Researchers Develop Mouse That Could Provide Advance Warning of Next Flu Pandemic

Researchers in Germany have developed a transgenic mouse that could help scientists identify new influenza virus strains with the potential to cause a global pandemic. The mouse is described in a study, “In vivo evasion of MxA by avian influenza viruses requires human signature in the viral nucleoprotein,” that will be published April 10 in The Journal of Experimental Medicine.

Influenza A viruses can cause devastating pandemics when they are transmitted to humans from pigs, birds, or other animal species. To cross the species barrier and establish themselves in the human population, influenza strains must acquire mutations that allow them to evade components of the human immune system, including, perhaps, the innate immune protein MxA. This protein can protect cultured human cells from avian influenza viruses but is ineffective against strains that have acquired the ability to infect humans.

To investigate whether MxA provides a similar barrier to cross-species infection in vivo, Peter Staeheli and colleagues at the Institute of Virology, Medical Center University of Freiburg, created transgenic mice that express human, rather than mouse, MxA. Similar to the results obtained with cultured human cells, the transgenic mice were resistant to avian influenza viruses but susceptible to flu viruses of human origin.

MxA is thought to target influenza A by binding to the nucleoprotein that encapsulates the virus’ genome, and mutations in this nucleoprotein have been linked to the virus’ ability to infect human cells. Staeheli and colleagues found that an avian influenza virus engineered to contain these mutations was able to infect and cause disease in the transgenic mice expressing human MxA.

MxA is therefore a barrier against cross-species influenza A infection, but one that the virus can evade through a few mutations in its nucleoprotein. Staeheli and colleagues think that their transgenic mice could help monitor the potential dangers of emerging viral strains. “Our MxA-transgenic mouse can readily distinguish between MxA-sensitive influenza virus strains and virus strains that can evade MxA restriction and, consequently, possess a high pandemic potential in humans,” Staeheli says. “Such analyses could complement current risk assessment strategies of emerging influenza viruses, including viral genome sequencing and screening for alterations in known viral virulence genes.”

Electron Microscopy Reveals How Vitamin A Enters The Cell

Using a new, lightning-fast camera paired with an electron microscope, Columbia University Medical Center scientists have captured images of one of the smallest proteins in our cells to be “seen” with a microscope.

The protein – called STRA6 – sits in the membrane of our cells and is responsible for transporting vitamin A into the cell interior. Vitamin A is essential to all mammals and is particularly important in making the light receptors in our eyes, and in the placenta and fetus where it’s critical for normal development.

Images of the protein – which revealed several unusual features –– were published in the August 26 issue of the journal Science, by structural biologist Filippo Mancia, PhD, assistant professor of physiology and cellular biophysics, who lead a team of other scientists including Wayne Hendrickson, Larry Shapiro, Joachim Frank and Bill Blaner at Columbia University Medical Center, Loredana Quadro at Rutgers University, Chiara Manzini at George Washington University and David Weber at the University of Maryland School of Medicine.

Until the new study, the way STRA6 transports vitamin A into the cell had been a mystery. Most transporters interact directly with the substances they transport. But STRA6 only interacts with Vitamin A via an intermediary protein that carries the greasy vitamin A in the bloodstream. Revealing the structure of STRA6 may not only give the researchers insight into Vitamin A transport, but also clues about how other related transporters may work.

A new type of camera technology was a key element to getting the images of STRA6. When paired with an electron microscope – the camera allows biologists to see tiny, never-seen-before structural details of the inner machinery of our cells.

“We can now get near atomic resolution because the new camera is much faster and allows us to take a movie of the molecules,” says Oliver Clarke, PhD, an associate research scientist in the Hendrickson lab at Columbia University Medical Center. “Even under the electron microscope, the molecules are moving around by a tiny amount, but when you take a picture of something moving, it comes out blurry. With such a movie, we can align the frames of the movie to generate a sharper image.”

Imaging the molecule also depended on painstaking biochemical procedures, developed by Yunting Chen, PhD, and associate research scientist in the Mancia lab, to generate large quantities of the protein and separate them from the cell’s other components. “It’s a very delicate protein, and we had to mimic its environment to keep it from getting out of shape,” she says. Those efforts took about two years to perfect.

The researchers used approximately 70,000 individual pictures of STRA6 to generate a 3-dimensional map of the protein, which was used to construct an atomic model accurate to the smallest detail.

The images and model reveal STRA6 is “a bit of a freak,” says Dr. Clarke. Even more surprising was the fact that STRA6 does not work alone, but is instead tightly associated with another protein, calmodulin, which plays a key role in calcium signaling.

Although Vitamin A moves through STRA6 to enter the cell, there is no channel in STRA6 like most transporters. Instead, vitamin A enters the top of STRA6, but then appears poised to exit through a side window that opens directly into the cell membrane, not the cell interior.

Though this needs to be verified, the mechanism may be a way to protect cells from too much vitamin A. “Vitamin A is actually somewhat toxic,” says Dr. Mancia. “Trapping vitamin A inside the membrane may keep control of the amount inside the cell.”

The new model of STRA6 advances the understanding of a critical cellular function and may help researchers understand how other, still mysterious cellular components, work.

Sophisticated ‘Mini-Brains’ Add to Evidence of Zika’s Toll on Fetal Cortex

Studying a new type of pinhead-size, lab-grown brain made with technology first suggested by three high school students, Johns Hopkins researchers have confirmed a key way in which Zika virus causes microcephaly and other damage in fetal brains: by infecting specialized stem cells that build its outer layer, the cortex.

The lab-grown mini-brains, which researchers say are truer to life and more cost-effective than similar research models, came about thanks to the son of two Johns Hopkins scientists and two other high school students who were doing summer research internships. They had the idea to make the equipment for growing the mini-brains with a 3-D printer. These so-called bioreactors, and the mini-brains they foster, should open other new and valuable windows into human brain development, brain disorders and drug testing — and perhaps even produce neurons for treatment of Parkinson’s disease and other disorders, the investigators say.

A report on the research appears online April 22 in the journal Cell.

“We have been working for three years to develop a better research model of brain development, and it’s fortunate we can now use this one to shed light on the major public health crisis posed by Zika infections,” says Hongjun Song, Ph.D., professor of neurology and neuroscience at the Johns Hopkins University School of Medicine’s Institute for Cell Engineering. “This more realistic, 3-D model confirms what we suspected based on what we saw in a two-dimensional cell culture: that Zika causes microcephaly — abnormally small brains and heads — mainly by attacking the neural progenitor cells that build the brain and turning them into virus factories.”

In recent years, researchers in various fields have begun to grow tiny organs from human stem cells to get a better view of development and disease, and speed the search for new drugs. But existing techniques for creating and working with mini-brains were limiting because of the organ’s complexity, Song says. Though the mini-brains themselves are about the size of a pinhead, the bioreactors where they grew were comparatively large, about the size of a soda can. That made working with the mini-brains expensive, given the high cost of the nutrients needed to cultivate human stem cells in the lab, he says, as well as the expense of chemical growth factors that guide the tissue to organize itself like a real brain. Few labs could afford to grow enough mini-brains to be useful for research, Song says, and those that did produced tissues with cells specialized for different parts of the brain mixed together at random.

Song and his wife and research partner, Guo-li Ming, M.D., Ph.D., professor of neurology, neuroscience, and psychiatry and behavioral sciences, found a way to improve the bioreactors from an unexpected source: their son and two other high school students, from New York and Texas, who spent a summer working in the lab. The students had worked with 3-D printers and thought they could be the key to producing a better bioreactor, one that would fit over commonly used 12-well laboratory plates and spin the liquid and cells inside at just the right speed to allow the cells to form brains.

Of course, it wasn’t that simple, Song says. Graduate student Xuyu Qian and postdoctoral fellow Ha Nam Nguyen, Ph.D., spent years determining factors such as what that optimum speed was, as well as which chemicals and growth factors should be added at what times to yield the desired result.

The group has so far used the new bioreactor, dubbed SpinΩ, to make three types of mini-brains mimicking the front, middle and back of a human brain. They used the forebrain, the first mini-brain with the six layers of brain cell types found in the human cortex, for the current study on Zika.

“One thing the mini-brains allowed us to do was to model the effects of Zika virus exposure during different stages of pregnancy,” says Ming. “If infection occurred very early in development, the virus mostly infected the mini-brains’ neural progenitor cells, and the effects were very severe. After a while, the mini-brains would stop growing and disintegrate. At a later stage, mimicking the second trimester, Zika still preferentially infected neural progenitor cells, but it also affected some neurons. Growth was slower, and the cortex was thinner than in noninfected brains.”

These different effects correspond to what clinicians have seen in infants born to women who contracted Zika during pregnancy, as well as miscarriages, she notes, namely that the earlier in pregnancy Zika infection occurs, the more severe its effects.

The research group’s next step will be to test drugs already FDA-approved for other conditions on the mini-brains to see whether one might provide some protection against Zika. And they included 3-D printing files for SpinΩ in the new paper so that researchers anywhere can print their own bioreactors with just a few hundred dollars in materials. Song says one possible future use could be to grow so-called dopaminergic neurons for transplant, to replace those that die off in Parkinson’s disease. “This is the next frontier of stem cell biology,” he says.

Penn and Rutgers Researchers Discover New Pathway That May Trigger Asthma

Asthma is an enormous public health problem that continues to grow larger, in part because scientists don’t fully understand how it is caused. Existing therapies don’t cure the disease and often don’t even significantly alleviate the symptoms. Now, scientists from the Perelman School of Medicine at the University of Pennsylvania and Rutgers University have identified a biological pathway that potentially explains why current asthma therapies don’t work well in many cases—and might be targeted to help those patients.

Asthma is a chronic condition that affects more than 25 million people in the United States alone, including more than 7 million children. It accounts for nearly 2 million ER visits annually and about 1.5 million patient-days of hospital inpatient care.

“Only 60 percent of asthma patients have an inflammatory or allergic component to their asthma and 40 percent of asthma patients wheeze in part due to intrinsic abnormalities of epithelial and smooth muscle cells,” said co-senior author Edward E. Morrisey, PhD, a professor of Cell and Developmental Biology and director of the Penn Center for Pulmonary Biology at Penn.

“Curiously, these patients are refractory to current therapies,” said co-senior author Reynold A. Panettieri, Jr., MD, Vice Chancellor of Translational Medicine and Science at Rutgers. “There’s a real need to understand the non-inflammatory aspects of asthma, and with this study we’re getting closer to that understanding.”

The study, which appears in the current issue of the Journal of Clinical Investigation, is a collaboration between the Morrisey laboratory and the laboratory of Panettieri, an asthma specialist who moved from Penn Medicine to Rutgers Biomedical and Health Sciences last summer.

Clues from Goblet Cells
The discovery of the possible new asthma pathway emerged from basic research by Morrisey and colleagues on the developmental biology of the epithelial layer of cells that line the lung and its airways. In experiments published in 2012, they found that the transcription factors Foxp1 and Foxp4—which can switch certain gene programs on or off—normally repress the production of mucus-secreting goblet cells in the lung epithelia of mice. Genetic inactivation of these two transcription factors caused goblet cells to differentiate abnormally.

Increased goblet cell differentiation in the airways is a hallmark of asthma. From this, the Morrisey lab investigated whether loss of the Foxp1/4 genes, specifically in the airway epithelium of the lung, also causes an asthma-like condition in mice.

In an initial set of experiments, the team, including first author Shanru Li, a staff scientist in the Morrisey lab, studied the physiological function of airways lacking the Foxp1/4 genes in adult mice. “We found that the airways of these mice did indeed behave like asthmatic human airways,” Morrisey said.
A classic sign of asthma is airway hyper-responsiveness (AHR)—an abnormally strong tendency for the smooth muscle cells underlying the airway epithelium to contract and cause a partial closure of the airway. The team found that the mice lacking airway Foxp1/4 showed significantly greater signs of AHR, compared to control mice, especially when experimentally challenged with airway irritants. “At the high doses of the challenge the Foxp1/4-knockout mice basically started to die because their airways closed off,” Morrisey said.
Intriguingly, the airway lining in the Foxp1/4-knockout mice did not show signs of the type of inflammation that is typically associated with asthma and targeted with standard asthma drugs.

Neuropeptide Y – The Key to Non-inflammatory Asthma?
To find out the principal cause of the hyper-responsiveness in the airways of these mice, the researchers looked at the gene expression patterns of the affected airway epithelial cells, comparing them to the patterns seen in control mice that have normal levels of Foxp1/4 expression.

“Because only Foxp1/4 genes were missing from the airway epithelium of these mutant mice, we hypothesized that epithelial cells in the mutants were secreting a factor that was causing the underlying smooth muscle cells to contract. Therefore, we looked specifically for expression changes in genes encoding molecules that could be secreted from the epithelial cells and be received by the airway smooth muscle cells,” Morrisey said.

One such molecule, neuropeptide Y (NPY), stood out well above the rest—it was not expressed in control airway epithelial cells but was expressed at high levels in the airway epithelia of the knockout mice.

NPY is a signaling molecule and neurotransmitter found abundantly in the nervous system and some other parts of the body. Its many biological actions include stimulating the constriction of blood vessels. Previous research has linked variants of its gene to increased asthma risk, but NPY hasn’t been known to have a direct role in asthma.

Morrisey’s team showed that NPY has a significant role in asthma by deleting the NPY gene while at the same time deleting the Foxp1/4 genes. This resulted in the airway hyper-responsiveness that is observed in Foxp1/4-mutant mice to return to almost normal levels. Importantly, since changes in NPY expression have been linked to asthma in humans, the investigators tested whether NPY could directly cause airway hyper-responsiveness in human lung tissue. These experiments showed that when normal human lung airways are exposed to NPY, they exhibit a marked increase in hyper-responsiveness to methacholine challenge. In all of these experiments, the inflammatory response remained unchanged, indicating that NPY did not cause alterations in the immune response that could cause asthmatic symptoms.

“These data strongly suggest that NPY can cause airway hyper-responsiveness in human lungs and could be a causative mechanism in human asthma,” Morrisey said.
“Further, the molecular mechanisms mediating airway hyper-responsiveness occur at the level of smooth muscle where NPY amplifies smooth muscle contraction at all mediators by activating Rho Kinase, a pivotal signaling molecule in the bronchoconstriction pathway,” Panettieri said. It also suggests that inhibiting NPY activity in people with asthma, perhaps with an inhaled medication, might help the millions of patients who get little or no benefit from current asthma therapies.

Pharmaceutical companies have already developed compounds that block NPY signaling for other applications such as obesity and hypertension. “Testing whether these NPY inhibitors would help human asthma patients would be worthwhile given the results of our studies” Morrisey said.
In addition to setting up tests of NPY-blocking drugs, he and his team hope to replicate their mouse-model findings in a larger animal model of asthma, which better simulates the human disease.

New Technology Aims to turn Complicated Lab Tests into Point-of-Care Tests for First Responders

Carbon monoxide (CO) poisoning can have tragic consequences if victims are not rescued or treated. First responders at the scene may not know immediately if a conscious patient is a victim of CO poisoning.

Normally, these type of tests are taken with an arterial blood sample (typically an artery in the arm), and are then sent off to a central lab, where a bench top unit uses spectroscopy and electrochemical sensor measurements to provide results.  It can take hours to get the results.

But what if a lab in a hospital is not an option and time may be ticking for first responders at a scene of a possible CO poisoning?

Rapid Response

Toronto-based ChroMedx, a medical technology company focused on the development of novel medical devices for in vitro diagnostics and point-of-care testing is working to address this. Their flagship device, the HemoPalm utilizes a small sample of blood via the finger, providing a result on the spot, with no specialist required. Central to the HemoPalm, is its ability to fully integrate CO-oximetry, measured through spectroscopy (the only method for CO-oximetry) which allows the user to measure total hemoglobin (Hb), Oxy-Hb, Deoxy-Hb, Met-Hb and carboxy-Hb, simultaneously with blood gases and electrolytes measured with electrochemical sensors.

The technology is especially beneficial to first responders, who can take the blood sample right at the scene and have the data available upon arrival to the hospital before the patient even arrives into the ER. The information can also be transmitted back to the hospital before the patient is wheeled into the ER triage.

Hospital Setting

In a hospital setting, the HemoPalm could simplifying sample collection and expediting patient results, both in the emergency department and the operating room, where the device could replace multiple machines currently in use. Another use would be for respiratory care, allowing respiratory therapists and visiting nurses to optimize treatment in the hospital and at home with comprehensive results of oxygen in the blood.

A second cartridge, the HemoPalm B, is being developed to measure bilirubin (which may be used to monitor liver function).  Bilirubin in high levels is an indication of jaundice, a potentially dangerous condition for newborns of which approximately 15% will develop this condition.  The heel-prick method of sampling, as provided by the HemoPalm system, makes sample collection easy with minimal trauma to the baby and minimal blood loss.   Once diagnosed, treatment is relatively straightforward, and pre-discharge testing could predict virtually all cases of jaundice and ensure proper parental response and care.

Future Tests

Other cartridges planned for development include testing for lactate, which is an indication of sepsis (blood poisoning), a common and deadly condition in the Emergency Department; creatinine, a measure of kidney function; and beta-hydroxybutyrate, elevated in diabetic emergencies.

Wayne Maddever, ChroMedx’s CEO said he expects to see HemoPalm in studies for FDA approvals within a year.  For more information, log on to http://www.chromedx.com