Category Archives: Cell Biology

Molecular structure of the cell nucleoskeleton revealed for the first time – Science Daily

Compared to bacteria, in eukaryotes the genetic material is located in the cell nucleus. Its outer shell consists of the nuclear membrane with numerous nuclear pores. Molecules are transported into or out of the cell nucleus via these pores. Beneath the membrane lies the nuclear lamina, a threadlike meshwork merely a few millionths of a millimeter thick. This stabilizes the cell nucleus and protects the DNA underneath from external influences. Moreover, the lamina plays a key role in essential processes in the cell nucleus -- such as the organization of the chromosomes, gene activity and the duplication of genetic material before cell division.

Detailed 3D image of the nuclear lamina in its native environment

Now, for the first time, a team of researchers headed by cell biology professor Ohad Medalia from the Department of Biochemistry at UZH has succeeded in elucidating the molecular architecture of the nuclear lamina in mammalian cells in detail. The scientists studied fibroblast cells of mice using cryo-electron tomography. "This technique combines electron microscopy and tomography, and enables cell structures to be displayed in 3D in a quasi-natural state," explains Yagmur Turgay, the first author of the study. The cells are shock-frozen in liquid ethane at minus 190 degrees without being pretreated with harmful chemicals, thereby preserving the cell structures in their original state.

"The lamin meshwork is a layer that's around 14 nanometers thick, located directly beneath the pore complexes of the nuclear membrane and consists of regions that are packed more or less densely," says Yagmur Turgay, describing the architecture of the nucleoskeleton. The scaffold is made of thin, threadlike structures that differ in length -- the lamin filaments. Only 3.5 nanometers thick, the lamin filaments are much thinner and more delicate than the structures forming the cytoskeleton outside the cell nucleus in higher organisms.

New approach for research on progeria and muscular dystrophy

The building blocks of the filaments are two proteins -- type A and B lamin proteins -- which assemble into polymers. They consist of a long stem and a globular domain, much like a pin with a head. Individual mutations in the lamin gene elicit severe diseases with symptoms such as premature aging (progeria), muscle wasting (muscular dystrophy), lipodystrophy and damage of the nervous system (neuropathies). "Cryo-electron tomography will enable us to study the structural differences in the nuclear lamina in healthy people and in patients with mutations in the lamin gene in detail in the future," concludes Ohad Medalia. According to the structural biologist, this method permits the development of new disease models at molecular level, which paves the way for new therapeutic interventions.

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In cleaning up misfolded proteins, cell powerhouses can break down – Science Daily

Working with yeast and human cells, researchers at Johns Hopkins say they have discovered an unexpected route for cells to eliminate protein clumps that may sometimes be the molecular equivalent of throwing too much or the wrong trash into the garbage disposal. Their finding, they say, could help explain part of what goes awry in the progression of such neurodegenerative diseases as Parkinson's and Alzheimer's.

Proteins in the cell that are damaged or folded incorrectly tend to form clumps or aggregates, which have been thought to dissolve gradually in a cell's cytoplasm or nucleus thanks to an enzyme complex called the proteasome, or in a digestive organelle called the lysosome.

But in experiments on yeast, which has many structures similar to those in human cells, the Johns Hopkins scientists unexpectedly found that many of those protein clumps break down in the cell's energy-producing powerhouses, called mitochondria. They also found that too many misfolded proteins can clog up and damage this vital structure.

The team's findings, described March 1 in Nature, could help explain why protein clumping and mitochondrial deterioration are both hallmarks of neurodegenerative diseases.

Rong Li, Ph.D., professor of cell biology, biomedical engineering and oncology at the Johns Hopkins University School of Medicine and a member of the Johns Hopkins Kimmel Cancer Center, who led the study, likens the disposal system to the interplay between a household's trash and a garbage disposal in the kitchen sink. The disposal is handy and helps keep the house free of food scraps, but the danger is that with too much trash, especially tough-to-grind garbage, the system could get clogged up or break down.

In a previous study, Li and her team found protein aggregates, which form abundantly under stressful conditions, such as intense heat, stuck to the outer surface of mitochondria. In this study, they found the aggregates bind to proteins that form the pores mitochondria normally use to import proteins needed to build this organelle. If these pores are damaged by mutations, then aggregates cannot be dissolved, the researchers report. These observations led the team to hypothesize that misfolded proteins in the aggregates are pulled into mitochondria for disposal, much like food scraps dropped into the garbage disposal. Testing this hypothesis was tricky, Li says, because most of the misfolded proteins started out in the cytoplasm, and most of those that enter mitochondria quickly get ground up.

As a consequence, Li and her team used a technique in which a fluorescent protein was split into two parts. Then, they put one part inside the mitochondria and linked the other part with a misfolded and clumping protein in the cytoplasm. If the misfolded protein entered the mitochondria, the two parts of the fluorescent protein could come together and light up the mitochondria. This was indeed what happened.

"With any experiment," Li says, "you have a hypothesis, but in your head, you may be skeptical, so seeing the bright mitochondria was an enlightening moment."

To see what might happen in a diseased system, the team then put into yeast cells a protein implicated in the neurodegenerative disease known as amyotrophic lateral sclerosis (ALS), or Lou Gehrig's disease. After a heat treatment that caused the ALS protein to misfold, it also wound up in the mitochondria. The researchers then did an experiment in which a lot of proteins in the cytoplasm were made to misfold and found that when too much of these proteins entered mitochondria, they started to break down.

The team wanted to make sure that the phenomenon it had observed in the yeast cells could also happen in human cells, so the scientists used the same split-fluorescent protein method to observe misfolded proteins to enter the mitochondria of lab-grown human retinal pigmented epithelial cells. As observed in yeast, misfolded proteins, but not those that were properly folded, entered and lit up mitochondria.

Biological systems are in general quite robust, but there are also some Achilles' heels that may be disease prone, Li says, and relying on the mitochondrial system to help with cleanup may be one such example. While young and healthy mitochondria may be fully up to the task, aged mitochondria or those overwhelmed by too much cleanup in troubled cells may suffer damage, which could then impair many of their other vital functions.

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In cleaning up misfolded proteins, cell powerhouses can break down - Science Daily

SelectScience Interview: Live Cell Analysis in Chronic Inflammation Research at the University of Oxford – SelectScience.net (blog)

Professor David R. Greaves, University of Oxford, UK, discusses the technology that is enabling him to research tissue repair and chronic inflammation in real time

David R. Greaves,University of Oxford, UK

Professor David R. Greaves is using the latest live cell imaging technology to carry out ground-breaking research into inflammation biology. Sonia Nicholas, Associate Editor for SelectScience, spoke to Professor Greaves to find out more.

SN: Please could you confirm your name, full job title and place of work.

DRG: Im Professor David R. Greaves, I am a University Lecturer in Cellular Pathology at the Sir William Dunn School of Pathology, University of Oxford, UK.

SN: Could you tell us about your job, what you do and what your responsibilities are?

DRG: I run a research laboratory working on macrophage biology and inflammation. I am very interested in macrophage chemotaxis as well as other aspects of macrophage cell biology such as phagocytosis and cytokine secretion.

In addition to doing biomedical research I run the BM Principles of Pathology course for second year medical students at the University of Oxford, I run a final year lecture course in Inflammation Biology, I run the British Heart Foundation 4-year Cardiovascular Sciences PhD program and I am a Tutorial Fellow in Medicine at Hertford College where I give tutorials in Biochemistry, Cell Biology, Endocrinology, Medical Genetics and Pathology.

Inflammation and disease

SN: Can you tell us more about your research into inflammation?

DRG: Inflammation is the normal physiological response to tissue injury and infection. Most of the time our inflammatory responses are of an appropriate magnitude, they are quickly resolved and any damage to our tissues is successfully repaired. Inflammation is important because it drives the development of many important human diseases including rheumatoid arthritis, cardiovascular disease, inflammatory bowel disease and many others. Recent research suggests chronic inflammation may be an important driver of major mood disorders including depression.

My research is aimed at identifying endogenous pathways that are involved in regulating the magnitude and duration of inflammatory responses. Recently, we have been looking at the role of two independent cell signaling pathways in regulating the inflammatory response. One is centered on endocannabinoids a class of lipids that signals via a G protein coupled receptor (GPCR) called CB2 and the other pathway is centered on an unusual cytokine called Chemerin (TIG-2) whose effects are mediated by three different GPCRs ChemR23, CCRL2 and GPR1.

Macrophages in healthy and inflamed tissues play an essential role in the initiation and resolution of inflammation. One important aspect of macrophage biology in the context of inflammation resolution is phagocytosis of cellular debris and phagocytosis of neutrophils that have undergone apoptosis. Macrophage phagocytosis of apoptotic cells (efferocytosis) has a profound effect on inflammation resolution. Macrophage efferocytosis changes the profile of macrophage cytokine secretion towards a more anti-inflammatory / pro-resolution phenotype, which in turn will enhance inflammation resolution. Failure to clear apoptotic neutrophils from a site of inflammation can lead to failure of resolution and a substantially worse outcome caused by secondary necrosis.

The IncuCyte Live-Cell Analysis System enables detailed analysis of immune cell biology monitor changes in morphology and measure cell health, chemotactic migration and phagocytosis in real time. Automatically visualize the differentiation of immortalized THP-1 cells into M0 macrophages and qualitatively analyze the differentiation of primary monocytes into M1 and M2 macrophage populations

A powerful research tool

SN: How does the IncuCyte technology help you to achieve your research goals?

DRG: We have now been using the IncuCyte Live-Cell Analysis Systemto study several different aspects of macrophage cell biology in a wide range of different applications. We have found this real time live cell imaging system to be easy to use and the associated image analysis software makes this a very powerful research tool.

SN: How did you monitor cell behavior before you installed the IncuCyte? What does this technology enable you to do that you couldnt do before?

DRG: All the macrophage biology experiments that we have published using the IncuCyte platform could have been performed using other imaging modalities but I think that the big advantage of the IncuCyte live cell imaging platform lies in the ease of use and ease of analysis compared to other cell imaging methods (confocal microscopy, flow cytometry and Imagestream). What we particularly like about the IncuCyte system is the ability to develop protocols to study both generalized and cell type specific behavior. For instance we can follow proliferation or apoptosis of macrophages, and we can study macrophage specific cell behavior such as apoptosis or chemotaxis. Data analysis is greatly facilitated by user friendly software.

SN: What is next for your research?

DRG: I want to start using the IncuCyte system to do scratch wound migration assays where we look for macrophage secreted factors that play a role in wound repair processes. Hopefully we can scale up this cell-based assay to look for novel chemicals, peptides and proteins that will enhance tissue repair in the context of inflammation resolution.

The long-term goal of research in my laboratory is to turn high quality basic science into new treatments that enhance wound repair and help resolve chronic inflammation. Our ability to study both murine and human macrophages on the IncuCyte platform will be important in future translational research programs.

SN: Do you have any advice for other researchers who are considering using IncuCyte technology?

DRG: Take your time in setting up the assays before you pile in to testing lots of different mediators, drugs etc. Every cell type is different so one size fits all protocols are unlikely to work first time!

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SelectScience Interview: Live Cell Analysis in Chronic Inflammation Research at the University of Oxford - SelectScience.net (blog)

Shedding light on the star of cell biology – Cherwell Online

In the twilight depths off the west coast of North America lives a small and graceful jellyfish floating apparently aimlessly through the void. Who would have known that this humble jellyAequorea victoriawas set to revolutionise cellular biology in the latter half of the twentieth century. Along the rim of the jellyfishs bell (the propulsive body) lies a ring of light-emitting organs which, in the blackness, produce an electric green glow that wouldnt be out place in a Ghostbusters film. This luminescence can be attributed to a chemical mechanism based around the molecule known as the Green Fluorescent Protein (GFP), synthesised by the jellyfish. Earning those involved in its discovery the Nobel Prize in 2008, GFP has been the key to unlocking the potential of biological imaging over the last 25 years.

The light organ houses two molecules essential for the light reaction: aequorin and GFP, working in conjunction. By catalyzing the degradation of the protein luciferin, aequorin causes blue light to be released. Rather than emitting this blue light, the photons are instead used an as energy source to activate the fluorescence of GFP. GFP has an excitation peak at the wavelengths of 395 nm and 475 nmcorresponding to blue and UV light. This means that it will most efficiency absorb light in this range of the spectrum. Absorbing this light leaves GFP in an unstable state with too much energy, being described as excited. Emission of green light at the wavelength of 508 nm, energetically lower than that it absorbed, returns it to its stable state.

Green light is rare in the ocean depths, meaning that an organism that can luminesce in such a way will be more obvious in its surroundings, allowing it to attract prey and confuse predators. But how is this relevant to cell biology in the laboratory? In 1992, American scientist Douglas Prasher sequenced and cloned the wild-type GFP gene. Over the following few years GFP became the darling of molecular genetics, a result of our ability to fuse the gene onto the beginning or the end of any other gene in any organism.

Related Do not go gentle into that good night

If inserted into an embryo, every cell in the body can inherit the GFP tagged protein. When the resulting organism is exposed to UV light it then glows green. This allows scientists to track both the distribution and the concentration of the protein throughout individual cells or through the organism as a whole, depending on which protein is tagged with GFP. We can see the trafficking of the proteins through the cell in real time, highlighting a host of cellular processes from protein packaging to the structure of the nuclear membrane.

Over the course of its history GFP has been constantly engineered and modified, transforming it into an increasingly more effective and versatile tool. A whole spectrum of different colours of fluorescent proteins have now been engineered. By using a red-producing variant of GFP, scientists have found success in diagnosing cancer since, due to its longer wavelength, red light can travel further through intervening tissue.

On a grander scale, one couldnt discuss GFP without bringing up the glow-in the dark rats, cats, rabbits, pigs, monkeysyou name it. Due to its obvious but relatively benign nature, GFP serves as one of the earliest genes used when trialling an organism with genetic modification, as a proof of the technology before more complex manipulation is attempted, with wide implications especially within medicine. We will soon reach the point where we can easily extract vaccines from cows milk, and produce disease resistant pigs.

The story of a simple jellyfish that has gone onto transform the very nature of molecular biology and medicine is a testament to the resourcefulness of science and humanity as a whole. It proves that the most useful of tools can have the most unlikely of origins, and should serve as a needed reality check. With every extinction, we say goodbye to another jewel in the biological crown, the vast wealth of unique genetic information that the organism possessed vanishing often forever. Who knows how many GFPs weve already lost.

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Shedding light on the star of cell biology - Cherwell Online

Hopkins scientists are engineering cells to eat deadly bacteria … – Baltimore Sun

Researchers at the Johns Hopkins University are working to engineer single-cell organisms that will seek out and eat bacteria that are deadly to humans.

Their work combines the fields of biology and engineering in an emerging discipline known as synthetic biology.

Although the work is still in its infancy, the researchers' engineered amoeba cells could be unleashed one day in hospitals to kill Legionella, the bacteria that cause Legionnaire's disease, a type of pneumonia; or Pseudomonas aeruginosa, a dangerous, drug-resistant bacteria associated with various infections and other life-threatening medical conditions in hospital patients.

Because amoebas are able to travel on their own over surfaces, the engineered cells also could be used to clean soil of bacterial contaminants, or even destroy microbes living on medical instruments. If the scientists are successful at making the cells perform tasks, it also could have important implications for research into cancer and other diseases.

"We're using this as a test bed for determining do we understand how cells work to the point where we can engineer them to perform certain tasks," said Douglas N. Robinson, a professor of cell biology and a member of the Hopkins team. "It's an opportunity to demonstrate that we understand what we think we understand. I think it's an opportunity to push what we're doing scientifically to another level."

The five-member team's work began in October after it received a four-year, $5.7 million federal contract from the Defense Advanced Research Projects Agency, known as DARPA.

Douglas said they want the engineered cells to respond to dangerous bacteria the way a human might respond to the smell of a freshly baked plate of cookies to immediately crave a cookie, walk into the kitchen and eat some.

Engineering cells to perform such tasks remains a work in progress.

"In practice it hasn't gone terribly well," said Pablo A. Iglesias, a professor of electrical and computer engineering and a member of the Hopkins team. "People manage to do things but it takes huge amounts of effort and it's more or less random. There has to be a lot of iterations before it works."

David Odde, a professor of biomedical engineering at the University of Minnesota, hailed the research as exciting, especially since antibiotic resistance is on the rise. He said the team would face many challenges.

"I think getting the cells to sense the bacteria robustly might be a challenge, and I'm sure they're aware of that," he said. "The cells have to sense something that the immune system has failed to sense."

The research could lead to new discoveries beyond what the team is focusing on, Odde said. They could learn more about how amoebas sense the bacteria and how that signals to them that they should move forward and eat, he said.

"How does the signaling inform the eating parts?" he said. "They might make new discoveries about how these cross systems talk to each other which will be really valuable for this project and many other projects."

The amoeba they are using, Dictyostelium discoideum, is commonly found in damp soil and naturally eats bacteria after sensing the biochemical scent of it. Since the amoeba eats bacteria, the researchers must program it to go after the kind of bacteria that they want it to eat, instead of other types of bacteria.

Robinson, the cell biology professor, will study how the amoeba's "legs" power movement. Peter Devreotes, another cell biology professor on the team, will study what happens in the amoeba's "brain" once it senses the bacteria nearby. Iglesias, a computational biologist, has expertise in control systems, once designing airplane controllers, and he will help design the biological controller used to steer the amoeba in the right direction.

The other two team members, Tamara O'Connor, an assistant professor in the Hopkins department of biological chemistry, and Takanari Inoue, an associate professor of cell biology, will try to ensure the amoeba goes after the right bacteria and link the amoeba's "brain" and "legs."

Andre Levchenko, a professor of biomedical engineering at Yale University, said that it might take a lot to "foolproof" the mechanism and that unexpected problems may arise, such as mutations in the cells.

"What would be interesting to see is how stable their new engineered organisms are. With anything that is alive and adaptable and dynamic, it's always a concern when you engineer it," Levchenko said. "I've been very impressed with this particular proposal. It's risky, but it does have a lot of elements that make me think it'll be very successful."

Dennis Discher, Director of the National Cancer Institute's Physical Sciences Oncology Center at the University of Pennsylvania, said that "the time is right" for this type of research.

"It's intriguing to not just think about cells in your body, but amoeba that usually are sort of good for nothing except basic biological science and repurpose them for other uses," he said.

Robinson said it may be hard to get the amoebas to move properly toward the bacteria they want it to eat because the controller could cause it to overshoot and end up too far away.

Iglesias said that under the contract with DARPA, the team will have to meet benchmarks every six months. The first benchmark was to prove that the amoeba's controller can be inserted successfully, which Iglesias said they have done.

The task was difficult because the amoebas are the size of a micron, or about 1/10th of the width of a human hair. They can also move fairly quickly, Iglesias said.

DARPA "wants you to think big and do something big, and I think in that respect it's pretty exciting," Iglesias said.

cwells@baltsun.com

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New method reveals how proteins stabilize the cell surface – Science Daily

To withstand external mechanical stress and handle trafficking of various substances, a cell needs to adjust its surrounding membrane. This is done through small indentations on the cell surface called caveolae. In order to stabilize its membrane, cells use the protein EHD2, which can be turned on and off to alternate between an inactive closed form and an active open form. The discovery, made by Ume University researchers and colleagues, was recently published in the journal PNAS.

Caveolae play a key role when cells adjust to their surrounding environment. An absence of these small indentations is associated with severe diseases where muscles and fat cells disintegrate or where cells of the blood vessels are malfunctioning. In a collaboration involving a broad spectrum of biophysical, biochemical and cell biological analysis, researchers have identified the mechanistic cycle of the protein EHD2 and how it regulates the dynamics of caveolae on the cell membrane.

"The fact that the EHD2 protein helps the cells to adjust to their environment could be critically important for how caveolae affect the ability of muscle cells to repair or the absorption and storing abilities of fat cells," says Richard Lundmark, who is researcher at the Department of Integrative Medical Biology at Ume University and corresponding author of the article.

The discovery was made by the research group of Richard Lundmark at the Department of Integrative Medical Biology and the Laboratory of Molecular Infection Medicine Sweden (MIMS), along with colleagues at Gothenburg University in Sweden and Albert-Ludwigs-Universitt Freiburg and Martin Luther University Halle-Wittenberg in Germany.

The researchers demonstrate how the molecule ATP serves as a fuel allowing EHD2 to bind to the cell membrane and assume an open state where parts of the protein are inserted into the cell membrane. This position allows for the formation of so-called oligomers from the protein, which stabilizes the membrane in a fixed state. When the ATP-molecules have been spent, the protein is released from the membrane and assumes an inactive and closed state. The EHD2 protein's internal domains keeps it in this inhibited form when it is not in contact with a cell membrane.

"This research shows how the mechanistic cycle of EHD2 that we describe plays a key role for the caveolae's ability to stabilize cell membranes," says Richard Lundmark.

In the article, the researchers also describe how they used a new method based on the absorption and reflection of infrared light. Together with advanced analytics, this new method can be used to study structures of the membrane-bound states of proteins, which is difficult to achieve using other techniques. Using this method, the researchers were able to show the drastic conformational change in EHD2 when it binds to a membrane.

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Hopkins scientists engineering cells to eat deadly bacteria – Baltimore Sun

Researchers at Johns Hopkins University are working to engineer single-cell organisms that will seek out and eat bacteria that are deadly to humans.

Their work combines the fields of biology and engineering in an emerging discipline known as synthetic biology.

Although the work is still in its infancy, the researchers' engineered amoeba cells could be unleashed one day in hospitals to kill Legionella, the bacteria that cause Legionnaire's disease, a type of pneumonia; or Pseudomonas aeruginosa, a dangerous, drug-resistant bacteria associated with various infections and other life-threatening medical conditions in hospital patients.

Because amoeba are able to travel on their own over surfaces, the engineered cells also could be used to clean soil of bacterial contaminants, or even destroy microbes living on medical instruments. If the scientists are successful at making the cells perform tasks, it also could have important implications for research into cancer and other diseases.

"We're using this as a test bed for determining do we understand how cells work to the point where we can engineer them to perform certain tasks," said Douglas N. Robinson, a professor of cell biology and a member of the Hopkins team. "It's an opportunity to demonstrate that we understand what we think we understand. I think it's an opportunity to push what we're doing scientifically to another level."

The five-member team's work began in October after it received a four-year, $5.7 million federal contract from the Defense Advanced Research Projects Agency, known as DARPA.

Douglas said they want the engineered cells to respond to dangerous bacteria the way a human might respond to the smell of a freshly-baked plate of cookies to immediately crave a cookie, walk into the kitchen and eat some.

Engineering cells to perform such tasks remains a work in progress.

"In practice it hasn't gone terribly well," said Pablo A. Iglesias, a professor of electrical and computer engineering and a member of the Hopkins team. "People manage to do things but it takes huge amounts of effort and it's more or less random. There has to be a lot of iterations before it works."

David Odde, a professor of biomedical engineering at the University of Minnesota, hailed the research as exciting, especially since antibiotic resistance is on the rise. He said the team would face many challenges.

"I think getting the cells to sense the bacteria robustly might be a challenge, and I'm sure they're aware of that," he said. "The cells have to sense something that the immune system has failed to sense."

The research could lead to new discoveries beyond what the team is focusing on, Odde said. They could learn more about how amoeba sense the bacteria and how that signals to them that they should move forward and eat, he said.

"How does the signaling inform the eating parts?" he said. "They might make new discoveries about how these cross systems talk to each other which will be really valuable for this project and many other projects."

The amoeba they are using, Dictyostelium discoideum, is commonly found in damp soil and naturally eats bacteria after sensing the biochemical scent of it. Since the amoeba eats bacteria, the researchers must program it to go after the kind of bacteria that they want it to eat, instead of other types of bacteria.

Robinson, the cell biology professor, will study how the amoeba's "legs" power movement. Peter Devreotes, another cell biology professor on the team, will study what happens in the amoeba's "brain" once it senses the bacteria nearby. Iglesias, a computational biologist, has expertise in control systems, once designing airplane controllers, and he will help design the biological controller used to steer the amoeba in the right direction.

The other two team members, Tamara O'Connor, an assistant professor in the Hopkins department of biological chemistry, and Takanari Inoue, an associate professor of cell biology, will try to ensure the amoeba go after the right bacteria and link the amoeba's "brain" and "legs."

Andre Levchenko, a professor of biomedical engineering at Yale University, said it might take a lot to "foolproof" the mechanism and that unexpected problems may arise, such as mutations in the cells.

"What would be interesting to see is how stable their new engineered organisms are. With anything that is alive and adaptable and dynamic, it's always a concern when you engineer it," Levchenko said. "I've been very impressed with this particular proposal. It's risky, but it does have a lot of elements that make me think it'll be very successful."

Dennis Discher, Director of the National Cancer Institute's Physical Sciences Oncology Center at the University of Pennsylvania, said "the time is right" for this type of research.

"It's intriguing to not just think about cells in your body, but amoeba that usually are sort of good for nothing except basic biological science and repurpose them for other uses," he said.

Robinson said it may be hard to get the amoeba to move properly toward the bacteria they want it to eat because the controller could cause it to overshoot and end up too far away.

Iglesias said that under the contract with DARPA, the team will have to meet benchmarks every six months. The first benchmark was to prove that the amoeba's controller can be inserted successfully, which Iglesias said they have done.

The task was difficult because the amoeba are the size of a micron, or about 1/10th of the width of a human hair. They can also move fairly quickly, Iglesias said.

DARPA "wants you to think big and do something big, and I think in that respect it's pretty exciting," Iglesias said.

cwells@baltsun.com

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Hopkins scientists engineering cells to eat deadly bacteria - Baltimore Sun

In a lab pushing the boundaries of biology, an embedded ethicist keeps scientists in check – STAT

T

he young scientists had a question. They were working with mouse embryos from which all living cells had been chemically dissolved away.

So far, so good, thought the bioethicist, as she listened to the presentation at a Harvard Medical School lab meeting.

The scientists were seeding the mouse scaffolds with human stem cells. Those cells were expected to turn into human liver cells and perhaps a mini human liver; and human kidney cells and perhaps mini human kidneys; andhumanheart and brain cells and

Wait.

Jeantine Lunshof insists she is not the ethics police. It says so on the door to her closet-sized office at Harvard. She doesnt find reasons to reflexively shut down experiments. She doesnt snoop around for deviations from ethical guidelines. But when scientists discuss their research in the twice-weekly lab meetings she attends, I will say, hmm, that raises some good questions, Lunshof said.

There is no shortage of good questions for Lunshof, who for the last three years has been embedded in the synthetic biology lab of George Church, the visionary whose projects include trying to resurrect the wooly mammoth and to write a human genome from scratch. Church is also famous for arguing that it is ethically acceptable to edit the genomes of human embryos if doing so will safely alleviate suffering, and for encouraging people to make their full genome sequence public, privacy be damned.

In the Church lab, Lunshof told STAT, you have incredibly interesting conversations.

George Church has a wild idea to upend evolution. Heres your guide

Rapid advances in genomics and stem cell biology are forcing researchers to regularly confront ethical quandaries that seem straight out of science fiction. The power to create organisms with cells, tissue, and even organs from different species,called a chimera, raises thornyquestions: What is the moral status of a primordial human brain nourished with a rudimentary heart and circulatory system, all inside a mouse scaffold? Can it feel pain? Should it not be created in the first place? Genome-editing presents otherchallenges: Where does therapy end and enhancement begin? Could genome-editing to prevent dwarfism, for example, go a little further and create a future NBA star? How should society balance competing values such as autonomy, like the freedom of parents to do everything they can for their children, and justice, as in not creating classes of genetic haves and have nots?

George is far ahead of everyone else in the kinds of experiments he undertakes, said John Aach, a senior scientist in Churchs lab who works closely with Lunshof. She performs a service in making them slow down to where the rest of the world is. Otherwise George might stumble. It doesnt take much to stumble and make a mess of things. Jeantine keeps things moving on the bioethics side as the science is moving ahead.

Lunshofs role is unusual if not unique. Genetics researchers will tap a bioethicist to join a grant or consult on a project, but it is rarely if ever the case that a genetics lab has a full-time bioethicist, said Brendan Parent, a bioethicist at New York University. He and others are unaware of any other such embeds. Instead, bioethicists and biologists tend to interact when they serve on committees convened by universities, scientific organizations, or government.

In contrast, Lunshof not only coauthors papers with Church and his colleagues, but also helps draft protocols for some of the cutting-edge science the lab conducts. By being present at the creation, she is able to flag ethical minefields before the lab finds itself bumbling acrossone.

She provides me with a comfort zone, Church said. I think much more about societal concerns that the labs research might raise. Shes here while were just starting to think about experiments, he added, and because of her we talk about [bioethics] earlier than most groups do. Jeantine is fearless in what she tackles.

The benefits of this collaboration extend beyond Church and his lab. Watching new biology emerge in real time has enabled Lunshof to develop much-needed new ways of thinking aboutbioethics, giving her field and the world outside the lab a fighting chance to keep up.

Its at the lab meetings every Monday and Thursday afternoon that Lunshof typically learns what might next land on her to-do list. The 50 or so scientists in attendance update Church on their research and others offer comments. The rows of chairs are generally all filled. Lunshof, in typically casual lab attire, rarely asks questions, instead taking notes and keeping track of who she needs to follow up with.

This week, researchers discussed plans to do cognitive testing on participants in a project centered on having their genomes sequenced. Lunshofs ears pricked up.

The combination of genetics and intelligence has long been a danger zone, largely because measurements of intelligence are imprecise and shaped by the dominant culture, as decades of debate about IQ tests have shown. The tests do not measure cognition, let alone intelligence, Lunshof said during the meeting, arguing for staying away from linking the genome to cognition or IQ. She urged the scientists to be more precise in describing what the tests measured: memory and mental processing speed. Correcting things later by saying, No, we are not measuring IQ, really were not, is very difficult, she said.

When I feel that something is a problem, I feel completely free to say, Dont go down that road, Lunshofsaid in an interview. She is not paid by Harvard. Born and raised in the Netherlands, sheis an assistant professor there, at University Medical Center Groningen, and she was awarded a Marie Curie fellowship to move to Boston and support her work in Churchs lab.

Audacious project plans to create human genomes from scratch

No one in the lab ever puts pressure on me to legitimize anything or to agree with what theyre doing, she said. I am always on the alert for things that could get into delicate areas.

Lunshofs collaboration with Church began in 2006. Itwas the start of the Personal Genome Project, an effort to sequence peoples full genomes and mine the data to link genetics to health. Church was causing consternation by proposing that people make their genome and their health history publicly available.

My first reaction was, this is totally crazy, Lunshof recalled. Anonymity and confidentiality were central to everything we do in biomedical ethics.

But then she thought, what if Church is right? He had argued that its impossible to guarantee that a DNA sample would remain anonymous. (Hewould beproved right in 2013.) So why not do away with that charade at the outset, and instead of making empty promises of anonymity, tell volunteers from the get-go that anyone could know who they were?

Lunshof had studied philosophy and Tibetan language and culture as an undergraduate, then had written a doctoral thesis on ethical issues in genomics. She also had earned a nursing degree, and worked at the Netherlands Cancer Institute in Amsterdam. In 2006, she stumbled on the PGP website and sent an email expressing her interest in it. Church replied within hours and their partnership was born.

Together, they developed a new form of patient consent for the Personal Genome Project. Called open consent, it was founded on principles new to the bioethics of genetic research. It tells participants they wont have privacy and confidentiality. Instead, consent is based on values such as reciprocity (scientists and volunteers interact as equals) and veracity. Lunshof is also a big believer in the ethical concept of citizenry, including allowing ones genetic data to be accessed by all qualified scientists tohelp advance medical progress and alleviate human suffering.

Because of advances in genetics and genomics, it made sense to abandon the traditional idea of medical confidentiality, Lunshof said, or at least not make it central.

That was a minority opinion. The National Institutes of Health, a main funder of Churchs lab, wasnt ready to embrace the idea of genetic privacy being violable, said Aach. It and the genetics community went in the other direction, saying we have to take steps to protect privacy, a huge and costly undertaking.

With the development of open consent, the Personal Genome Project took off, and now has more than 5,000 participants in the United States alone.

When I feel that something is a problem, I feel completely free to say, Dont go down that road.

Bioethicist Jeantine Lunshof

The ethics debate around genomics intensified with publication of a breakthrough 2012 paperon CRISPR, the revolutionary new genome-editing technology. After Church and his team got CRISPR to edit the genomes of human cells, later that year, they and others quickly faced two quandaries: Should CRISPR ever be used to enhance peoples genetic inheritance? Should it be used to edit the genomes of human eggs, sperm, or early embryos, producing changes that could be inherited by offspring and, maybe, generations of designer babies?

For many scientists and ethicists, the line-in-the-sand position on such germline editing and genetic enhancement has long been no. Lunshof had other ideas.

From the bioethics standpoint, she told STAT one afternoon at a Harvard Medical School cafe, it is not clear why altering genes [for enhancement] is by definition unethical. Some philosophers have consistently argued that there is a duty to at least consider genetic enhancement.

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In the real world, Prospective parents decide to use or not to use reproductive technologies, Lunshof argued, and that could one day include germline genome editing.

That reflects the balancing act she brings to the ethical puzzles she tries to unravel. Sometimes two core values are in conflict. In the case of germline editing and enhancement, parental autonomy (to make reproductive choices) might clash with the idea that all children are entitled to an equal start in the world. But the latter is honored in the breach more than the observance, Lunshof says, and so should not be allowed to trump parental autonomy.

Last week, a report from the National Academy of Sciences and the National Academy of Medicine opened the door to germline editing. It opposed enhancement, but called the line between enhancement and therapy blurry. Lunshof beat them to it: The criteria for what is therapy and what is enhancement are fluid, she wrote two years ago.

For all the passions that germline editing incites, its effects would be small: It requires in vitro fertilization, so few parents would use it (unless reproductive sex goes the way of flip phones). Other applications of CRISPR could be more consequential. One could alter ecosystems. Calledgene drive, it is a technology for editing the genomes of an organism in a way that causes the change to be inherited by every offspring, contrary to usual inheritance patterns.

As scientists in Churchs lab and elsewhereinvolved the public in conversations about testing gene drives in wild populations of mice or mosquitoes, Lunshof recently raised a novel bioethics question: If a bioneer community says yes to gene drive, it sets a precedent and could lead people in other places to allow it, too, she said. How much would this community be held morally accountable for genetic interventions elsewhere that go wrong?

The ethical minefield created by the possibility of seeding mouse embryo scaffolds with human stem cells, and possibly growing a functional, if mini, human brain, has been trickier to navigate. Youd grow human organs, Lunshof said. My question was, what if this worked?

There didnt seem to be any government or other rules against it. Scientists using stem cells from embryos are supposed to clear experiments with an Embryonic Stem Cell Research Oversight (ESCRO) Committee, which many research universities have established. But Churchs lab proposed to use stem cells produced by reverting adult cells back to an embryo-like state. And although there are rules against creating human chimeras, it wasnt clear whether this thing would be a chimera: It wouldnt be a single living entity, though it might have living human cells or even organs.It seemed there was no bureaucracy to stop the experiment.

Lunshof spent hours with the two scientists who were planning the experiment. She primes the lab to be sensitive to ethical issues even when they dont know what to be sensitive about, Aach said. She proposed asking the ESCRO committee. Church agreed. It decided that the experiment did not violate any known guideline but asked him to keep the committee informed as the experiments progressed.

As it happens, the experiments didnt work and the lab moved on smack into another ethical conundrum.

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This time, postdoctoral fellow Eswar Iyer was using a process called micropatterning to create special surfaces on glass slides. Placed on them, human stem cells formed a precisely shaped little colonythat differentiated into one or another organ.

Iyer described this work at a 2015 lab meeting. Twophrases made Lunshof sit up: embryo-like features and generation of cerebral organoids.

It was the de-cellularized mouse dilemma all over again, but with glass slides instead of mouse scaffolding, and, again, no rules seemed to apply. There are federal prohibitions against allowing an embryo to develop past the point where it forms a structure called a primitive streak, which happens on the 15th day after fertilization. At this point the embryo can no longer split (into twins) and is therefore widely regarded as a morally significant individual. But human cells or tissues developing on the micropatterned surfaces never form a primitive streak; only whole embryos do.

The question, Lunshof said, was, What is the threshold where a synthetic entity is enough of an embryo that the same moral questions must be considered?

That question loomed even larger with those cerebral organoids, primordial mini-brains that are even more realistic and much more embryo-like, Church said.

Cerebral organoids, too, fall through the cracks of the rules on embryo research, Lunshof said, but we know were doing things that involve the same ethical issues that inspired the rules, such as when human life begins, when something has a moral status, and whether since this is brain tissue the thing is sentient.

After the lab meeting, Iyer dropped by her office. The 10-minute visit he expected lasted two-and-a-half hours. Lunshof not only asked him to explain every detail of every slide he had shown. Their conversation also ranged into Western and Eastern philosophy (Iyer is a Hindu), especially views on when life begins. They agreed to keep talking.

Lunshof gave Church a rundown of the discussion, began looking for scholarly papers that might shed light on the ethically-uncharted territory, and figured out what rules are applicable. She also took the helm of a working group on the ethics of embryo-like entities.

One result is a paper to be published in eLife, an online biology journal. In it, Lunshof, Aach, Iyer, and Church propose that research limits for these entities be based as directly as possible on the generation of morally concerning features. (The entities are called SHEEFs: Synthetic Human Entities with Embryo-like Features.) For instance: How human are the cerebral organoids? Do they feel pain? How could youtell?

Just because the thing cannot develop into a baby is not a valid reason to green-light the experiments, Lunshof said. She believes that if human cells are highly organized and display functional interactivity as a blood supply in a cerebral organoid would then one must at least consider the possibility that the SHEEF has moral status.

Lunshof also initiated a discussion of SHEEFs with Harvards stem-cell oversight committee, which led to a meeting last November at Harvard Law School. There, Church explained that it is possible to get blood vessels to infuse cerebral organoids, which allows us to go to larger and larger organoids. So far, he said, we can see beautiful structures very similar to advanced cerebral [tissue]. There is essentially no limit to the technology, so we need to focus on the ethics and the humanity as guides to how far to take the science.

Which means Lunshof is unlikely to run out of good questions.

Sharon Begley can be reached at sharon.begley@statnews.com Follow Sharon on Twitter @sxbegle

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In a lab pushing the boundaries of biology, an embedded ethicist keeps scientists in check - STAT

Molecular biology: Fingerprinting cell identities – Science Daily

Every cell has its own individual molecular fingerprint, which is informative for its functions and regulatory states. Researchers from Ludwig-Maximilians-Universitaet (LMU) in Munich 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 analyze 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.

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Molecular biology: Fingerprinting cell identities - Science Daily

Protein once thought exclusive to neurons helps some cancers grow, spread, defy death – Medical Xpress

February 21, 2017 Dr. Ping-Hung Chen, Dr. Sandra Schmid, Dr. Marcel Mettlen and other research team members determined that aggressive cancer cells adapt nerve cell mechanisms to maintain or squelch signals needed to survive and grow. Credit: UT Southwestern

How we think and fall in love are controlled by lightning-fast electrochemical signals across synapses, the dynamic spaces between nerve cells. Until now, nobody knew that cancer cells can repurpose tools of neuronal communication to fuel aggressive tumor growth and spread.

UTSouthwestern Medical Center researchers report those findings in two recent studies, one in the Proceedings of the National Academy of Sciences (PNAS) and the second in Developmental Cell

"Many properties of aggressive cancer growth are driven by altered cell signaling," said Dr. Sandra Schmid, senior author of both papers and Chair of Cell Biology at UTSouthwestern. "We found that cancer cells are taking a page from the neuron's signaling playbook to maintain certain beneficial signals and to squelch signals that would harm the cancer cells."

The two studies find that dynamin1 (Dyn1) - a protein once thought to be present only in nerve cells of the brain and spinal cord - is also found in aggressive cancer cells. In nerve cells, or neurons, Dyn1 helps sustain neural transmission by causing rapid endocytosis - the uptake of signaling molecules and receptors into the cell - and their recycling back to the cell surface. These processes ensure that the neurons keep healthy supplies at the ready to refire in rapid succession and also help to amplify or suppress important nerve signals as necessary, Dr. Schmid explained.

"This role is what the cancer cells have figured out. Aggressive cancer cells have usurped the mechanisms that neurons use for the rapid uptake and recycling of neural transmitters. Instead of neural transmitters, the cancer cells use Dyn1 for rapid uptake and recycling of EGF (epidermal growth factor) receptors. Mutations in EGF receptors are drivers of breast and lung cancers," she said of the Developmental Cell study.

In order to thrive, cancer cells must multiply faster than nearby noncancerous cells. EGF receptors help them do that, she explained.

Cancer cell survival is another factor in disease progression. In the PNAS study, the Schmid lab found that aggressive cancer cells appear to have adapted neuronal mechanisms to thwart a key cancer-killing pathway triggered by activating "death receptors" (DRs) on cancer cells. Specifically, aggressive cancer cells appear to have adapted ways to selectively activate Dyn1 to suppress DR signaling that usually leads to cancer cell death.

"It is amazing that the aggressive cancers use a signaling pathway to increase the activity of EGF and also turn on Dyn1 pathways to suppress cancer death - so you have this vicious circle," said Dr. Schmid, who holds the Cecil H. Green Distinguished Chair in Cellular and Molecular Biology.

She stressed that less aggressive cancers respond to forms of chemotherapy that repress EGF signaling and/or die in response to the TRAIL-DR pathway. However, aggressive lung and breast cancer cells have adapted ways to commandeer the neuronal mechanisms identified in these studies.

The hope is that this research will someday lead to improved strategies to fight the most aggressive cancers, she said. Currently, her laboratory is conducting research to identify Dyn1 inhibitors as potential anticancer drugs using a 280,000-compound library in a shared facility at UTSouthwestern.

"Cancer is a disease of cell biology. To grow, spread, and survive, cancer cells modify normal cellular behavior to their advantage. They can't reinvent the underlying mechanisms, but can adapt them. In these studies, we find that some cancer cells repurpose tools that neurons use in order to get a competitive advantage over nearby normal cells," she said.

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Protein once thought exclusive to neurons helps some cancers grow, spread, defy death - Medical Xpress