Researchers discover general recipe for making antimicrobial agents that kill bacteria

Many antimicrobial peptides in our immune system kill bacteria by punching holes in their membranes. Scientists have been researching antimicrobial peptides for more than 30 years, and there is currently a large effort to mimic their antimicrobial action in order to fight antibiotic-resistant bacteria and emerging pathogens.

Now, a research team led by Gerard Wong, a professor of bioengineering at the UCLA Henry Samueli School of Engineering and Applied Science, has discovered an important pattern in the amino acid content of antimicrobial peptides and has shown that it is consistent with all 1,080 known peptides in the antimicrobial database.

The discovery of this pattern allows for the formulation of a general recipe for making antimicrobial peptides. The recipe is based on physical principles behind the generation of membrane curvature, specifically the type of curvature that facilitates membrane pore formation in bacterial membranes. Knowing this rule will greatly facilitate engineering efforts aimed at making new antibiotics.

The discovery and development of new antibacterials is costly and time consuming. Moreover, it is well known that bacteria also evolve immunity to new drugs quickly. This discovery allows for the creation new antibacterial drugs without starting from scratch: A general recipe can be followed, rather than using simple trial and error. Consequently, this will greatly accelerate drug discovery.

More information: The research was recently published in the peer-reviewed Journal of the American Chemical Society and is available online at http://pubs.acs.org/doi/full/10.1021/ja200079a

Provided by University of California Los Angeles (news : web)

Low sodium salt substitute; good for heart, diabetes and high blood patients

 

A group of researchers from Universiti Putra Malaysia (UPM) successfully produced Cardio-mate or a salt substitute from plants with low sodium content which proves to be friendly for heart, diabetes and high blood pressure patients who are at risk of food salt.

Lead researcher Prof. Dr. Suhaila Mohamed from the Institute of Bioscience, said the product that takes form of pills and powders contain anti-oxidant features and high calcium and it also doubles as flavor enhancer.

"Cardio-mate has the ability to decrease blood sugar, control excessive blood pressure and protecting the eyes, liver, heart, kidney, bone, skin, brain to blood vessels.

"This antimicrobial product also delays food decomposition and contains soluble dietary fiber and high protein as well as health properties to prevent breast cancer, cervical cancer and prostate cancer," he said at a Putra Cipta Press Conference organized by Research Management Centre (RMC) and Corporate Communications (BKK) held here.

She said the product also helps control body weight and delays the deteriorating diseases among aged individuals.

Prof. Suhaila said Cardio-mate is suitable for sauce dipping, snacks, ice cream, beverages, and cosmetics, as powdered cheese substitute, salt, soy sauce and variety of foods.

"Cardio-mate is used by sprinkling it on foods after cooking and it must not be used whilst food being cooked," she added.

Cardio-Mate has passed the toxicity test and proclaimed as an herbal supplement, not a drug product.

The research conducted since 2002 was financed by the Research University Grant Scheme (RUGS), Ministry of Science, Technology and Innovation (MOSTI) and Ministry of Higher Education (KPT).

She is assisted by eight other researchers, namely Dr. Patricia Matanjun; Dr. Farideh Namvar; Dr. Noordin Mohamed Mustapha, Fatemeh Shamsabadi; Assoc. Dr. Rasedee Abdullah; Samaneh Ghasemi Fard; Dr. Juliana Md Roslan Jaffri and Rosalina Tan Roslan Tan.

Patented in 2006, the product has received international recognition such as the gold medal in the International Exhibition of Inventions New Products Ideas (IENA) 2008 in Nuremberg, Germany and the 18th International Invention Innovation Industrial Design and Technology Exhibition (ITEX), 2007.

Provided by Universiti Putra Malaysia

For testing skin cream, synthetic skin may be as good as the real thing

New research suggests that currently available types of synthetic skin may now be good enough to imitate animal skin in laboratory tests, and may be on their way to truly simulating human skin in the future.

Researchers compared the response of synthetic skins to rat skin when they were both exposed to a generic skin cream treatment, and the results indicated they both reacted similarly.

The scientists used high-resolution images of two types of synthetic skin and samples of rat skin to discover similarities on microscopic scales.

The findings have implications for the treatment of burn victims.

When a person's body is severely burned, he or she may not have enough healthy skin remaining to attempt healing the burns through skin cell regeneration with his or her own skin. In this case, synthetic skin or animal skin provides a potential substitute. But the use of animal skin comes with a variety of problems.

"In addition to ethical issues, animal skin is hard to obtain, expensive, and gives highly variable results because of individual skin variability," said Bharat Bhushan, Ohio Eminent Scholar and the Howard D. Winbigler Professor of mechanical engineering at Ohio State University.

"Animal skin will vary from animal to animal, which makes it hard to anticipate how it might affect burnt victims, individually," Bhushan said. "But, synthetic skin's composition is consistent, making it a more reliable product," he continued.

Bhushan's research will appear in the June 5 issue of the Journal of Applied Polymer Science.

Bhushan and his colleague Wei Tang, an engineer at China University of Mining and Technology, compared two different types of synthetic skin to rat skin. The first synthetic skin was a commercially available skin purchased from Smooth-On, Inc. of Easton, Pennsylvania. The second synthetic skin was produced in Bhushan's lab. Ohio State's University Lab Animal Resources provided the rat skin samples.

Whether a synthetic skin feels and acts like real skin is very important, Bhushan explained. The skin must stand up to environmental effects such as sunlight or rain, while maintaining its texture and consistency. Scientists have continued to improve the practical and aesthetic properties of synthetic skin, which suggests it may soon be ready to replace animal skin and, farther in the future, human skin.

"Right now, our main concern is to determine whether the synthetic skin behaves like any real skin. Then, scientists can go on to more complex problems like modeling synthetic products that behave exactly like human skin," Bhushan said.

Bhushan is an expert at measuring effects on tiny scales, such as a nanometer, or billionth of a meter, which is important in skin research.

"Cellular events, like the effective and accurate delivery of drugs and the absorption of skincare products – these things occur at the nanoscale," explained Bhushan.

Using a highly sensitive microscope, known as an atomic force microscope, Bhushan and Tang were able to view the skin and the affects of an applied skin cream on a scale of about 100 nanometers. The average width of a human hair is approximately 1,000 times larger.

Despite the difference in surface features between the two synthetic skins and rat skin, the skin-cream had a comparable affect on all three samples. "The skin cream reduced the surface roughness, increased the skin's ability to absorb moisture from the environment, and softened the skin surface," said Bhushan.

Even before the addition of the skin cream, the synthetic and rat skins appeared comparable. Although the synthetic skins lacked hair follicles, they had similar roughness, meaning the distance between the highest point and lowest points on the skins' surfaces were similar.

"After treatment with skin cream, the trends of the peak-to-valley distance of the two synthetic skins and rat skin were the same, and both of them decreased. This indicates the skin cream treatment smoothed the skin surface," said Bhushan.

Bhushan explains that their future work will involve improving testing methods for measuring certain properties such as surface roughness. They also want to test a different skin cream.

Provided by The Ohio State University (news : web)

Researchers create elastic material that changes color in UV light

Researchers from North Carolina State University have created a range of soft, elastic gels that change color when exposed to ultraviolet (UV) light – and change back when the UV light is removed or the material is heated up.

The gels are impregnated with a type of photochromic compound called spiropyran. Spiropyrans change color when exposed to UV light, and the color they change into depends on the chemical environment surrounding the material.

The researchers made the gels out of an elastic silicone substance, which can be chemically modified to contain various other chemical compounds – changing the chemical environment inside the material. Changing this interior chemistry allows researchers to fine-tune how the color of the material changes when exposed to UV light.

"For example, if you want the material to turn yellow when exposed to UV light, you would attach carboxylic acid," explains Dr. Jan Genzer, Celanese Professor of Chemical and Biomolecular Engineering at NC State and co-author of a paper describing the research. "If you want magenta, you'd attach hydroxyl. Mix them together, and you get a shade of orange."

Photochromic compounds are not new, but this is the first time they've been incorporated into an elastic material, without impairing the material's elasticity.

The researchers were also able to create patterns by using a shaped mold to change the chemical make-up of specific regions in the material. For example, applying hydroxyl around a star-shaped mold (like a tiny cookie cutter) on the material would result in a yellow star-shaped pattern appearing on a dark magenta elastic when it is exposed to UV light.

"There are surely applications for this material – it's flexible, changes color in UV light, reverts to its original color in visible light, and can be patterned," Genzer says. "At this stage we have not identified the best application yet."

More information: The paper, "Photochromic materials with tunable color and mechanical flexibility," was published this month in Soft Matter, a journal of the Royal Society of Chemistry.

Provided by North Carolina State University (news : web)

Biosensors: A handy kit

A silicon-based microfluidic chip that distinguishes different viral strains shows potential for the quick on-site diagnosis of infectious diseases.

The control of infectious diseases such as the 2009 H1N1 pandemic influenza hinges on handy analytical tools that can rapidly and accurately identify infected patients at the doctor’s office or at an airport. For this reason, there has been much interest in technologies that could enable replacement of the bulky instruments used at present with point-of-care testing devices. Linus Tzu-Hsiang Kao and co-workers at the A*STAR Institute of Microelectronics and the Genome Institute of Singapore have now developed a silicon-based microfluidic system that is able to sense and differentiate the H1N1 virus from other seasonal influenza strains in ultrasmall specimens.

The detection and characterization of viral strains is now routinely performed using an assay method called real-time reverse transcription polymerase chain reaction (RT-PCR), a method that typically calls for specialized laboratory instruments and skilled personnel. Kao’s team, however, was able to integrate the PCR function into a compact two-module microfluidic chip using standard semiconductor technology. “The system will be suitable for use as a portable diagnostic tool for on-the-spot screening of highly contagious viruses, such as the influenza A H1N1 strain,” says Kao.

Because untreated influenza samples usually contain minute amounts of viral RNA mixed with other nucleic acids and proteins, the researchers designed an ‘on-chip’ PCR module that amplifies target sequences for both H1N1 and seasonal viruses at the same time. The key to their compact screening technology, however, is the silicon-nanowire sensing module used for virus identification. The nanowires in the module are modified with nucleic acid-containing polymers that specifically bind the target DNA, which results in a change in electrical resistance in proportion to the concentration of target DNA present in the sample.

The team fabricated the PCR module, which includes a reaction chamber connected to small aluminum heaters and temperature sensors through tiny channels, directly into a silicon chip using an etching technique. They then constructed the silicon nanowires by optical lithography and finally immobilized the nucleic acid-containing polymers.

Experiments revealed that the small size of the PCR chamber gave it a uniform temperature distribution (see image), providing an ideal environment for efficient RNA amplification. The PCR module also responded much faster to heating/cooling cycles than standard instruments because of the small sample volume—leading to quicker diagnoses.

The team is currently planning to improve the sample extraction module. “We are in the process of building a fully automated and integrated prototype, which will allow us to proceed to clinical validation with our collaborators,” says Kao.

More information: Kao, L. T.-H. et al. Multiplexed detection and differentiation of the DNA strains for influenza A (H1N1 2009) using a silicon-based microfluidic system. Biosensors and Bioelectronics 26, 2006–2011 (2011).

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

Biosensors: Sweet and simple

 

Surface-enhanced Raman spectroscopy (SERS) is a highly sensitive and versatile analytical tool that is widely used in biosensing applications. In conventional Raman spectroscopy, molecules are detected by their characteristic scattering of laser light, but the sensitivity of the standard method is relatively low. By detecting the same Raman scattering from molecules adsorbed to rough metal surfaces, however, the sensitivity can be enhanced remarkably, even allowing the detection of single molecules (see image). Unfortunately, the mechanism of this enhancement is not well understood and is strongly dependent on the combination of surface and molecular target.

Malini Olivo and co-workers at the A*STAR Singapore Bioimaging Consortium and Institute of Microelectronics have now developed a new class of surface that provides a much-needed sensitivity enhancement for the detection of glucose. The new substrate promises the fast, direct and accurate detection of glucose in solution at physiological concentrations.

Olivo and her co-workers have been investigating SERS for the measurement of glucose in biological samples. Glucose has very low Raman scattering efficiency and existing substrates for SERS fail to bring the method’s sensitivity of detection up to a level suitable for detecting the typical concentrations in real samples.

Instead of the commonly used rough metal substrates, the researchers turned to silicon, which they etched to form a well-defined pattern of nanogaps. They then coated the patterned silicon with thin layers of silver and gold. In tests comparing the new substrate with commercial substrates for glucose detection, Olivo and her team found that the silicon-based substrate gave the sensitivity boost they were looking for, which they attribute to the uniformity of roughness provided by the nanogap pattern.

“We were actually very surprised by our substrate’s high reproducibility,” say Olivo. “The best reproducibility reported previously for glucose was only about 10%. However, due to the special design and pattern of our substrate, we achieved reproducibility of about 3–4%, which is outstanding.” The nanogap substrate also provided good sensitivity for the detection of glucose in the physiologically important 0–25 millimolar range.

Olivo and her co-workers are already building on their success with work on an analogous system for sensing proteins. “We would like to translate similar SERS substrate platforms to optical fibers in order to develop a minimally invasive in vivo SERS platform for clinical diagnostics,” she says. The researchers have high hopes that small sensors based on this SERS platform may one day be implanted into patients for real-time glucose sensing.

More information: Dinish, U. S., et al. Development of highly reproducible nanogap SERS substrates: Comparative performance analysis and its application for glucose sensing. Biosensors and Bioelectronics 26, 1987–1992 (2011).

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

Study suggests enzyme crucial to DNA replication may provide potent anti-cancer drug target

Enlarge

During DNA replication of the lagging strand, numerous Okazaki fragments must be joined. The newer fragment ends in a short flap call the 3? overhang, while the previous fragment leaves a long 5? flap after its primer is removed. The junction opens when the template strand is bent 100 degrees. FEN1 grasps the DNA at the bend, threads the flap through an archway, and trims the flap to match the overhang.

(PhysOrg.com) -- An enzyme essential for DNA replication and repair in humans works in a way that might be exploited as anti-cancer therapy, say researchers at The Scripps Research Institute and Lawrence Berkeley National Laboratory.

The research, published in the April 15, 2011 issue of the journal Cell, focused on a member of a group of enzymes called flap endonucleases, which are essential to the life of a cell. The findings show new, clearly defined crystal structures of the enzyme FEN1 in action—demonstrating it functions in a way opposite to accepted dogma.

"This work represents a seminal advance in the understanding of FEN1," said team leader John Tainer, professor and member of the Skaggs Institute for Chemical Biology at Scripps Research and senior scientist at Lawrence Berkeley National Lab. "The research produced very accurate structures showing DNA before and after being cut by FEN1 activity, providing a basis for understanding a whole superfamily of enzymes that must cut specific DNA structures in order for DNA to be replicated and repaired."

This superfamily includes important targets for the development of new cancer interventions, Tainer added. Many cancers show high levels of FEN1 expression, which in some cases is correlated to tumor aggression. For these cases, FEN1-specific inhibitors may have chemotherapeutic potential.

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Much of the FEN1 structure was solved by Sakurai et al, but how FEN1 works was not apparent in the DNA-free structure. The presence of DNA appears to induce the transition from disorder to order; FEN1 positions the 5? flap and the 3? overhang mainly by grasping the double-strand portions of DNA on either side of the 100-degree bend.

"A better understanding of FEN1 structure and function may have long-term positive benefits to human health," noted co-author Andy Arvai, a scientific associate at Scripps Research.

Working rapidly with exquisite precision

In order for DNA to replicate, it has to unwind its double helix, which is formed out of two strands of amino acids coiled together. This unwinding is done by a replication fork whereby the two strands are separated. These strands, which form two branching prongs of the replication fork, serves as a template for production of a new complementary strand.

That task is fairly straightforward on what is known as the "leading" of the two strands. The replication fork moves along from the so-called 3' (three prime) end to the 5' (five prime) end, and DNA polymerase synthesizes a 5' to 3' complementary strand.

But because the two strands are anti-parallel, meaning they are oriented in opposite directions, the work of DNA polymerase, which can only work in the 5' to 3' direction, is more difficult on the so-called lagging strand. This strand needs to be replicated in pieces, which are known as Okazaki fragments, located near the replication fork. These fragments include a "primer," a strand of RNA that serves as a starting point for DNA synthesis.

This is where FEN1 comes in—it removes that RNA primer on the 5' flap, which occurs every 100 base pairs or so on the lagging strand, said Tainer. It's an enormous job that has to be done rapidly and accurately in order to glue the ends of replicated DNA on the lagging strand together to eventually provide an intact chromosome. "To replicate one DNA double helix in one cell you have to cut off a 5' flap so that you don't have one base pair too many or one base pair too few, and you have to do this accurately with 50 million Okazaki primers in each cell cycle," Tainer said. "It has always been a mystery as to how FEN1 can precisely cut this flap so efficiently and so rapidly. It's an amazing, efficient molecular machine for precisely cutting DNA."

To determine what FEN1 looked like in action, Arvai led the difficult but ultimately successful effort to grow crystals of the human FEN1 protein bound to DNA. The team then used X-ray crystallography to determine the atomic structure of the complex. Using Lawrence Berkeley National Laboratory's Advanced Light Source beamline, called SIBYLS, the scientists solved three different crystal structures.

The end result was a highly detailed and accurate model showing the structures of DNA before and after being cut by FEN1.

Earlier crystal structures suggested that FEN1 first grabs onto the flap of the 5' single stranded DNA, slides down to the joint where DNA is duplicated, and cuts and patches the primer there. But the new study found that, in fact, FEN1 binds, bends, frays, and then cuts the DNA.

"It binds duplex DNA, bends it into a single-stranded DNA right at the flap, flips out two base pairs, and cuts between them," said Tainer. "This gives FEN1 very precise control—a sophistication we had not expected."

Clues to cancer control

Researchers know that mutations in FEN1 can predispose humans to cancer growth because errors in flap removal can create unstable DNA that promotes cell growth and division. And studies in mice have shown that when one of two inherited FEN1 genes are knocked out, the mice are predisposed to cancer development if their DNA is damaged.

While other DNA repair systems can help compensate for FEN1 mistakes, or for missing FEN1 activity, "you need a lot of FEN1 for DNA repair and replication to work properly," Tainer said.

This suggests that, in tumors already missing one set of repair proteins, selectively inhibiting the function of FEN1 in rapidly replicating cells may prove to be an effective anti-cancer therapy. "The Achilles heel of cancer cells is defective DNA repair pathways," said Tainer, "because that makes them more sensitive to traditional therapies, such as chemotherapy and radiation. If cancer can't repair the damage these therapies do to tumors, they will die."

This is the paradox of DNA repair: while a defect in DNA repair can cause cancer, knocking out a number of backup repair systems may make tumors vulnerable to anti-cancer therapies.

"My hope is that our finding of how FEN1 works mechanistically might provide a foundation for a next-generation cancer drug," said Tainer. "We need to cut as many lifelines as possible in cancer cells in order to provide an effective treatment."

More information: "Human Flap Endonuclease Structures, DNA Double-Base Flipping, and a Unified Understanding of the FEN1 Superfamily," by Susan E. Tsutakawa et al. Cell.

Provided by The Scripps Research Institute (news : web)