Into the (mis)fold: a diagnostic tool for proteins

Alzheimer’s disease is the most common form of dementia, currently affecting more than 35 million people worldwide. Although many genetic and hereditary factors are thought to contribute to the telltale deterioration of memory and cognitive functions resulting from Alzheimer’s, a central aspect to this disease is an accumulation of misfolded proteins in the brain.

Now, scientists at Berkeley Lab have engineered a universal, highly sensitive technique for detecting misfolded proteins in biological fluids. This groundbreaking nanoscience capability could help pinpoint Alzheimer’s in its early stages and enable researchers to discover new therapies for this devastating disease.

When a protein doesn’t fold into its normal shape, it also doesn’t perform its normal functions. This disruption in behavior could lead to proteins that aggregate into plaques or deposits and become toxic to cells. In Alzheimer’s disease, aggregates of a protein called beta-amyloid form in the central nervous system, causing damage to cells in the brain and triggering dementia.

An analytical capability for measuring tiny clusters of these proteins—before irreversible damage occurs—would be a powerful tool in the early detection of Alzheimer’s and other misfolded protein diseases. However, despite significant research efforts, there are currently no diagnostic tools available to selectively detect small-scale aggregates of misfolded proteins in biological fluids, such as blood or spinal fluid.

This video is not supported by your browser at this time.

“This collaboration illustrates how a biomedical problem can also be a nanoscience problem, in which a chemical reagent is needed to recognize partially aggregated proteins,” said Ron Zuckermann, Director of the Biological Nanostructures Facility at the Molecular Foundry, a nanoscience user facility at Berkeley Lab. “We were faced with the challenge of synthesizing a material that’s capable of specifically detecting this aggregated protein and not any of the other proteins in the blood.”

Zuckermann is a pioneer in the development of peptoids, synthetic polymers that behave like naturally occurring proteins but can withstand aggressive chemical and biological environments without degrading. His group previously discovered peptoids capable of self-assembling into nanoscale jaws, nanosheets, and nanoscale ropes that braid themselves.

“Peptoids are ideal for this application as they are similar to proteins in structure, but different enough that they aren’t degraded by enzymes in the blood,” added Zuckermann. “We can now engineer materials that are capable of specific recognition yet can evade destruction.”

Using the Foundry’s state-of-the-art robotic synthesis capabilities, the team prepared a panel of peptoids designed to capture a misfolded prion protein, an abnormal, infectious form of a cellular protein found in the brain. By attaching these peptoids to tiny magnetic beads, the team could then use a magnet to isolate misfolded proteins directly from blood samples. The most selective and sensitive of these peptoids, coined aggregate-specific reagent, or ASR1, could capture not only the prion aggregates, but aggregates associated with Alzheimer’s disease as well.

“Our study shows how basic research capabilities can be translated into a practical application,” said Zuckermann. “The potential for this tool to serve as a diagnostic in other misfolded protein diseases, such as Parkinson’s and Type II diabetes, is wide open, and I’m excited to continue this collaboration.”

More information: This research is reported in a paper titled, “A universal method for detection of amyloidogenic misfolded proteins,” appearing in the journal Biochemistry and available in Biochemistry online.

Abstract
Diseases associated with the misfolding of endogenous proteins, such as Alzheimer’s disease and type II diabetes, are becoming increasingly prevalent. The pathophysiology of these diseases is not totally understood, but mounting evidence suggests that the misfolded protein aggregates themselves may be toxic to cells and serve as key mediators of cell death. As such, an assay that can detect aggregates in a sensitive and selective fashion could provide the basis for early detection of disease, before cellular damage occurs. Here we report the evolution of a reagent that can selectively capture diverse misfolded proteins by interacting with a common supramolecular feature of protein aggregates. By coupling this enrichment tool with protein specific immunoassays, diverse misfolded proteins and sub-femtomole amounts of oligomeric aggregates can be detected in complex biological matrices. We anticipate that this near-universal approach for quantitative misfolded protein detection will become a useful research tool for better understanding amyloidogenic protein pathology as well as serve as the basis for early detection of misfolded protein diseases.

Provided by Lawrence Berkeley National Laboratory (news : web)

Praxair Expands Gulf Coast Hydrogen Supply Capabilities

 Praxair, Inc. is expanding its hydrogen capacity in Texas and Louisiana and extending its Mississippi River corridor hydrogen pipeline system under long-term supply agreements for 270 million standard cubic feet per day (scfd) with Valero Energy Corporation.

In Louisiana, Praxair is installing a new, world-class 135 million scfd hydrogen plant at Valero’s St. Charles refinery. Under a long-term agreement, Valero will consume 120 million scfd of hydrogen and approximately 400,000 pounds per hour of steam at its new 50,000 barrels-per-day hydrocracker. Start-up is expected in the fourth quarter of 2012.

Praxair is extending its Louisiana pipeline by 50 miles, connecting the new hydrogen plant in St. Charles to Praxair’s existing complex in Geismar, Louisiana. The pipeline extension is scheduled to be in operation by the second quarter of 2012.

The new facilities will allow Praxair to supply other customers in Louisiana, providing a hydrogen supply alternative to refineries and chemical plants located in the lower Mississippi River corridor.

“These projects represent a significant investment in supply for our customers in the lower Mississippi River corridor as they upgrade and expand their own production capabilities,” said Mike Jordan, vice president, South Region, for Praxair’s North American Industrial Gases business unit. “They put the southern Louisiana region in a position similar to Texas in terms of competitive hydrogen supply alternatives.”

Additionally, Praxair is installing a new 135 million scfd hydrogen plant at Valero’s Port Arthur, Texas, refinery. Plant start-up is scheduled for the first quarter of 2013. Under the new contracts, Praxair will supply 150 million scfd of hydrogen and approximately 200,000 pounds per hour of steam to Valero’s 50,000 barrels-per-day hydrocracker. Hydrogen will be supplied from the new plant in conjunction with Praxair’s one billion scfd pipeline and storage complex.

 

UF develops method to make plastic from discarded plant material

Plastic may compete with paper in the grocery line, but it doesn’t have to compete with the world’s food supply, according to University of Florida researchers.

They’ve developed a way to produce plastic that doesn’t use valuable natural resources, such as food or fuel, for raw materials.

The new method uses a strain of bacteria to create bioplastic from discarded plant material, such as yard waste.

Bioplastic, or plastic from renewable resources, is produced when an organism such as a bacterium creates lactic acid while fermenting carbohydrates. The lactic acid can then be converted into long chains of molecules to form plastic.

Current bioplastic production uses food carbohydrates, such as cane sugar or corn starch, as raw materials. Traditional plastic production requires petroleum.

Keelnatham Shanmugam, a UF microbiology and cell science professor, Lonnie Ingram, a distinguished professor in microbiology and cell science, and their co-workers made the development. Their research is published in the May issue of the Journal of Industrial Microbiology and Biotechnology.

“As we start using more and more bioplastics, we are infringing upon the use of food material,” said Shanmugam. “We’d like to switch away from food-based carbohydrates to non-food-based carbohydrates for producing plastics.”

Using discarded plant material to produce plastic helps keep commodity prices down. The plastic produced from the process is both biodegradable and recyclable, Shanmugam said.

In the study, the researchers tested the bacterium — Bacillus coagulans strain 36D1 — for its ability to produce lactic acid in a variety of conditions typical of bioplastic production. The bacterium was collected from a geyser in Calistoga, Calif., which was one of the many places the researchers sampled for bacteria.

Previous attempts to produce lactic acid from discarded plant materials using microorganisms have not yielded enough lactic acid and weren’t cost effective.

However, Shanmugam and Ingram found that by adding calcium carbonate to the process, they achieved lactic acid yields as high as those achieved by organisms that fermented food carbohydrates.

Additionally, the heat-tolerant bacterial strain also cut production costs significantly by allowing the process to run at a higher temperature, which reduced the amount of expensive, plant-digesting enzymes required by up to four times.

Cost savings are also achieved by eliminating the need for food carbohydrates as raw materials since discarded plant waste is less expensive. For example, using straw as a raw material is 13 times less expensive than sugar and five times less expensive than corn or wheat.

Mark Ou, a UF biological scientist and the study’s lead author, said increases in oil prices over the last several years have led to more interest in petroleum alternatives for plastic production.

“If we can save some of our oil and turn our plants into our plastic cups and packaging, then we can increase our national security by reducing our dependence on foreign oil,” Ou said.

Provided by University of Florida (news : web)

Evonik starts up new monosilane plant in Japan

 Evonik Industries and its partner Taiyo Nippon Sanso Corporation (TNSC) officially started up the new integrated production facility for monosilane and AEROSIL® in Yokkaichi, 400 kilometers south of Tokyo (Japan). Evonik has thus realized a future-oriented project to serve the growing photovoltaic and electronic markets. The project volume was around €150 million and the facility was the company’s largest single project in 2010. Evonik has a long-term agreement to supply monosilane to TNSC. Monosilane is used in the production of thin film solar cells, flat-screen displays and semiconductors for the electronics industry. Applications for AEROSIL® include processing into plastics, colorants and coatings.

At the opening ceremony in Yokkaichi, Klaus Engel, Chairman of Evonik's Executive Board, commented: “This substantial investment in Japan expands our significant market and technology position in the future-oriented solar energy market. It also makes a further important contribution to the global resource efficiency megatrend.”

The process used to produce monosilane at this new plant was developed by Evonik, which already operates a facility using this technology in Rheinfelden (Germany). The new plant in Yokkaichi will serve the Asian market with electronic-grade monosilane. 

 

New substance may allow successful transplantation of 'marginal' livers

New research raises the possibility that the critically short supply of livers for organ donation could be expanded by treating so-called "marginal" livers with a substance that protects them from damage after being connected to recipients' blood supplies. The report appears in ACS' journal Molecular Pharmaceutics.

Ram Mahato and colleagues note that the need for liver transplants has grown over the years, though the number of available livers has not. Currently, more than 16,000 people are waiting for a liver in the U.S., but less than 7,000 liver transplants were performed during the entire year of 2010. This shortage has led organ transplant teams to consider using marginal, or damaged, livers, such as those with cholestasis — a build-up of bile. But transplanting a damaged liver has risks, including a higher risk that the organ will fail. To overcome this challenge, the researchers utilized a hedgehog-signaling inhibitor to increase the odds of a successful liver transplant.

They found that a compound called cyclopamine prevented further injury to cholestatic livers after the blood supply was cut off then returned — a situation similar to what transplanted livers undergo. The research was performed in rats, which are stand-ins for humans in the laboratory. It provided "convincing evidence" that cyclopamine may protect cholestatic livers from additional damage after a transplant procedure and improve clinical outcomes for the patients.

More information: “Cyclopamine attenuates acute warm ischemia reperfusion injury in cholestatic rat liver: Hope for marginal livers”, Mol. Pharmaceutics, Article ASAP DOI: 10.1021/mp200115v

Abstract
Cholestasis is a significant risk factor for immediate hepatic failure due to ischemia reperfusion (I/R) injury in patients undergoing liver surgery or transplantation. We recently demonstrated that inhibition of Hedgehog (Hh) signaling with cyclopamine (CYA) before I/R prevents liver injury. In this study we hypothesized that Hh signaling may modulate I/R injury in cholestatic rat liver. Cholestasis was induced by bile duct ligation (BDL). Seven days after BDL, rats were exposed to either CYA or vehicle for 7 days daily before being subjected to 30 min of ischemia and 4 h of reperfusion. Expression of Hh ligands (Sonic Hedgehog, Patched-1 and Glioblastoma-1), assessment of liver injury, neutrophil infiltration, cytokines, lipid peroxidation, cell proliferation and apoptosis were determined. Significant upregulation of Hh ligands was seen in vehicle treated BDL rats. I/R injury superimposed on these animals resulted in markedly elevated serum alanine transaminase (ALT), aspartate transaminase (AST), total bilirubin accompanied with increased neutrophil recruitment and lipid peroxidation. Preconditioning with CYA reduced the histological damage and serum liver injury markers. CYA also reduced neutrophil infiltration, proinflammatory cytokines such as TNF-? and IL-1ß expression of ?-smooth muscle actin and type 1 collagen resulting in reduced fibrosis. Furthermore CYA treated animals showed reduced cholangiocyte proliferation, and apoptosis. Hepatoprotection by CYA was conferred by reduced activation of protein kinase B (Akt) and extracellular signal regulated kinase (ERK). Endogenous Hh signaling in cholestasis exacerbates inflammatory injury during liver I/R. Blockade of Hh pathway represents a clinically relevant novel approach to limit I/R injury in cholestatic marginal liver.

Provided by American Chemical Society (news : web)

Hard or soft: at the touch of a button New nano material switches properties as required

 A world premiere: a material which changes its strength, virtually at the touch of a button. This transformation can be achieved in a matter of seconds through changes in the electron structure of a material; thus hard and brittle matter, for example, can become soft and malleable. What makes this development revolutionary, is that the transformation can be controlled by electric signals. This world-first has its origins in Hamburg. Jörg Weißmüller, a materials scientist at both the Technical University of Hamburg and the Helmholtz Center Geesthacht, has carried out research on this groundbreaking development, working in cooperation with colleagues from the Institute for Metal Research in Shenyang, China.

The 51-year-old researcher from the Saarland referred to his fundamental research, which opens the door to a multitude of diverse applications, as “a breakthrough in the material sciences”. The new metallic high-performance material is described by Prof. Dr. Jörg Weißmüller and the Chinese research scientist Hai-Jun Jin in the latest issue of the renowned scientific journal “Science". Their research findings could, for example, make future intelligent materials with the ability of self healing, smoothing out flaws autonomously.

The firmness of a boiled egg can be adjusted at will through the cooking time. Some decisions are, however, irrevocable – a hard-boiled egg can never be reconverted into a soft-boiled one. There would be less annoyance at the breakfast table if we could simply switch back and forth between the different degrees of firmness of the egg.

Similar issues arise in the making of structural materials such as metals and alloys. The materials properties are set once and for all during production. This forces engineers to make compromises in the selection of the mechanical properties of a material. Greater strength is inevitably accompanied by increased brittleness and a reduction of the damage tolerance.

Professor Weißmüller, head of the Institute of Materials Physics and Technology at the Technical University of Hamburg and also of the department for Hybrid Material Systems at the Helmholtz Center Geesthacht, stated: “This is a point where significant progress is being made. For the first time we have succeeded in producing a material which, while in service, can switch back and forth between a state of strong and brittle behavior and one of soft and malleable. We are still at the fundamental research stage but our discovery may bring significant progress in the development of so-called smart materials.”

In order to produce this innovative material, material scientists employ a comparatively simple process: corrosion. The metals, typically precious metals such as gold or platinum, are placed in an acidic solution. As a consequence of the onset of the corrosion process, minute ducts and holes are formed in the metal. The emerging nanostructured material is pervaded by a network of pore channels.

The pores are impregnated with a conductive liquid, for example a simple saline solution or a diluted acid, and a true hybrid material of metal and liquid is thus created. It is the unusual “marriage”, as Weißmüller calls this union of metal and water which, when triggered by an electric signal, enables the properties of the material to change at the touch of a button.

As ions are dissolved in the liquid, the surfaces of the metal can be electrically charged. In other words, the mechanical properties of the metallic partner are changed by the application of an electric potential in the liquid partner. The effect can be traced back to a strengthening or weakening of the atomic bonding in the surface of the metal when extra electrons are added to or withdrawn from the surface atoms. The strength of the material can be as much as doubled when required. Alternatively, the material can be switched to a state which is weaker, but more damage tolerant, energy-absorbing and malleable.

Specific applications are still a matter for the future. However, researchers are already thinking ahead. In principle, the material can create electric signals spontaneously and selectively, so as to strengthen the matter in regions of local stress concentration. Damage, for instance in the form of cracks, could thereby be prevented or even healed. This has brought scientists a great step closer to their objective of ‘intelligent’ high performance materials.

Original publication:
Hai-Jun Jin and Jörg Weissmüller; A Material with Electrically Tunable Strength and Flow Stress; Science 3 June 2011, Vol. 332 no. 6034 pp. 1179-1182

 

Development of a FRET sensor for real-time imaging of intracellular redox dynamics

In work published in the June 2011 issue of Experimental Biology and Medicine, Kolossov, Spring and their co-investigators - a multidisciplinary team within the Institute for Genomic Biology at the University of Illinois - have transferred the concept of redox-sensitive Green Fluorescent Proteins (GFPs) to a quantitative Förster resonance energy transfer (FRET) imaging platform.

For the FRET-based sensors, a change in redox induces a conformational change in a redox-sensitive switch that links two fluorescent proteins (the donor and acceptor), changing their distance, which in turn causes a detectable change in FRET efficiency. In its oxidized state the wavelength spectrum of the sensor's fluorescence emission is red-shifted (due to increased acceptor fluorescence), independent of variations in the local sensor concentration or in the intensity of the excitation light. As explained by Robert Clegg, a pioneer in the development of novel applications of optical microscopy in the biological sciences and key collaborator on the study, "FRET-based sensors circumvent the complications associated with imaging methods based on fluorescence intensity, since the increase in the FRET acceptor molecule's fluorescence can only take place if there is a change in the efficiency of energy transfer. This specific and discriminatory feature of FRET is one of the driving motives behind our development of a FRET-based assay rather than relying only on changes in the fluorescent intensity of a single component."

The current publication builds on the authors' previous work, where they reported a series of first-generation redox-sensitive linkers flanked by FRET donor and acceptor GFP-variants. As summarized by co-author Vladimir Kolossov, "The major advance in the current study is an improved dynamic range of the spectroscopic signal; in other words, a greater difference between fully reduced and oxidized states. Increasing the dynamic range leads to better discrimination between the redox states of the probe in complex biological specimens. Furthermore, the highly oxidative midpoint potential of the novel probe is ideal for measuring glutathione redox potentials in oxidative compartments of mammalian cells."

Recently, a different innovative ratiometric probe - a redox-sensitive GFP (roGFP) - has been developed in another lab. The measurement with the roGFP sensor involves the ratio of intensities of two sequential images, acquired at two different excitation wavelengths. Two thiol groups form/break a disulfide bond that modulates the peak excitation wavelength of the roGFP chromophore in response to the redox environment. Bryan Spring, a co-author, notes, "The roGFP and the FRET-based sensors have contrasting characteristics. The FRET-based sensor may prove advantageous for intravital microscopy studies, because only a single laser line is required. In contrast, roGFP requires sequential scanning of two laser lines, which slows the frame rate of image acquisition; also, the images must be compensated for the different laser intensities in order to correct for wavelength-dependent tissue scattering, and the measurement relies on the optical alignment of two excitation light beams. However, the roGFP probe is sensitive to a different range of oxidation-reduction potentials than our FRET probe, possibly leading to complementary applications." Spring adds, "We look forward to further exciting innovations for optimizing the performance of oxidation-reduction-based sensors."

Dr. Rex Gaskins, who led the project remarked, "Distinct advantages of the FRET-based approach include: (1) the ability to quantify the change in redox state; (2) independence of sensor concentration; and (3) modularity, the ability to precisely tune the redox sensitivity and range by exchange of the switch or the fluorophore modules in the probe. We expect that newly developed redox-sensitive probes could potentially be critical to a better understanding of the pharmacologic and toxicological actions of chemotherapeutic drugs and oxidants."

Dr. Steven R. Goodman, Editor-in-Chief of Experimental Biology and Medicine, said "This multidisciplinary group has developed a novel FRET-based biosensor which is a major advance in the measurement of oxidative stress in living cells in real-time. This will allow the measurement of intraorganellar glutathione potentials in living cells".

Provided by Society for Experimental Biology and Medicine (news : web)

Gifts from the Gila monster

Who would have thought that Gila monster saliva would be the inspiration for a blockbuster new drug for Type 2 diabetes? Or that medicines for chronic pain, heart attacks, high blood pressure and stroke would emerge from venom of the Magician's cone snail, the saw-scaled viper, the Brazilian lancehead snake and the Southeastern pygmy rattlesnake? These are just some of the sources contributing to the emergence of potential new drugs based on "peptides" that is the topic of the cover story in the current edition of Chemical & Engineering News (C&EN), ACS' weekly newsmagazine. Peptides are short sequences of amino acids that are the building blocks of proteins.

C&EN Senior Correspondent Ann Thayer explains that peptides play central roles in many key body processes involved in health and disease. As drugs, they have several advantages over traditional medications, including higher potency and lower toxicity. However, efforts to enlist peptides more extensively in medical treatment have stumbled due to peptides' short duration of action, tendency to be digested by enzymes in the stomach and other problems.

Thayer describes advances in overcoming those problems with 60 peptide drugs sold worldwide having sales of $13 billion in 2010 and scores of others in the pipeline. Some of the most successful of those already in use are based on peptides found naturally in animals like the Gila monster. A companion article describes how manufacturers are stepping up production, collaborating with peptide discovery companies. Peptide drugs are now longer and more complex than those developed earlier, but improvements have made production faster and more cost-effective.

More information: “Improving Peptides”: http://pubs.acs.or … 22cover.html

Provided by American Chemical Society (news : web)