingly indestructible RIPK1 protein.

This suggests that cutting RIPK1, thereby disarming it, is crucial to controlling cell death and inflammation. Recognizing this cause-effect relationship, Kastner's team named the resulting disease cleavage-resistant RIPK1-induced auto-inflammatory (CRIA) syndrome.

Although the researchers made the connection between CRIA syndrome and RIPK1 mutations, they still needed to understand the molecular mechanisms involved in the disease. To do this, Kastner and his team collaborated with Najoua Laloui, Ph.D., and John Silke, Ph.D., at the Walter and Eliza Hall Institute in Australia, who made specialised mouse models with similar RIPK1 mutations as seen in CRIA patients.

The Australian team discovered that mouse embryos with two mutant copies of RIPK1 (and no normal copy) died in the uterus due to excessive cell death signals, which further confirmed the importance of cutting RIPK1 to limit its function in normal cells. However, mice bearing one mutant copy of RIPK1 and one normal copy, as is the case for CRIA patients, were mostly normal but had heightened responses to a variety of inflammatory stimuli, which the researchers think may suggest a possible mechanism for how the human disease occurs.

Finding answers
Kastner and his team worked to find a treatment for CRIA syndrome. Seven patients with the condition were given therapies that are known to reduce inflammation. While drugs such as etanercept and anakinra, which are routinely used to treat autoinflammatory and chronic diseases such as rheumatoid arthritis, had little effect on the patients, one biological drug did.

Tocilizumab, a drug that suppresses the immune system, reduced the severity and frequency of CRIA syndrome symptoms in five out of seven patients in some cases with life-changing effects.

Hirotsugu Oda, M.D., Ph.D., a post-doctoral researcher in Kastner's laboratory and co-first author of the paper, said: "As a physician-scientist, the most thrilling experience to me was to hear the mother of a CRIA patient say that her son was a completely different, healthy child after the tocilizumab treatment. Through the genetic diagnosis, we were able to contribute to the treatment of a few patients. This is, after all, the ultimate goal."

Surprised by this serendipitous result, researchers are now trying to understand the detailed molecular mechanism that enables tocilizumab to treat CRIA. Specific inhibitors of RIPK1, which are under development, may also hold promise in both CRIA and other seemingly intractable inflammatory conditions.

Najoua Lalaoui, Steven E. Boyden, Hirotsugu Oda, Geryl M. Wood, Deborah L. Stone, Diep Chau, Lin Liu, Monique Stoffels, Tobias Kratina, Kate E. Lawlor, Kristien J. M. Zaal, Patrycja M. Hoffmann, Nima Etemadi, Kristy Shield-Artin, Christine Biben, Wanxia Li Tsai, Mary D. Blake, Hye Sun Kuehn, Dan Yang, Holly Anderton, Natasha Silke, Laurens Wachsmuth, Lixin Zheng, Natalia Sampaio Moura, David B. Beck, Gustavo Gutierrez-Cruz, Amanda K. Ombrello, Gineth P. Pinto-Patarroyo, Andrew J. Kueh, Marco J. Herold, Cathrine Hall, Hongying Wang, Jae Jin Chae, Natalia I. Dmitrieva, Mark McKenzie, Amanda Light, Beverly K. Barham, Anne Jones, Tina M. Romeo, Qing Zhou, Ivona Aksentijevich, James C. Mullikin, Andrew J. Gross, Anthony K. Shum, Edwin D. Hawkins, Seth L. Masters, Michael J. Lenardo, Manfred Boehm, Sergio D. Rosenzweig, Manolis Pasparakis, Anne K. Voss, Massimo Gadina, Daniel L. Kastner, John Silke. Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease. Nature, 2019; 577 (7788): 103 DOI: 10.1038/s41586-019-1828-5