New contrast agents detect bacterial infections with high sensitivity and specificity

A new family of contrast agents that sneak into bacteria disguised as glucose food can detect bacterial infections in animals with high sensitivity and specificity. These agents -- called maltodextrin-based imaging probes -- can also distinguish a bacterial infection from other inflammatory conditions.

"These contrast agents fill the need for probes that can accurately image small numbers of bacteria in vivo and distinguish infections from other pathologies like cancer," said Niren Murthy, an associate professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University. "These probes could ultimately improve the diagnosis and treatment of bacterial infections, which remains a major challenge in medicine."

The imaging probes were described in the July 17, 2011 advance online edition of the journal Nature Materials.

Coulter Department postdoctoral fellows Xinghai Ning and Seungjun Lee led the project. University of Georgia Complex Carbohydrate Research Center postdoctoral associate Zhirui Wang; and Georgia State University Department of Biology associate professor Eric Gilbert and student Bryan Subblefield also contributed to the work.

In the United States in 2010, bacterial infections caused 40,000 deaths from sepsis and were the leading cause of limb amputations. A major limitation preventing the effective treatment of bacterial infections is an inability to detect them inside the body with accuracy and sensitivity. To image bacterial infections, probes must first deliver a large quantity of the contrast agent into bacteria.

"Most existing imaging probes target the bacterial cell wall and cannot access the inside of the bacteria, but maltodextrin-based imaging probes target a bacterial ingestion pathway, which allows the contrast agent to reach a high concentration within bacteria," said Murthy.

Maltodextrin-based imaging probes consist of a fluorescent dye linked to maltohexaose, which is a major source of glucose for bacteria. The probes deliver the contrast agent into bacteria through the organism's maltodextrin transporter, which only exists in bacterial cells and not mammalian cells.

"To our knowledge, this represents the first demonstration of a targeting strategy that can deliver millimolar concentrations of an imaging probe within bacteria," noted Murthy.

In experiments using a rat model, the researchers found that the contrast agent accumulated in bacteria-infected tissues, but was efficiently cleared from uninfected tissues. They saw a 42-fold increase in fluorescence intensity between bacterial infected and uninfected tissues. However, the contrast agent did not accumulate in the healthy bacterial microflora located in the intestines. Because systemically administered glucose molecules cannot access the interior of the intestines, the bacteria located there never came into contact with the probe.

They also found that the probes could detect as few as one million viable bacteria cells. Current contrast agents for imaging bacteria require at least 100 million bacteria, according to the researchers.

In another experiment, the researchers found that the maltodextrin-based probes could distinguish between bacterial infections and inflammation with high specificity. Tissues infected with E. coli bacteria exhibited a 17-fold increase in fluorescence intensity when compared with inflamed tissues that were not infected.

Additional laboratory experiments showed that the probes could deliver large quantities of imaging probes to gram-positive and gram-negative bacteria for internalization. Both types of bacteria internalized the maltodextrin-based probes at a rate three orders of magnitude faster than mammalian cells.

"Maltodextrin-based probes show promise for imaging infections in a wide range of tissues, with an ability to detect bacteria in vivo with a sensitivity two orders of magnitude higher than previously reported," said Murthy.

Provided by Georgia Institute of Technology (news : web)

New 3-D photonic crystal has both electronic, optical properties

In an advance that could open new avenues for solar cells, lasers, metamaterials and more, researchers at the University of Illinois have demonstrated the first optoelectronically active 3-D photonic crystal.

"We've discovered a way to change the three-dimensional structure of a well-established semiconductor material to enable new optical properties while maintaining its very attractive electrical properties," said Paul Braun, a professor of materials science and engineering and of chemistry who led the research effort.

The team published its advance in the journal Nature Materials.

Photonic crystals are materials that can control or manipulate light in unexpected ways thanks to their unique physical structures. Photonic crystals can induce unusual phenomena and affect photon behavior in ways that traditional optical materials and devices can't. They are popular materials of study for applications in lasers, solar energy, LEDs, metamaterials and more.

However, previous attempts at making 3-D photonic crystals have resulted in devices that are only optically active that is, they can direct light but not electronically active, so they can't turn electricity to light or vice versa.

The Illinois team's photonic crystal has both properties.

"With our approach to fabricating photonic crystals, there's a lot of potential to optimize electronic and optical properties simultaneously," said Erik Nelson, a former graduate student in Braun's lab who now is a postdoctoral researcher at Harvard University. "It gives you the opportunity to control light in ways that are very unique to control the way it's emitted and absorbed or how it propagates."

To create a 3-D photonic crystal that is both electronically and optically active, the researchers started with a template of tiny spheres packed together. Then, they deposit gallium arsenide (GaAs), a widely used semiconductor, through the template, filling in the gaps between the spheres.

Enlarge

This graphic shows the method for epitaxial growth of 3-D photonic crystals. Credit: Erik Nelson

The GaAs grows as a single crystal from the bottom up, a process called epitaxy. Epitaxy is common in industry to create flat, two-dimensional films of single-crystal semiconductors, but Braun's group developed a way to apply it to an intricate three-dimensional structure.

"The key discovery here was that we grew single-crystal semiconductor through this complex template," said Braun, who also is affiliated with the Beckman Institute for Advanced Science and Technology and with the Frederick Seitz Materials Research Laboratory at Illinois. "Gallium arsenide wants to grow as a film on the substrate from the bottom up, but it runs into the template and goes around it. It's almost as though the template is filling up with water. As long as you keep growing GaAs, it keeps filling the template from the bottom up until you reach the top surface."

The epitaxial approach eliminates many of the defects introduced by top-down fabrication methods, a popular pathway for creating 3-D photonic structures. Another advantage is the ease of creating layered heterostructures. For example, a quantum well layer could be introduced into the photonic crystal by partially filling the template with GaAs and then briefly switching the vapor stream to another material.

Once the template is full, the researchers remove the spheres, leaving a complex, porous 3-D structure of single-crystal semiconductor. Then they coat the entire structure with a very thin layer of a semiconductor with a wider bandgap to improve performance and prevent surface recombination.

To test their technique, the group built a 3-D photonic crystal LED the first such working device.

Now, Braun's group is working to optimize the structure for specific applications. The LED demonstrates that the concept produces functional devices, but by tweaking the structure or using other semiconductor materials, researchers can improve solar collection or target specific wavelengths for metamaterials applications or low-threshold lasers.

"From this point on, it's a matter of changing the device geometry to achieve whatever properties you want," Nelson said. It really opens up a whole new area of research into extremely efficient or novel energy devices.

More information: "Epitaxial Growth of Three-Dimensionally Architectured Optoelectronic Devices", Nature Materials (2011).

Provided by University of Illinois at Urbana-Champaign (news : web)

Scientists create vaccine against heroin high

Researchers at The Scripps Research Institute have developed a highly successful vaccine against a heroin high and have proven its therapeutic potential in animal models.

The new study, published recently online ahead of print by the American Chemical Society's Journal of Medicinal Chemistry, demonstrates how a novel vaccine produces antibodies (a kind of immune molecule) that stop not only heroin but also other psychoactive compounds metabolized from heroin from reaching the brain to produce euphoric effects.

"In my 25 years of making drug-of-abuse vaccines, I haven't seen such a strong immune response as I have with what we term a dynamic anti-heroin vaccine," said the study's principal investigator, Kim D. Janda, the Ely R. Callaway, Jr. Chair in Chemistry and a member of The Skaggs Institute for Chemical Biology at Scripps Research. "It is just extremely effective. The hope is that such a protective vaccine will be an effective therapeutic option for those trying to break their addiction to heroin."

"We saw a very robust and specific response from this heroin vaccine," said George F. Koob, chair of the Scripps Research Committee on the Neurobiology of Addictive Disorders and a co-author of the new study. "I think a humanized version could be of real help to those who need and want it."

A Worldwide Epidemic

While injection drug abuse is a debilitating worldwide epidemic, heroin abuse and addiction are especially destructive, with costs estimated at $22 billion in the United States due to loss of productivity, criminal activity, medical care, and social welfare, the authors say in their study.

Heroin abuse and addiction are also driving forces in the spread of HIV through needle sharing.

Using an approach termed "immunopharmacotherapy," Janda and his Scripps Research colleagues previously created vaccines that used immune molecules to blunt the effects of other abused drugs such as cocaine, methamphetamine, and nicotine. Human clinical trials are under way for the cocaine and nicotine vaccines.

Attempts by other researchers over the past four decades to create a clinically viable heroin vaccine, however, have fallen short, in part due to the fact that heroin is an elusive target metabolized into multiple substances each producing psychoactive effects.

An Innovative Approach

To overcome this problem, in the new study the Scripps Research team used a "dynamic" approach, targeting not only heroin itself, but also the chemical it quickly degrades into, 6-acetylmorphine (6AM), and morphine.

"Heroin is lipophilic and is rapidly degraded to 6AM," said G. Neil Stowe, a research associate in Janda's laboratory who is first author of the new study. "Both readily cross the blood-brain barrier and gain access to the opioid receptors in the brain."

The researchers linked a heroin-like hapten (a small molecule that elicits an immune response) to a generic carrier protein called keyhole limpet hemocyanin or KLH, and mixed it with Alum, an adjuvant (vaccine additive), to create a vaccine "cocktail." This mixture slowly degraded in the body, exposing the immune system to different psychoactive metabolites of heroin such as 6AM and morphine.

"Critically, the vaccine produces antibodies to a constantly changing drug target," said Stowe. "Such an approach has never before been engaged with drug-of-abuse vaccines."

To compare the results of a non-dynamic approach, the team also prepared a vaccine simply targeting morphine, a substance related to heroin. Both vaccines were then injected into rats and the effects were examined in Koob's laboratory.

Promising Results

The results showed that the rats rapidly generated robust polyclonal antibodies in response to the dynamic heroin vaccine.

In addition, the study found that addicted rats were less likely to "self-administer" heroin by pressing on a lever after several booster shots of the vaccine. Only three of the seven rats that received the heroin vaccine self-administered heroin. In contrast, all of the control rats, including those given the morphine vaccine, self-administered the drug.

The effect of the heroin vaccine "was very dramatic; as dramatic as we have ever seen in experiments of this kind," said Koob. "To have an animal vaccinated and not show a response to heroin is pretty amazing."

The team also found that the heroin vaccine was highly specific, meaning that it only produced an antibody response to heroin and 6AM, and not to the other opioid-related drugs tested, such as oxycodone as well as drugs used for opioid dependence—methadone, naltrexone, and naloxone. "The importance of this," said Janda, "is that it indicates these vaccines could be used in combination with other heroin rehabilitation therapies."

The Scripps Research team has recently begun an exciting collaboration with researchers at the Walter Reed Army Institute of Research to see if it is feasible to develop a dual-purpose vaccine against HIV and for the treatment of heroin addiction in a single shot, Janda said.

More information: A Vaccine Strategy that Induces Protective Immunity Against Heroin, http://pubs.acs.or … 21/jm200461m

Provided by The Scripps Research Institute (news : web)

Zinc 'sparks' fly from egg within minutes of fertilization

At fertilization, a massive release of the metal zinc appears to set the fertilized egg cell on the path to dividing and growing into an embryo, according to the results of animal studies supported by the National Institutes of Health.

The zinc discharge follows the egg cell’s steady accumulation of zinc atoms in the developmental stages before fertilization. The researchers documented the discharge by bathing the eggs in a solution that gives off light when exposed to zinc. They referred to the zinc discharge and accompanying light flash as zinc sparks.

“The discovery of egg cells’ massive intake and later release of zinc defines a new role for this element in biology,” said Louis DePaolo, chief of the Reproductive Sciences Branch at the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), one of the NIH institutes supporting the study. “We anticipate the findings will one day lead to information useful for the treatment of infertility as well as the development of new ways to prevent fertilization from occurring.”

The study’s authors suggest that zinc acts as a switch, turning off the process of cell division while the egg matures and turning it on again after fertilization.

“These findings suggest zinc is essential for developing a healthy egg and, ultimately, a healthy embryo,” said Teresa Woodruff, Ph.D., one of the article’s senior authors.

The study’s first author is Alison M. Kim, Ph.D., of Northwestern University, Evanston, Ill. The other authors are Miranda L. Bernhardt, Betty Y. Kong, Richard W. Ahn, Dr. Woodruff and Thomas V. O’Halloran, Ph.D., of Northwestern, and Stefan Vogt, Ph.D., of Argonne National Laboratory, Washington, D.C.

Their findings appear in the July issue of ACS Chemical Biology.

In this study, the researchers observed egg cells from mice and from monkeys. To conduct the study, they devised a microscope that would allow them to view the concentration and distribution of zinc atoms in individual cells. With the aid of the chemical that gives off light when exposed to zinc, the researchers documented the first zinc sparks 20 minutes after fertilization. Most fertilized eggs released two or three rounds of sparks, but the researchers saw as few as one and as many as five within the first two hours after fertilization. The sparks flared every 10 minutes, on average.

Previous research had shown that fertilization triggers cyclical changes in the level of calcium in the egg cell. The researchers noted that the zinc sparks always occurred after a peak in calcium levels inside the cell.

“The number, timing and intensity of these sparks could tell us something important about the quality of the egg and will be an important area for future research,” said Dr. O’Halloran, the article’s other senior author. “It’s may also be worth investigating whether the amount of zinc in a woman’s diet plays a role in fertility.”

Additional experiments helped confirm a role for zinc in the fertilization process. Typically, once the egg is released from the ovary, it must get rid of excess chromosomes in two stages as it prepares to fuse with the sperm. The team’s earlier research showed that the early accumulation of zinc is essential for properly completing the first stage, Dr. O’Halloran explained. The latest results suggest that zinc may act as a brake in between these stages, as the egg awaits fertilization. If the cell is fertilized, the zinc release appears to lift the brake. The cell discards its excess genetic material and begins to divide.

The researchers also showed that even unfertilized eggs would start to divide if zinc levels were artificially reduced, mimicking release. In addition, when fertilized cells were forced to take on additional zinc, the process was reversed.

“We have shown that zinc appears to regulate this precisely calibrated, intricate process,” Dr. Woodruff said. “The findings give us new insights into what these cells need to grow and mature properly.”

Provided by National Institutes of Health