Poisonous cure: Toxic fungi may hold secrets to tackling deadly diseases

Take two poisonous mushrooms, and call me in the morning, said no doctor ever. Credit: Photo by G.L. Kohuth

Take two poisonous mushrooms, and call me in the morning. While no doctor would ever write this prescription, toxic fungi may hold the secrets to tackling deadly diseases.

A team of Michigan State University scientists has discovered an enzyme that is the key to the lethal potency of poisonous mushrooms. The results, published in the current issue of the journal Chemistry and Biology, reveal the enzyme's ability to create the mushroom's molecules that harbor missile-like proficiency in attacking and annihilating a single vulnerable target in the human liver.

The team revealed how the enzyme contributes to the manufacture of chemical compounds known as cyclic peptides, a favorite type of molecule that pharmaceutical companies use to create new drugs. These findings could lead to single-minded medicines with zero side effects, said Jonathan Walton, professor of plant biology and co-lead author.

"Mushrooms are prolific chemical factories, yet only a few of their peptides are poisonous," he said. "These toxins survive the high temperatures of cooking and the acids of digestion, and yet they're readily absorbed by the bloodstream and go directly to their intended target. These are the exact qualities needed for an effective medicine."

Walton published the paper with fellow MSU scientists Hong Luo, Sung-Yong Hong, R. Michael Sgambelluri and Evan Angelos. Working with the mushroom species Amanita, Walton and his teammates disassembled one of its poisonous peptides, which can be compared to a laser-guided missile with a nuclear warhead.

By removing the molecular equivalent of the deadly warhead, they now have a sturdy, precise delivery system that can supply medicine -- rather than poison -- to a single target. By taking a laser, rather than a shotgun approach, scientists could develop medicines capable of curing disease without the patient suffering any side effects.

The enzyme the team discovered is called POPB, and it converts toxins from their initial linear shape into cyclic peptides, fortress-like molecular circles comprising eight amino acids.

Harnessing the distinct properties of POPB will allow scientist to create billions of variant molecules, which can be tested against many different medical targets such as pathogenic bacteria and cancer.

"We've found some variables that are key," said Walton, an AgBioResearch scientist. "By making more variants, we can add or replace molecules that may or may not work. To date we've created a library of a hundred or so, and we eventually plan to create millions."

The challenge of the next stage of research, though, is testing the variants against diseases such as cancer. The bottleneck lies in the screening process. While Walton's team has discovered a missile capable of carrying a million different potential medicines, as of yet the payload that will be effective remains a mystery.

Source: Michigan State University

Native fungus suggested as another tool for restoring ghostly whitebark pine forests

Siberian slippery jack is a native fungus that may help in the effort to restore whitebark pine forests.
Credit: Cathy Cripps

Cathy Cripps doesn't seem to worry about the grizzly bears and black bears that watch her work, but she is concerned about the ghosts and skeletons she encounters.

The ghosts are whitebark pine forests that have been devastated by mountain pine beetles and white pine blister rust, said the Montana State University scientist who studies fungi that grow in extreme environments. The skeletons are dead trees that no longer shade snow or produce pine cones. The round purple pine cones hold the seeds that feed bears, red squirrels and Clark's nutcracker birds. Shade at the top of watersheds keeps snow from melting too fast in the spring, preventing trout streams from drying up too early in the summer.

Fortunately, she has found hope in a native fungus called Siberian slippery jack, or Suillus sibiricus, said Cripps, a mycologist in MSU's Department of Plant Sciences and Plant Pathology.

Cripps conducted a three-year study in collaboration with Waterton Lakes National Park in Canada that showed a 10 to 15 percent increase in the survival rate of whitebark pine seedlings when Siberian slippery jack spores are injected into the soil around them. The injection takes place in nurseries before the seedlings are transplanted in the mountains.

That increase is significant and good news for those trying to reinstate whitebark pine trees to the north-central Rocky Mountains and Pacific Northwest, Cripps said. The whitebark pine is a keystone species that grows at high elevations where other trees cannot, but it has been declared an endangered species in Canada and awaits the designation in the United States.

"That (jump in survival rates) might not sound like a big difference, but a small amount is a big deal considering the labor-intensive process," Cripps said.

Cyndi Smith, scientist emeritus at Waterton, said "The positive results have encouraged Waterton Lakes National Park to continue inoculating both whitebark and limber pine seedlings, to give them the best opportunity we can to establish and survive to maturity."

Participants in the research project, in addition to Cripps' and Smith's teams, were the U.S. Forest Service, National Park Service and volunteers from the United States and Canada.

Explaining how the collaboration began, Smith said, "Cathy gave a presentation on some of her Yellowstone work at the annual science meeting of the Whitebark Pine Ecosystem Foundation in Hailey, Idaho, in 2006. I was really taken with the idea that the ecosystem may have lost the beneficial fungi because our forests have been dead and dying for so long and that perhaps there was a way to reverse that trend so I approached Cathy with the idea.

"I'm a big believer in collaboration, whether locally or internationally, but certainly working with someone of Cathy's academic stature has been very helpful when I have applied for funding for whitebark and limber pine projects," Smith said, adding that, "Cathy's enthusiasm is very infectious, and it is a delight to work with her."

To carry out the research project, the participants placed cages around whitebark pine trees to collect pine cones without interference from wildlife. Then they tested the cones to see if they were resistant to white pine blister rust, removed seeds by hand from the resistant cones, grew the seeds into seedlings and shipped them to the nursery in Glacier National Park.

Cripps and former graduate student Erin Lonergan drove to Waterton Lakes 

National Park and throughout the Greater Yellowstone area, hiked to the tops of mountains and collected the Siberian slippery jack and other fungi from whitebark pine forests. Then they returned to MSU where they used a coffee grinder to process the spongy outer layer of the fungi. They added water to create a spore slurry, stored the mixture at MSU and later injected about 3 million spores into the soil around each seedling temporarily housed at the Glacier National Park nursery.

A few months later, volunteers planted more than 1,000 seedlings into MSU's test plots. Most of those test plots were located in Waterton, while others were in the neighboring Glacier National Park. The two parks together comprise Waterton-Glacier International Peace Park.

Two years into the study, Lonergan, Cripps and Smith reported success in the journal, American Forests. One year later, they announced their final results in the spring/summer 2014 issue of Nutcracker Notes, a small journal hosted by the Whitebark Pine Ecosystem Foundation.

"We wanted to get the word out that results after three years showed that inoculation with these native fungi significantly improved the survival of rust resistant seedlings, especially when inoculated seedlings were planted in burned areas near shelter objects such as stumps and logs," Cripps said.

High nitrogen fertilizations and fungicides prevent the inoculations from working, Cripps said. The age of the seedlings is important because they need to grow plenty of side roots before being inoculated.

When successful, the injected fungi slip like tiny socks over the ends of every root of the whitebark pine seedling and form a relationship that benefits both the tree and the fungi, Cripps said. The fungi help the seedling take in more nutrients and water from the soil. The tree produces the sugars that feed the fungi.

"Instead of being bad guys, these fungi are beneficial," Cripps said of the Siberian slippery jack. "They help plants take up nitrogen and phosphorus from the soil. That's a big deal."

Her study is one of many research projects involving whitebark pine forests, but it's unique because it focuses on beneficial native fungi, Cripps said. She added that land managers might want to incorporate MSU's findings into their overall strategy for restoring whitebark pine forests. She noted that large-scale inoculations are already planned for nurseries in Canada. She said inoculating beneficial fungi into nursery stock is common in Europe.

"As we work to save the vital whitebark pine from disappearing from the landscape, it is essential to use all available tools," Cripps said. "Ectomycorrhizal fungi are an integral part of forest integrity, ecology and health. Showing respect for these mighty microbes might just mean the difference between the restoration and death of a forest."

"Ectomycorrhizal fungi" refers to beneficial fungi that form a symbiotic relationship with the roots of trees. Siberian slippery jack is one of those fungi, and it only associates with five-needle pines. White pine blister rust is another type of fungus, one of the "bad guys."

Source: Montana State University

Bacteria could be rich source for making terpenes

Odoriferous terpene metabolites: A phylogenetic tree of terpene synthases shows the synthases (bold face or underlined) found by researchers in Japan and at Brown University using bacterial sequences. Credit: Image courtesy of Brown University

If you've ever enjoyed the scent of a pine forest or sniffed a freshly cut basil leaf, then you're familiar with terpenes. The compounds are responsible for the essential oils of plants and the resins of trees. Since the discovery of terpenes more than 150 years ago, scientists have isolated some 50,000 different terpene compounds derived from plants and fungi. Bacteria and other microorganisms are known to make terpenes too, but they've received much less study.

New research at Brown University, published in the Proceedings of the National Academy of Sciences, shows that the genetic capacity of bacteria to make terpenes is widespread. 

Using a specialized technique to sift through genomic databases for a variety of bacteria, the researchers found 262 gene sequences that likely code for terpene synthases -- enzymes that catalyze the production terpenes. The researchers then used several of those enzymes to isolate 13 previously unidentified bacterial terpenes.

The findings suggest that bacteria "represent a fertile source for discovery of new natural products," the researchers write.

David Cane, a professor of chemistry at Brown and one of the authors on the new paper, began working about 15 years ago to understand how bacteria make terpenes.

"At that time, the first genomic sequences of certain classes of bacteria were just beginning to come out," he said. "We had this idea that maybe you could find the enzymes responsible for making terpenes by looking at the sequences of the genes that were being discovered."

To do that, Cane searched through the genome data gathered for a group of bacteria called Streptomyces, looking for sequences similar those known to produce terpene synthases in plants and fungi. Eventually, he found that Streptomyces did indeed have genes encoding terpene synthases and that those enzymes could be used to make terpenes.

The verified bacterial sequences found by Cane and others enabled researchers to refine subsequent searches for additional terpene synthase genes. "Instead of using plant sequences or fungal sequences as your search query, we can now use bacterial sequences, which should yield a greater degree of similarity," he said. "So now we're fishing in the right waters with the right kind of bait, and you can find more matches."

This latest paper made use of the third generation of iterative searches and a powerful search technique developed by Haruo Ikeda of Kitasato University in Japan. Previous work had identified 140 probable sequences for terpene synthases. This latest work expanded that to 262.

The next step was to verify that these sequences did indeed code for enzymes capable of making terpenes. Testing all 262 wasn't practical, so the team chose a few they thought might give them the best chance of finding terpene compounds that hadn't previously been identified. They looked for sequences that didn't seem to fit clearly into previously known categories of terpenes.

After they had selected a few, the team made use of a genetically engineered Streptomyces bacterium as a bio-refinery to generate the terpene products.

"What Professor Ikeda did, in collaboration with us, is develop a variant of a very well-studied Streptomyces system," Cane said. "He eliminated the genes that were responsible for making most of its native products, but he left behind all of the capacity to provide the starting materials and handle the accumulation of products."

By taking some of the gene sequences they found and splicing them into their test organism, the researchers could let the organisms generate the product using the instructions from the newly introduced gene. Using this method, they were able to make 13 previously unknown terpenes, their structures verified by mass spectrometry and nuclear magnetic resonance spectroscopy.

"It's a big step forward in the area in that it provides a paradigm for how one could go about discovering many new substances," Cane said. "It's a good example of how one can use sequence analysis to identify genes of interest and then apply molecular genetic and microbiological techniques to produce the chemical substances of interest."

The work also suggests that there may be many new terpene products as yet undiscovered hiding in the genomes of bacteria.

Source: Brown University

Soil mineral surfaces linked to key atmospheric processes

Pictured are, from left, are David Bish, Melissa Donaldson and Jonathan Raff. Credit: Indiana University

Research by Indiana University scientists finds that soil may be a significant and underappreciated source of nitrous acid, a chemical that plays a pivotal role in atmospheric processes such as the formation of smog and determining the lifetime of greenhouse gases.

The study shows for the first time that the surface acidity of common minerals found in soil determines whether the gas nitrous acid will be released into the atmosphere. The finding could contribute to improved models for understanding and controlling air pollution, a significant public health concern.

"We find that the surfaces of minerals in the soil can be much more acidic than the overall pH of the soil would suggest," said Jonathan Raff, assistant professor in the School of Public and Environmental Affairs and Department of Chemistry. "It's the acidity of the soil minerals that acts as a knob or a control lever, and that determines whether nitrous acid outgasses from soil or remains as nitrite."

The article, "Soil surface acidity plays a determining role in the atmospheric-terrestrial exchange of nitrous acid," will be published this week in the journal Proceedings of the National Academy of Sciences. Melissa A. Donaldson, a Ph.D. student in the School of Public and Environmental Affairs, is the lead author. Co-authors are Raff and David L. Bish, the Haydn Murray Chair of Applied Clay Mineralogy in the Department of Geological Sciences.

Nitrous acid, or HONO, plays a key role in regulating atmospheric processes. Sunlight causes it to break down into nitric oxide and the hydroxyl radical, OH. The latter controls the atmospheric lifetime of gases important to air quality and climate change and initiates the chemistry leading to the formation of ground-level ozone, a primary component of smog.
Scientists have known about the nitrous acid's role in air pollution for 40 years, but they haven't fully understood how it is produced and destroyed or how it interacts with other substances, because HONO is unstable and difficult to measure.

"Only in the last 10 years have we had the technology to study nitrous acid under environmentally relevant conditions," Raff said.

Recent studies have shown nitrous acid to be emitted from soil in many locations. But this was unexpected because, according to basic chemistry, the reactions that release nitrous acid should take place only in extremely acidic soils, typically found in rain forests or the taiga of North America and Eurasia.

The standard method to determine the acidity of soil is to mix bulk soil with water and measure the overall pH. But the IU researchers show that the crucial factor is not overall pH but the acidity at the surface of soil minerals, especially iron oxides and aluminum oxides. At the molecular level, the water adsorbed directly to these minerals is unusually acidic and facilitates the conversion of nitrite in the soil to nitrous acid, which then volatilizes.

"With the traditional approach of calculating soil pH, we were severely underestimating nitrous acid emissions from soil," Raff said. "I think the source is going to turn out to be more important than was previously imagined."

The research was carried out using soil from a farm field near Columbus, Ind. But aluminum and iron oxides are ubiquitous in soil, and the researchers say the results suggest that about 70 percent of Earth's soils could be sources of nitrous acid.

Ultimately, the research will contribute to a better understanding of how nitrous acid is produced and how it affects atmospheric processes. That in turn will improve the computer models used by the U.S. Environmental Protection Agency and other regulatory agencies to control air pollution, which the World Health Organization estimates contributes to 7 million premature deaths annually.

"With improved models, policymakers can make better judgments about the costs and benefits of regulations," Raff said. "If we don't get the chemistry right, we're not going to get the right answers to our policy questions regarding air pollution."

Source: Indiana University

Improving taste of alcohol-free beer with aromas from regular beer

Some aromatic substances from alcoholic beer can be extracted and added to alcohol-free varieties.

Consumers often complain that alcohol-free beer is tasteless, but some of the aromas it is lacking can be carried across from regular beer. Researchers from the University of Valladolid (Spain) have developed the technique and a panel of tasters has confirmed its effectiveness.

The alcohol in beer acts as a solvent for a variety of aromatic compounds; therefore, when it is eliminated, as in non-alcoholic beers, the final product loses aromas and some of its taste. It is difficult to recover these compounds, but researchers from the University of Valladolid have done just this using a pervaporation process.

"This technique consists in using a semipermeable membrane to separate two fractions from alcoholic beer: one liquid phase in which alcohol is retained, and another gaseous phase, where the aromatic compounds come in," Carlos A. Blanco, one of the authors explains. "Then, this gaseous phase can be condensed, the aromatic compounds extracted and added to non-alcoholic beer."

To conduct the study, the scientists used a special beer (with 5.5% alcohol) and another reserve beer (6.5%) from which they extracted three aromatic compounds: ethyl acetate, isoamyl acetate and isobutyl alcohol. They then added these substances to two 'almost' alcohol-free beers on the market: low-alcohol beer (less than 1% ABV) and alcohol-free beer (less than 0.1% ABV)..

A panel of experts tasted them. 90% of tasters preferred enriched low-alcohol beer instead of their original factory counterparts, and this percentage rose to 80% for alcohol-free beer. The figures have been published in the 'Journal of Food Engineering'.

"In light of these results, we conclude that the taste is improved, and thus the quality of this 'alcohol-free' beer, as the majority of panellists preferred the beer with aromas to the original," Blanco confirms.

The researchers recognise that this technique cannot yet capture all the aromas and tastes associated with alcoholic beer, but it does show progress in making 'alcohol-free' varieties more palatable for the consumer.

Spain is the primary producer and consumer of alcohol-free beer in the European Union. Around 13% of the beer sold in this country is alcohol-free, consumption of which has increased in recent years due to driving restrictions and for health reasons.

Source: Plataforma SINC