March 8, 2017 Enterovirus 71 infected cells: with the cell nuclei stained blue while the virus proteins are stained green. Credit: A*STAR Institute of Molecular and Cell Biology

The unavailability of antiviral medicines and vaccines has made outbreaks of hand, food and mouth disease (HFMD) caused by enterovirus 71 (EV71), a serious threat that affects millions worldwide. Now, an A*STAR comprehensive study has identified which human proteins in a cell are hijacked by EV71 and which try to resist its invasion. Clarifying these host-pathogen interactions could reveal new targets for antiviral therapeutics.

EV71 infections mainly affect children and can lead to aseptic meningitis, and long-term neurological complications, including polio-like paralysis. Since the EV71 genome encodes for just 11 proteins, it has cleverly evolved to exploit human cells to its advantage and guarantee its successful replication.

To check which human proteins facilitate or hinder EV71 replication, scientists at the A*STAR Institute of Molecular and Cell Biology have developed a gene 'atlas'. They screened 21,121 human genes, using a technique called small interfering RNA (siRNA). The team reported an extensive list of known and unknown classes of genes that play a role during EV71 infection.

Among the 256 so-called 'host factors' identified, several proteins help regulate the length of different stages of the cell cycle, like aurora kinase B (AURKB) and cyclin-dependent kinase 6 (CDK6). Interestingly, the virus seems to manipulate these proteins to favor its own replication. For example, by evicting CDK6 out of its workplace, the nucleus of the cell, EV71 could extend certain stages of the cell cycle to its own benefit.

Another sly mechanism used by this virus is to interfere with the cellular quality control process that discards abnormal or wrongly manufactured proteins. In this way, viral proteins can be produced inside the human cell, undisturbed.

The scientists focused on two host factors that were both shown to assist EV71 replication: N-glycanase 1 (NGLY1) and valosin-containing protein (VCP). Drugs that inhibit these two host factors also reduce the number of EV71-infected cells. VCP is probably held inside vesicular structures used by the virus to copy its genome, but it remains unknown how EV71 benefits from NGLY1.

"This is the first genome-wide siRNA screening for EV71-human factors interaction and reveals the complex interplay between the virus and the proteins of a specific human cell line," points out Justin Jang Hann Chu, lead author of the study. "Some host factors we found are shared with picornaviruses and enteroviruses infections, while others are completely new and need to be further explored. This information opens a new chapter in the development of antiviral strategies for HFMD."

Explore further: Novel mechanism for invasion of EV71 virus demonstrated

More information: Kan Xing Wu et al. Human genome-wide RNAi screen reveals host factors required for enterovirus 71 replication, Nature Communications (2016). DOI: 10.1038/ncomms13150

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Genetic screening to fight the common childhood virus that causes hand, foot and mouth disease - Phys.Org

March 7, 2017 by Carrie Wells, The 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 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 one-tenth 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.

Explore further: Amoeba feast on backpacks

2017 The Baltimore Sun Distributed by Tribune Content Agency, LLC.

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Scientists engineering cells to eat deadly bacteria - Phys.Org

Researchers at Columbia University have retracted a 2013 paper in The Journal of Clinical Investigation, after uncovering abnormalities in the stem cell lines that undermined the conclusions in the paper.

Last year, corresponding author Dieter Eglidiscoveredhe could notreproduce key data in the 2013 paper because almost all the cell lines first author Haiqing Hua used contained abnormalities, casting doubt on the overall findings. When Egli reached out to Hua foranswers, Hua could not explain the abnormalities. As a result, Hua and Egli agreed the paper should be retracted.

Since some of the details of how the paper ended up relying on abnormal cells remain unclear, the university confirmed to us that it is investigating the matter.

Heres the retraction notice for iPSC-derived cells model diabetes due to glucokinase deficiency, cited 42 times:

The corresponding authors were made aware of karyotype abnormalities through a routine quality control test of pluripotent stem cells used in the studies reported in this paper. After extensive internal review and genetic analysis, they found that the karyotypes of some of the cells used for the experiments reported were abnormal and that the normal karyotypes shown in Figure 1 and Supplemental Figure 2 were not from cell lines used in the study. They also cannot confirm the endonuclease-mediated correction of the mutant GCK G299R allele. H. Hua takes responsibility for the characterization and presentation of cell line karyotypes and the genetic manipulations. Because of these discrepancies, the authors wish to retract the article. They apologize for these errors and for any inconvenience caused to others.

In the fall of 2009, Hua joined Rudolph Leibels diabetes and nutrition lab at Columbia University, under the co-supervision ofEgli, who brought an expertise in stem cell biology. Hua told us:

The aim of my research project was to leverage the expertise of both Dr. Egli (on stem cell biology) and Dr. Leibel (on diabetes) and to demonstrate the concept that the islet cells generated in the lab from diabetic patients through stem cell technology would present comparable dysfunction as the islet cells in the patients body. Because we chose patients with genetic mutations that cause diabetes, we were hoping to demonstrate that correction of the mutations would restore the normal function of the islet cells.

But, Hua noted, he wasnt and still isnt an expert in stem cell biology, so he had to learn on the job:

When I began the project, I never worked with cells before and had no experience or understanding of cell line karyotype.

Hua started by generating several cell lines from a diabetic patient. To check that the genetic makeup of these cell lines were the same, he sent several for analysis to a contracted service, which examines 20 cells per cell line and generates a report:

I did karyotype analysis for the cell lines right after I derived them, probably in 2011, before I started to do any experiments on them. The reports came back with some cells being normal and some being abnormal. To be fair, I thought what I learned from Dr. Egli was that it is a normal phenomenon that some cells are abnormal as long as the number is not high.

Indeed, Egli, an assistant professor of stem cell biology at Columbia University Medical Center, confirmed that pluripotent stem cells are often prone to undergo abnormalities:

Karyotypic abnormalities are common, and occur in many cells upon extended cultures, so this is not in and of itself a concern. Often one can go back to earlier cultures that are normal.

Hua published the work in 2013, along with a relatedpaper in Diabetes in 2014, -Cell Dysfunction Due to Increased ER Stress in a Stem Cell Model of Wolfram Syndrome. Hua believes, at a conceptual level, both papers achieved the goal of demonstrating that the correction of the mutations would restore the normal function of the islet cells.

In 2014, Hua told usthat he moved back to China for family reasons.

Last year, other investigators asked Egli to share the cells lines from the 2013 study. To ensure he was providing high quality material, Egli sent what he believed to be normal cell lines from the study for quality control testing. Egli said thats when he learned many of the cell lines contained abnormalities.

To suss out the problem, Egli went back to the cell lines stored in the lab to look for normal cells:

Dr. Hua had already left the University at that time and so I personally started to look for karyotypically normal cells. There were no normal cells to be found.

Egli explained what the abnormalities meant for the study results:

You could best describe the abnormalities of the [cell] lines [Hua] used as mumbo-jumbo. There were multiple rearrangements in the chromosomes in the cell lines and thus you wouldnt know if the effects you saw were due to gene modifications or simply due to those rearrangements. Essentially, the abnormal cell lines question the entire paper, and its very unlikely the paper would have been accepted at the journal.

When Egli failed to reproduce the data from the 2013 paper, he contacted Hua to find out where the normal cell lines were. But Hua was not sure in fact, he told us it was a surprise to learn that most of the cell lines he had used contained abnormalities, adding:

another layer of complication is that when cells became karyotype abnormal, they could behave like cancer cells, namely they could start as minor portion in the culture but later on took over and became majority. So another mistake we made was that we didnt perform karyotype analysis at the end of the study to make sure that after all the experiments we did, the cells were still normal.

A spokesperson at Columbia University verified that the university is conducting an investigation into the issues:

I could confirm that there is an ongoing investigation.

When Hua was informed of these issues, he suggested the study be retracted:

Immediately, I proposed to Dr. Egli and Dr. Leibel that we should retract the publication because we were not certain about the conclusion any more.

Hua takes responsibility for what happened, adding:

So this was done at very early phase of my research, and I was busy with a lot of parallel projects since I was the first post-doc of Dr. EgliBecause I wasnt understanding the problem correctly, I put up the figures with normal karyotype as first figure for the publication and continued my research with one particular cell line.

Egli also talked about the experience of retracting a paper:

Retracting a paper is not a rewarding process, and often reports stay in the literature even if they should not. Retracting the paper exposes us to the possibility of damage. I took proactive steps to investigate and retract because I wanted to correct the record. This would not have happened without my initiative involving 2-3 months of benchwork.

Hua described this as a truly unfortunate and painful chapter, which he hopes others can learn from:

The health of academic world and advance of science really depends on correction of previous mistakes and clearance of uncertainties. [A]voiding overwhelming multitasking is important. At the first year of my research, I was setting the lab together with Dr. Egli and meanwhile performed more than 100 experiments. Each of them would took more than 10 days and I was really stacking all the experiments. This particular project was about one fourth of my effort at that time. My biggest recommendation or reflection would be that it is very very very important to quality control and characterize starting materials of a project. Many people, including myself, are more focused on rushing the project forward and do not realized that if the starting materials are flawed, anything built on them has no solid foundation.

Hat tip: Rolf Degen

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Unexplained abnormalities in stem cells prompt Columbia researchers to pull diabetes paper - Retraction Watch (blog)

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

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)

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

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

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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Hopkins scientists are engineering cells to eat deadly bacteria ... - Baltimore Sun

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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Materials provided by Umea University. Note: Content may be edited for style and length.

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

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