Protein study gives fresh impetus in fight against superbugs

Researchers have mapped the complex molecular structure of an enzyme found in many bacteria. These molecules – known as restriction enzymes – control the speed at which bacteria can acquire resistance to drugs and eventually become superbugs.

The study, carried out by an international team including scientists from the University of Edinburgh, focused on E. coli, but the results would apply to many other infectious bacteria.

After prolonged treatment with antibiotics, bacteria may evolve to become resistant to many drugs, as is the case with superbugs such as MRSA.

Bacteria become resistant by absorbing DNA – usually from other bugs or viruses – which contains genetic information enabling the bacteria to block the action of drugs. Restriction enzymes can slow or halt this absorption process. Enzymes that work in this way are believed to have evolved as a defence mechanism for bacteria.

The researchers also studied the enzyme in action by reacting it with DNA from another organism. They were able to model the mechanism by which the enzyme disables the foreign DNA, while safeguarding the bacteria's own genetic material. Restriction enzymes' ability to sever genetic material is widely applied by scientists to cut and paste strands of DNA in genetic engineering.

The study was carried out in collaboration with the Universities of Leeds and Portsmouth with partners in Poland and France. It was supported by the Biotechnology and Biological Sciences Research Council and the Wellcome Trust and published in Genes and Development journal.

Dr David Dryden, of the University of Edinburgh's School of Chemistry, who led the study, said: "We have known for some time that these enzymes are very effective in protecting bacteria from attack by other species. Now we have painted a picture of how this occurs, which should prove to be a valuable insight in tackling the spread of antibiotic-resistant superbugs."

Provided by University of Edinburgh

New membrane lipid measuring technique may help fight disease

Could controlling cell-membrane fat play a key role in turning off disease?

Researchers at the University of Illinois at Chicago think so, and a biosensor they've created that measures membrane lipid levels may open up new pathways to disease treatment.

Wonhwa Cho, distinguished professor of chemistry, and his coworkers engineered a way to modify proteins to fluoresce and act as sensors for lipid levels.

Their findings are reported in Nature Chemistry, online on Oct. 9.

"Lipid molecules on cell membranes can act as switches that turn on or off protein-protein interactions affecting all cellular processes, including those associated with disease," says Cho. "While the exact mechanism is still unknown, our hypothesis is that lipid molecules serve sort of like a sliding switch."

Cho said once lipid concentrations reach a certain threshold, they trigger reactions, including disease-fighting immune responses. Quantifying lipid membrane concentration in a living cell and studying its location in real time can provide a powerful tool for understanding and developing new ways to combat a range of maladies from inflammation, cancer and diabetes to metabolic diseases.

"It's not just the presence of lipid, but the number of lipid molecules that are important for turning on and off biological activity," said Cho.

While visualizing lipid molecules with fluorescent proteins isn't new, Cho's technique allows quantification by using a hybrid protein molecule that fluoresces only when it binds specific lipids. His lab worked with a lipid known as PIP2 -- an important fat molecule involved in many cellular processes. Cho's sensor binds to PIP2 and gives a clear signal that can be quantified through a fluorescent microscope.

The result is the first successful quantification of membrane lipids in a living cell in real time.

"We had to engineer the protein in such a way to make it very stable, behave well, and specifically recognizes a particular lipid," Cho said. He has been working on the technique for about a decade, overcoming technical obstacles only about three years ago.

Cho hopes now to create a tool kit of biosensors to quantify most, if not all lipids.

"We'd like to be able to measure multiple lipids, simultaneously," he said. "It would give us a snapshot of all the processes being regulated by the different lipids inside a cell."

Provided by University of Illinois at Chicago (news : web)

New membrane lipid measuring technique may help fight disease

Could controlling cell-membrane fat play a key role in turning off disease? Researchers at the University of Illinois at Chicago think so, and a biosensor they've created that measures membrane lipid levels may open up new pathways to disease treatment. Wonhwa Cho, distinguished professor of chemistry, and his coworkers engineered a way to modify proteins to fluoresce and act as sensors for lipid levels.

Their findings are reported in Nature Chemistry, online on Oct. 9.

"Lipid molecules on cell membranes can act as switches that turn on or off protein-protein interactions affecting all cellular processes, including those associated with disease," says Cho. "While the exact mechanism is still unknown, our hypothesis is that lipid molecules serve sort of like a sliding switch."

Cho said once lipid concentrations reach a certain threshold, they trigger reactions, including disease-fighting immune responses. Quantifying lipid membrane concentration in a living cell and studying its location in real time can provide a powerful tool for understanding and developing new ways to combat a range of maladies from inflammation, cancer and diabetes to metabolic diseases.

"It's not just the presence of lipid, but the number of lipid molecules that are important for turning on and off biological activity," said Cho.

While visualizing lipid molecules with fluorescent proteins isn't new, Cho's technique allows quantification by using a hybrid protein molecule that fluoresces only when it binds specific lipids. His lab worked with a lipid known as PIP2 -- an important fat molecule involved in many cellular processes. Cho's sensor binds to PIP2 and gives a clear signal that can be quantified through a fluorescent microscope.

The result is the first successful quantification of membrane lipids in a living cell in real time.

"We had to engineer the protein in such a way to make it very stable, behave well, and specifically recognizes a particular lipid," Cho said. He has been working on the technique for about a decade, overcoming technical obstacles only about three years ago.

Cho hopes now to create a tool kit of biosensors to quantify most, if not all lipids.

"We'd like to be able to measure multiple lipids, simultaneously," he said. "It would give us a snapshot of all the processes being regulated by the different lipids inside a cell."

Other authors on the paper are postdoctoral researcher Youngdae Yoon, who developed the sensor; Park J. Lee, a doctoral student who developed microscope tools to enable the lipid quantification; and doctoral student Svetlana Kurilova, who worked on the protein cell delivery.

The above story is reprinted (with editorial adaptations ) from materials provided by University of Illinois at Chicago.

Journal Reference:

Youngdae Yoon, Park J. Lee, Svetlana Kurilova, Wonhwa Cho. In situ quantitative imaging of cellular lipids using molecular sensors. Nature Chemistry, 2011; DOI: 10.1038/nchem.1163

Reverse engineering materials with a rapid, non-destructive laser-based technique can aid the fight against counterfeits

In business, protecting a company’s intellectual property can mean the difference between market success and bankruptcy. As the threat of competition from illegal copies of patented technology grows, high-tech firms are themselves turning to reverse engineering to spot potential patent infringements. Effendi Widjaja and Marc Garland from the A*STAR Institute of Chemical and Engineering Sciences have now developed a technique that promises to revolutionize reverse-engineering protocols—a rapid, non-destructive approach for mapping the composition of multilayered materials.

Multilayer films are used in the lamination of electronic components as well as sport equipment, providing multifunctional protection that single-layer films cannot, such as water and corrosion resistance, for example. Identifying the composition of these films, however, usually involves chopping the sample up and dissolving it in solvents, a destructive and labor-intensive process. Widjaja and Garland have developed a non-destructive reverse-engineering strategies based on vibrational spectroscopy and advanced signal processing.

Scientists have long known that when excited by light, molecules emit vibrational signals that provide detailed information on their chemical and structural environments. Raman spectroscopy is a laser-based version of such a techniques that can be used with very little sample preparation and provides high precision. The difficulty in applying such analyses to multilayer films has been the volume of data generated—particles in a multilayer films have a wide range of overlapping vibrational signals, resulting in a complex readout that is hard to assign to individual substances.

Widjaja and Garland overcame this challenge by developing an algorithm called band-target entropy minimization (BTEM), in which rigorous statistical equations are used to ascertain the simplest sets of ‘pure’ component patterns within a vibrational spectrum. The program is extraordinarily sensitive to trace components within a material: it has been show in previous studies that even substances contributing less than 1% to the total vibrational signal information can be recovered by BTEM.

The team tested their strategy by attempting to reverse engineer the multilayer structure of a commercial packaging envelope. After shining the Raman beam onto the sample, the BTEM algorithm detected seven underlying patterns in the spectra. These patterns correspond to two forms of paper fiber, three inorganic crystalline materials and two polymers. By distributing each pure signal across the sample’s dimensions, the team successfully reconstructed the spatial distributions of the components in each laminated layer.

The speed and precision of the Raman/BTEM analysis, Widjaja and Garland note, makes the technique a valuable weapon in the fight against patent infringement.

More information: Widjaja, E. & Garland, M. Reverse engineering of multi-layer films. Materials Today 14, 114–117 (2011).

Provided by Agency for Science, Technology and Research (A*STAR)

Poplar tree leaf bud extract could fight skin aging

Antioxidants are popular anti-aging ingredients in skin creams, and now scientists are reporting a new source of these healthful substances — leaf buds of poplar trees. Their study appears in the ACS' Journal of Agricultural and Food Chemistry.

Xavier Vitrac and colleagues note that there's a long history of using poplar buds to treat various health problems, such as colds, sinusitis, sunburn and arthritis. A substance found in beehives that is made from poplar buds (called propolis) also appears to have similar disease-fighting benefits. Propolis' effects seem to be due to poplar bud compounds, but very little is known about these substances. To see whether poplar buds are a good source of antioxidants for skin creams, the researchers decided to test an extract from the buds.

The group found that poplar bud extract had moderate antioxidant activity, and it demonstrated anti-aging effects on cells in the laboratory. "The collective antioxidant properties and transcriptional effect of this extract suggest potential anti-aging properties which could be utilized in cosmetic and nutraceutical formulations," the scientists say.

More information: “Phenolic Composition and Antioxidant Properties of Poplar Bud (Populus nigra) Extract: Individual Antioxidant Contribution of Phenolics and Transcriptional Effect on Skin Aging” J. Agric. Food Chem., 2011, 59 (9), pp 4527–4536 DOI: 10.1021/jf104791t

Abstract
The Populus species possess great potential for therapeutical applications, especially for their known anti-inflammatory properties. The antioxidant properties of propolis, a hive product collected by honey bees mainly from poplar bud exudates, suggest that poplar buds also possess antioxidant properties. Here is reported the characterization of the antioxidant properties of an aqueous poplar bud (Populus nigra) extract. It presented a high total phenolic content, and moderate antioxidant properties as determined by ORAC assay. The main phenolic compounds identified were phenolic acids and flavonoid aglycons. These phenolic compounds were analyzed by ORAC assay for their individual antioxidant activity, in order to determine the major contributors to the total antioxidant activity of the extract. Thanks to their high antioxidant activity, caffeic and p-coumaric acids were identified as the major antioxidant components. Representing only 3.5% of its dry weight, these compounds represented together about 50% of the total antioxidant activity of the extract. The antioxidant properties of poplar bud extract and the phenolic compounds identified were also analyzed by cellular antioxidant activity assay (CAA), which was weakly correlated with ORAC assay. The transcriptional effect of poplar bud extract on skin aging was evaluated in vitro on a replicative senescence model of normal human dermal fibroblasts, using a customized DNA macroarray specifically designed to investigate skin aging markers. Among the detected genes, poplar bud extract significantly regulated genes involved in antioxidant defenses, inflammatory response and cell renewal. The collective antioxidant properties and transcriptional effect of this extract suggest potential antiaging properties which could be utilized in cosmetic and nutraceutical formulations.

Provided by American Chemical Society (news : web)

Cystic fibrosis bacteria could help fight back against antibiotic resistance

A bacteria which infects people with cystic fibrosis could help combat other antibiotic-resistant microbes, according to a team from Cardiff and Warwick Universities.

Continuous use of existing antibiotics means that resistant bacteria are now causing major health problems all over the world. New antibiotics are urgently needed to combat the emergence of multidrug-resistant bacteria such as the MRSA superbug.

Now a surprising source of hope has emerged in the form of Burkholderia, a group of bacteria which can cause severe lung infections in people with the genetic disorder cystic fibrosis. However, the Cardiff and Warwick team has now discovered antibiotics from Burkholderia are effective against MRSA and even other cystic fibrosis infecting bacteria.

Dr Eshwar Mahenthiralingam, of Cardiff University's School of Biosciences, Cardiff University, has been studying Burkholderia for the last decade. Using forensic fingerprinting tests to genetically identify the bacteria, Dr Mahenthiralingam's research group has tracked strains all over the world and helped develop guidelines to prevent it spreading.

By the summer of 2007, Dr Mahenthiralingam had built up a large collection of Burkholderia bacteria. He and his team then decided to screen them for antibiotics active against other bacteria, particularly drugs with the potential to kill other bacteria that infect cystic fibrosis patients. Over the next two years, Dr Mahenthiralingam's team discovered that around one quarter of Burkholderia bacteria have very strong antibiotic activity on multidrug-resistant pathogens such as MRSA. One particular strain, Burkholderia ambifaria, was found to produce two very potent antibiotics active on resistant bacteria, in particular Acinetobacter baumanii.

The chemical structures of the antibiotics, called enacyloxins, were determined by Professor Gregory Challis and Dr. Lijiang Song at the University of Warwick, demonstrating that they belong to one of the most successful families of natural product drugs, the polyketides. Other examples of polyketides include erythromycin, which is used to cure many bacterial infections, and doxorubin, used as an anti-cancer drug. Professor Challis commented: "The combination of enzymes used by Burkholderia to make the enacyloxins is very unusual. Our insights into this process should allow us to use cutting edge synthetic biology techniques to produce novel enacyloxin analogues with improved pharmaceutical properties."

The team's findings have now been published in the journal Chemistry and Biology. Dr Mahenthiralingam commented: "Burkholderia are soil bacteria like Streptomyces, which are the source of most of our current antibiotics. Our research therefore offers real hope of a completely new source for the identification and engineering of highly potent antibiotics. With antibiotic resistant bacteria causing great suffering around the world, these new sources are urgently needed."

The chemical structures of the antibiotics, called enacyloxins, were determined by Professor Gregory Challis and Dr. Lijiang Song at the University of Warwick, demonstrating that they belong to one of the most successful families of natural product drugs, the polyketides. Other examples of polyketides include erythromycin, which is used to cure many bacterial infections, and doxorubin, used as an anti-cancer drug. Professor Challis commented: "The combination of enzymes used by Burkholderia to make the enacyloxins is very unusual. Our insights into this process should allow us to use cutting edge synthetic biology techniques to produce novel enacyloxin analogues with improved pharmaceutical properties."

Provided by Cardiff University (news : web)

NIST, ASTM land a one-two punch to fight explosives terrorism

 Trace-explosives detectors (TEDs) are an increasingly common sight at airports and on loading docks, and emergency response personnel carry them to evaluate suspicious packages. A new test material developed by the National Institute of Standards and Technology (NIST) in cooperation with ASTM International enables users of these products to evaluate their performance and reliability.

The new testing material, NIST Standard Reference Material (SRM) 2906, Trace Explosives Calibration Solutions, was designed to meet the specifications of ASTM E 2520-07, Standard Practice for Verifying Minimum Acceptable Performance of Trace Explosive Detectors. ASTM is one of the leading industrial organizations for the development of voluntary consensus standards.

The NIST reference material contains calibration solutions of three high explosives: RDX (an ingredient in Composition C-4), PETN, and TNT. Under the test protocol, users sequentially apply a single drop of explosive solution and a solvent blank to swipes, the solvents are allowed to evaporate, and the instrument is tested. A simple ‘yes-no’ alarm checklist is used to determine TED performance.

SRM 2906 includes four ampoules of each of the three explosives and a blank along with a dropper bottle for each. NIST researchers formulated the concentrations of these solutions to be near, but above, the detection limit of commercial swipe-type detectors, which are commonly based on ion mobility spectrometry. When tested with the solutions, properly functioning TEDs should provide an alarm response.

This SRM fully satisfies the need for independent test materials with low uncertainties in concentrations necessary for reliable TED evaluation. Equipment vendors may use the SRM to improve and optimize their designs and demonstrate to their customers how well their machines function. Buyers may use the SRM to make sound procurement decisions. The combination of a validated standard practice and SRM will provide TED users with a reliable means of verifying initial and continuing field performance of their equipment, contributing to the fight against explosives terrorism.

More information: http://www.nist.go … rm/index.cfm

Provided by National Institute of Standards and Technology (news : web)

Does fluoride really fight cavities by 'the skin of the teeth'?

In a study that the authors describe as lending credence to the idiom, "by the skin of your teeth," scientists are reporting that the protective shield fluoride forms on teeth is up to 100 times thinner than previously believed. It raises questions about how this renowned cavity-fighter really works and could lead to better ways of protecting teeth from decay, the scientists suggest. Their study appears in ACS's journal Langmuir.

Frank Müller and colleagues point out that tooth decay is a major public health problem worldwide. In the United States alone, consumers spend more than $50 billion each year on the treatment of cavities. The fluoride in some toothpaste, mouthwash and municipal drinking water is one of the most effective ways to prevent decay. Scientists long have known that fluoride makes enamel — the hard white substance covering the surface of teeth — more resistant to decay. Some thought that fluoride simply changed the main mineral in enamel, hydroxyapatite, into a more-decay resistant material called fluorapatite.

The new research found that the fluorapatite layer formed in this way is only 6 nanometers thick. It would take almost 10,000 such layers to span the width of a human hair. That's at least 10 times thinner than previous studies indicated. The scientists question whether a layer so thin, which is quickly worn away by ordinary chewing, really can shield teeth from decay, or whether fluoride has some other unrecognized effect on tooth enamel. They are launching a new study in search of an answer.

More information: "Elemental Depth Profiling of Fluoridated Hydroxyapatite: Saving Your Dentition by the Skin of Your Teeth?" Langmuir.

Provided by American Chemical Society (news : web)