New Microscope Decodes Complex Eye Circuitry

ScienceDaily (Mar. 16, 2011) — The properties of optical stimuli need to be conveyed from the eye to the brain. To do this efficiently, the relevant information is extracted by pre-processing in the eye. For example, some of the so-called retinal ganglion cells, which transmit visual information to the brain via the optic nerve, only react to light stimuli moving in a particular direction. This direction selectivity is generated by inhibitory interneurons that influence the activity of the ganglion cells through their synapses.

Cells and synapses reconstructed from serial block face electron microscopy data. A single starburst amacrine cell (yellow, note synaptic varicosities) and two direction-selective ganglion cells (green). Even though there is substantial dendritic overlap with both cells, all connections (magenta) go to the right ganglion cell.

Using a novel microscopy method developed at the Institute, scientists from the Max Planck Institute for Medical Research in Heidelberg have now discovered that the distribution of the synapses between ganglion cells and interneurons follows highly specific rules. Only those dendrites that extend from the cell body of the amacrine cell in a direction opposite to the preferred direction of the ganglion cell connect with the ganglion cell.

The sensory cells in the retina of the mammalian eye convert light stimuli into electrical signals and transmit them via downstream interneurons to the retinal ganglion cells which, in turn, forward them to the brain. The interneurons are connected to each other in such a way that the individual ganglion cells receive visual information from a circular area of the visual field known as the receptive field. Some ganglion cells are only activated, for example, when light falls on the centre of their receptive fields and the edge remains dark (ON cells). The opposite is the case for other ganglion cells (OFF cells). And there are also ganglion cells that are activated by light that sweeps across their receptive fields in a particular direction; motion in the opposite (null-) direction inhibits activation.

Starburst amacrine cells, which modulate the activity of the ganglion cells through inhibitory synaptic connections, play an important role in this direction selectivity. The same research group at the Max Planck Institute in Heidelberg demonstrated a number of years ago that starburst amacrine cells are activated by moving stimuli. Each branch in the circular dendrite tree reacts preferentially to stimuli that move away from the cell body; movements in the opposite direction, towards the cell body, inhibit its activity. In the central area around the cell body dendrites function only as receivers of synaptic signals, while the dendrites on the periphery act as transmitters as well -- and, therefore, double as axons. Whether these dendrites cause the direction selectivity in the ganglion cells or whether the ganglion cells "compute" it using other signals was unclear up to now.

Max Planck researchers Kevin Briggman, Moritz Helmstaedter and Winfried Denk have now discovered that, although the cells themselves are symmetrical, the synapses between retinal ganglion cells and starburst amacrine cells are distributed asymmetrically: seen from the ganglion cell, the starburst cell dendrites connected with it run in the direction opposite to the preferred direction of motion. "Ganglion cells prefer amacrine-cell dendrites that run along the null-direction," says Winfried Denk.

According to previous studies by Winfried Denk and his research group, the electrical characteristics of the dendrites, which emerge starlike from the cell bodies of amacrine cells, play a crucial role here. The further they are located from the centre of the cell toward the edge, the easier they are to excite; therefore, stimuli are transmitted preferentially in this direction. This mechanism does not require but is helped by inhibitory influences between neighbouring amacrine cells, known as lateral inhibition. "A ganglion cell can thus differentiate between movements from different directions simply by making connections with certain starburst amacrine cell dendrites -- namely those that prevent activation of the ganglion cell in null-direction through their inhibitory synapses. These are precisely the amacrine cell dendrites that run along this direction," explains Winfried Denk.

Functional and structural analysis

This discovery was made possible by combining two different microscopy methods. The scientists succeeded, first, in determining the preferred motion direction of the ganglion cells using a two-photon fluorescence microscope. A calcium-sensitive fluorescent dye indicated in response to which stimuli calcium flows into the cells -- a process that signals electrical activity in cells.

They then measured the exact trajectory of all of the dendrites of these ganglion cells and those of connected amacrine cells with the help of a new electron microscopy method known as serial block face electron microscopy. This process enabled them to produce a volumetric image by repeatedly scanning the surface of a tissue sample using the electron beam of a scanning electron microscope. A thin "slice" is shaved off the sample surface after each scan is complete, using an extremely sharp diamond knife. These slices are thinner than 25 nanometers, just about one thousandth of the thickness of a human hair.

The high three-dimensional resolution of this method enabled the scientists to trace the fine, densely packed branched dendrites of retinal neurons and clearly identify the synapses between them. The complete automation of the imaging process enables them to record data sets with thousands and even tens of thousands of sections "while on holiday or attending a conference," says Winfried Denk. "For the first time, minute cell structures can now be viewed at a high resolution in larger chunks of tissue. This procedure will also play an indispensable role in the clarification of the circuit patterns of all regions of the nervous system in the future."

New Way to Test Cancer Drugs

ScienceDaily (Mar. 16, 2011) — A Purdue University scientist's nanopolymer would make it easier and cheaper for drug developers to test the effectiveness of a widely used class of cancer inhibitors.

W. Andy Tao uses nanopolymers and chemical reactions that cause color changes in a solution to detect activity related to cancer cell formation.

W. Andy Tao, an associate professor of biochemistry analytical chemistry and a member of the Purdue Center for Cancer Research team, created the Purdue-patented pIMAGO nanopolymer that can be used to determine whether cancer drugs have been effective against biochemical processes that can lead to cancer cell formation. The nanopolymers would attach themselves to target proteins that would later be detected by a relatively simple laboratory procedure called chemiluminescence.

Tymora Analytical, a company Tao started in the Purdue Research Park, will manufacture the pIMAGO nanopolymers. The 'p' stands for phosphor, and the IMAGO comes from the Greek word for image.

Tao's pIMAGO nanopolymers are coated in titanium ions and would attract and bond with phosphorylated proteins, ones in which a phosphate group has been added to a protein activating an enzyme called kinase. Kinase, when overactive, is known to cause cancer cell formation, and many cancer drugs are aimed at inhibiting kinase activity.

"It is universal. You can detect any kind of phosphorylation in a protein," said Tao, whose findings were reported in the early online version of the journal Analytical Chemistry. "It is also cheaper and would be more widely available."

The nanopolymers would be added to a solution of proteins, a chemical agent to start phosphorylation and a drug to inhibit kinase activity. Phosphorylated proteins would only be present if the drug is ineffective.

Avidin-HRP -- the protein Avidin bound with the enzyme horseradish peroxidase -- would be added. Avidin would bind with a vitamin B acid called biotin that is also on the nanopolymers' surfaces. A chemical called a substrate, added later, would cause a reaction with HRP, causing the solution to change color.

A lightly colored solution would mean there had been little kinase activity and few phosphorylated proteins and that the drug was effective. A darker solution would signal more kinase activity and a less effective drug.

"This could have a lot of applications in pharmaceuticals for drug discovery," Tao said.

Screening kinase inhibitors using antibodies can be cost-prohibitive for many laboratories because antibodies are in short supply and aren't available for many types of cells. Radioisotope tests are highly regulated and possibly dangerous because of radiation involved.

"We want to develop this as a commercial application to replace radioisotopes and antibodies as a universal method for screening kinase inhibitors," Tao said.

The National Science Foundation and the National Institutes of Health funded the research.

Unprecedented View of Protein Folding May Help Develop Brain Disease Therapies

ScienceDaily (Mar. 16, 2011) — When vital proteins in our bodies are misfolded, debilitating diseases can result. If researchers could see the folding happen, they might be able to design treatments for some of these diseases or even keep them from occurring. But many of our most critical proteins are folded, hidden from sight, inside tiny molecular chambers. Now researchers at Stanford have gotten the first-ever peek inside one of these protein-folding chambers as the folding happened, and the folding mechanism they saw surprised them.

Biology Professor Judith Frydman and graduate student Nicholai Douglas, who was first author on the paper published in Cell. 

Misfold an origami swan and the worst that happens is you wind up with an ugly paper duckling. Misfold one of the vital proteins in your body -- each of which must be folded in a particular way to perform its function -- and the result can be a debilitating neurodegenerative disease such as Alzheimer's or Huntington's.

There are no cures for such brain-wasting diseases, but now Stanford researchers have taken an important step that may one day aid in developing therapies for them. They have literally popped the lid off one of the microscopic chambers in which many of life's most crucial proteins are folded, witnessing a surprising mechanism as the heretofore hidden folding process happened before their eyes.

Virtually all proteins need to be folded, whether in primitive organisms such as bacteria or multicellular creatures such as humans. Many are guided through the process by molecules called chaperones, of which a specialized subset -- chaperonins -- folds many of the most complex proteins.

Folding in bacteria has been studied in detail, but Judith Frydman, a professor of biology who led the Stanford research, said this is the first time anyone has seen the folding process performed in higher organisms.

"The mechanism of folding we saw in the chaperonin is very different from what we expected and from what has been seen in bacteria," Frydman said. "It was really surprising, and we are still amazed that it worked. This chaperonin appears to provide a unique chemical environment."

Chaperonins are shaped like a barrel, with two ring-shaped chambers arranged one atop the other. At the open end of each ring is a lid that opens and closes in a spiraling fashion, like the aperture of a camera, something Frydman's team discovered in 2008 while studying the chaperonin called TRiC. Since then, they've been working to solve the puzzle of how a protein gets folded once the chaperonin has grabbed it, pulled it into the chamber and the aperture has closed. A paper describing their findings was published earlier this year in Cell.

Frydman said there were two likely ways in which a protein, initially a linear chain of molecules (amino acids), could theoretically be folded inside the chamber.

One is by mechanical means, with the chamber holding onto the protein and physically pushing it into the right shape.

"The other one is that when the lid closes, the chaperonin lets go of the protein, but some special chemical properties in this chamber somehow make it fold," she said. "Our evidence is that this mechanism is the correct one."

The only way to know which mechanism was doing the work was to see inside the chamber while the folding was happening, but simply opening up the lid wouldn't work, because the shape of the entire chamber changes in accordance with the motion of the lid. When the lid spirals open, the walls of the chamber spiral open, too, and the protein floats away.

To see what was happening, Frydman's team devised a chemical "trick" by which they could remove the lid on the chamber, but still get the walls of the chamber to close in, as if the lid were spiraling.

When they "closed" the lidless chamber, the chaperonin simply released the protein that had been destined to be folded. Like a long balloon that slipped from a child's grip before it could be folded into a giraffe, the protein simply drifted off.

The challenge then became figuring out how the protein was getting released.

"One of the reasons why the mechanical model of pushing the protein into shape without letting go had been proposed was because there was no obvious way for this chaperonin to let go of the protein," Frydman said.

When a protein gets grabbed for folding by TRiC, it is held by eight binding sites along the walls of the chamber. Between each binding site is a tiny loop. Frydman's team suspected that during the closing process, the loops might move to somehow "shave off" the protein and release it into the folding chamber. One of her students made mutations in the loop. When the researchers did experiments in which TRiC chaperonins equipped with mutated loops were closed, the protein stayed put. It also failed to fold.

"That suggests that the way this chaperonin folds its proteins is by releasing them in a closed chamber that has very special chemical properties," Frydman said.

"This mechanism of release is completely different from what has been seen in any other chaperone. That was very, very surprising."

The experimental work described in the Cell paper was done using a simpler version of TRiC, from a single-celled organism, than would be found in multi-cellular organisms, Frydman said, because the simpler version is much easier to manipulate.

"Now we are interested in going back to the eukaryotic [multi-cellular] complex, where every binding site in the folding chamber is different and every release loop is different," Frydman said. "I think this really opens up a lot of interesting avenues to explore how this works in higher organisms. Since TRiC helps fold many disease-linked proteins, and is central to protect cells from misfolding diseases such as Huntington's disease, this work could have many therapeutic applications."

New Vaccine Candidate Shows Strong Potential to Prevent Highly Contagious Norovirus

ScienceDaily (Mar. 16, 2011) — Scientists have shown that an experimental vaccine against the human norovirus -- the bug behind about 90 percent of highly contagious nonbacterial illnesses that cause diarrhea and vomiting -- can generate a strong immune response in mice without appearing to cause the animals any harm.

Using a novel viral vector-based method to grow and deliver the vaccine that has shown promise in other agents designed to fight such infections as HIV and hepatitis C, the researchers are the first to test this vaccine design method's effectiveness against the human norovirus.

Animals receiving the vaccine developed high levels of antibodies, a robust white blood cell response and an additional immune response in the area of the body most affected by this particular infection -- the gastrointestinal system.

The researchers say this study supports the use of viral vector-based techniques as a new way to develop vaccines for human norovirus and other viruses that cannot grow in cell cultures. It also suggests that these Ohio State University scientists could be well on their way to developing a safe vaccine against a highly problematic pathogen that causes millions of gastrointestinal illnesses every year in the United States.

"The mice in our study developed a much higher antibody response to our vaccine candidate than they did to a more traditional vaccine. That's one of the keys, to have a sustained antibody response, so that when the disease comes along, you can neutralize the virus and protect yourself," said Jianrong Li, assistant professor of food science and technology at Ohio State and senior author of the study.

Li co-authored the study with Yuanmei Ma, a graduate student in food science and technology. The research appears in the current issue of the Journal of Virology.

The Centers for Disease Control and Prevention estimates that more than 21 million cases of acute gastroenteritis -- characterized by diarrhea, vomiting and stomach pain -- each year are caused by norovirus infection. Human norovirus is transmitted primarily through fecal-oral contact, either through contaminated food or water or direct person-to-person spread. This virus is famous for being so contagious that as few as 10 viral particles may be enough to cause symptoms. No vaccine or anti-viral drug is currently available for human norovirus.

That kind of pathogenic power makes the virus a high priority for vaccine developers, said Li, who also serves on Ohio State's environmental health sciences faculty. But the process is complicated by two primary problems: The virus cannot grow in cell cultures, and no small animal models exist to mimic the infection.

Without the ability to grow the norovirus in cell cultures, the researchers instead inserted a human norovirus capsid gene -- capsid refers to the virus's outer shell -- into a specific location on the genome of a different virus. This process creates what is known as a recombinant virus -- a new viral strain formed by recombining genetic material from other viruses.

The viral host for this vaccine candidate is called vesicular stomatitis virus, or VSV, a bullet-shaped virus that has been an attractive vector for vaccine designers, Li said. The resulting recombinant viral vector functions as both the vehicle to deliver the vaccine as well as the agent that produces virus-like particles that mimic the human norovirus itself.

In this work, vaccination with the recombinant virus caused the norovirus capsid particles to grow continuously in animals, triggering a specific immune response. When the scientists tested these particles for their antigenic potential to look like foreign intruders in the body, the particles were neutralized by antibodies specifically designed to fight the human norovirus.

"So it looks like the virus and acts like the virus, but it's not, and that is how a vaccine designed with virus-like particles should function," Li said. "The virus-like particles can be continually produced in animals or humans for several weeks and stimulate strong immune responses. That's the advantage of using VSV."

Li said the VSV-based recombinant is also considered a powerful application because it can essentially be used as a bioreactor to facilitate large-scale production of these specific virus-like particles. In addition, it saves time: The viral vector developed virus-like particles within two days.

For comparison purposes in this study, Li and Ma also created a more traditional vaccine candidate by inserting a human norovirus gene into a different type of virus: a baculovirus, which is rod-shaped. It took six days for these viruses to grow enough to be used as a vaccine candidate, and the production level was comparatively low.

The scientists then conducted an animal study to observe what kind of immune response the VSV-based norovirus vaccine candidate could generate. Mice received either the VSV-based vaccine or various types of control substances for comparison, including one group that received the vaccine created with the more traditional technique. The substances were given orally or through the nose.

Weekly blood samples showed that two weeks after receiving the vaccines, the mice given the VSV-based norovirus vaccine had developed and sustained a high level of antibodies against the human norovirus -- about 25 times higher levels of antibodies than those induced by the traditionally prepared vaccine candidate.

"This might be the most important advantage of the VSV-based norovirus vaccine candidate: It prepares a high concentration of norovirus-specific antibodies that can assist with virus detection, disease diagnosis and therapy," Li said.

In addition, the mice vaccinated with VSV-based vaccine generated a T cell immune response that was two times higher than the T cell response produced in mice receiving the traditional vaccine candidate. The immune response involving T cells, a type of white blood cell, plays an important role in efficient clearance of norovirus infection.

Li said the mucosal immune response -- that involving areas covered by mucous membranes -- was similar in the two vaccine types the mice received. The scientists tested fecal samples and vaginal antibody levels in the mice, and found the levels comparable between the groups of mice receiving the two different types of vaccine.

VSV is known to infect animals, especially cattle and pigs. Human infection with VSV is very rare. The VSV-based norovirus vaccine led to minimal weight loss but caused no symptoms of illness in mice. This showed that the virus strain was attenuated, or had lost its ability to spread, because of the additional gene inserted into its genome, Li explained.

Because mice will not develop traditional norovirus symptoms, this study did not involve a test of the vaccine against the pathogen itself. Li said his further research plans include enhancing the vaccine candidate by inserting additional genes into VSV along with the human norovirus gene, which is expected to make the vaccine more potent but still safe. And he then hopes to test the vaccine candidate in a larger animal model, such as so-called germ-free pigs, animals that have never been exposed to any pathogens. These animals develop diarrhea in response to norovirus infection, as do humans.

Pig Model of Cystic Fibrosis Improves Understanding of Disease

ScienceDaily (Mar. 16, 2011) — It's been more than 20 years since scientists first discovered the gene that causes cystic fibrosis (CF), yet questions about how the mutated gene causes disease remain unanswered.

University of Iowa researchers have created a pig model that genetically replicates the most common form of cystic fibrosis. The pigs develop disease symptoms, including gastrointestinal abnormalities and lung disease, which mimic CF in humans. The image shows cells that line the bronchial airways of non-CF (top) and CF (bottom) pigs. Hair-like cilia protrude from the tops of cells. In non-CF, air fills the airway lumen above the cells. In CF, inflammatory cells, mucus and bacteria sit in the airway lumen. In CF, more of the airway cells have a reddish-purple color indicating increased mucus production. 

Using a newly created pig model that genetically replicates the most common form of cystic fibrosis, University of Iowa researchers have now shown that the CF protein is "misprocessed" in the pigs and does not end up in the correct cellular location. This glitch leads to disease symptoms, including gastrointestinal abnormalities and lung disease in the pigs, which mimic CF in humans. The findings are published in the March 16 issue of the journalScience Translational Medicine.

The findings match earlier laboratory experiments that suggested the gene mutation disrupts the process whereby the CF protein is folded into its correct shape and shipped to the membranes of cells that line the airways and other organs.

When it is correctly located at the cell membrane, this protein -- called cystic fibrosis transmembrane conductance regulator (CFTR) -- forms a channel to allow chloride ions to move in and out of cells. This ion movement is a critical component of the system that maintains salt and water balance across cell membranes in the lung as well as other organs and supports normal membrane function including eradicating bacteria from cell surfaces.

The new study shows that in pigs, the CFTR protein behaves the same way in a living animal as it does in experimental cell systems, suggesting that these experimental systems are useful for learning about the CFTR protein's properties. The cell systems and the new pig model may also be helpful in testing therapies designed to increase the amount of protein that gets to the cell membrane, or boost the activity of the protein that is located at the membrane.

"Instead of just trying to treat the symptoms of CF, current research is moving toward therapies that target mutations in the CFTR gene," said David Stoltz, M.D., Ph.D., UI assistant professor of internal medicine and senior study author. "For example, there already are drugs known as "correctors" being tested. These drugs help CFTR move from inside the cell to its correct location on the cell surface.

"The pig model could help us develop and test more corrector drugs, and it will also help us better understand why the protein is misprocessed in the first place," Stoltz added. "If we understand what is going wrong, we may be able to develop new therapies that can target the problem and allow more of the CFTR to make it to the cell surface, which may alleviate the disease symptoms."

In 2008, the UI team and colleagues at University of Missouri created pigs that were missing the CFTR protein. These animals developed CF disease symptoms that closely mimicked the human disease. In the new pig model, the animals have two copies of the CFTR gene containing the most common CF-causing mutation, which is known as the delta F508 mutation. These pigs also develop CF symptoms similar to the human disease. In particular, the CF pigs are born with gastrointestinal disease and develop lung disease over time.

By studying the protein in the pigs, the researchers were able to show that most of the CFTR protein is misprocessed and gets degraded, but a small amount of the protein does get to the cell membrane where it is able to form active chloride channels. However, the level of activity is only about 6 percent of the activity found in normal pigs with fully functional CFTR channels. The study shows that this small amount of CFTR activity is not sufficient to prevent CF disease in the pigs.

CF is a recessive disease, meaning a person with one mutated copy and one good copy of the CFTR gene is a "carrier" but does not have CF. This suggests that 50 percent of normal CFTR activity is sufficient for health. The question has always been, 'Is there a minimal amount of active CFTR that would be enough to protect people from the disease symptoms?'

"We know that people with 50 percent CFTR function have no disease, and now we know that 6 percent of full activity is not enough to prevent disease in the pigs," Stoltz said. "We still don't know how much CFTR is enough to prevent the disease, but this model animal could give us a way to investigate."

In addition to Stoltz, the UI research team included senior author Michael Welsh, M.D., UI professor of internal medicine and molecular physiology and biophysics and a Howard Hughes Medical Institute investigator, and co-first authors, Lynda Ostedgaard, Ph.D.; David Meyerholz, D.V.M., Ph.D.; and Jeng-Haur Chen, Ph.D.

This work was a collaboration between UI scientists and scientists at the University of Missouri including Dr. Randall Prather and members of his research team.

Researchers from the UI Departments of Internal Medicine, Pathology, Surgery and Pediatrics were also part of the team.

The study was funded in part by grants from the National Institutes of Health and the Cystic Fibrosis Foundation.