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# News

New protein manufacturing process unveiled

Scientists now have a way to study special proteins associated with disease

Researchers from Northwestern University and Yale University have developed a user-friendly technology to help scientists understand how proteins work and how to fix them when they are broken. Such knowledge could pave the way for new drugs for a myriad of diseases, including cancer.

The human body has a nifty way of turning its proteins on and off to alter their function and activity in cells: phosphorylation, the reversible attachment of phosphate groups to proteins. These “decorations” on proteins provide an enormous variety of function and are essential to all forms of life. Little is known, however, about how this dynamic process works in humans.

Using a special strain of E. coli bacteria, the researchers have built a cell-free protein synthesis platform technology that can manufacture large quantities of these human phosphoproteins for scientific study. This will enable scientists to learn more about the function and structure of phosphoproteins and identify which ones are involved in disease.

“This innovation will help advance the understanding of human biochemistry and physiology,” said Michael C. Jewett, a biochemical engineer who led the Northwestern team.

The study was published Sept. 9 by the journal Nature Communications.

Trouble in the phosphorylation process can be a hallmark of disease, such as cancer, inflammation and Alzheimer’s disease. The human proteome (the entire set of expressed proteins) is estimated to be phosphorylated at more than 100,000 unique sites, making study of phosphorylated proteins and their role in disease a daunting task.

“Our technology begins to make this a tractable problem,” Jewett said. “We now can make these special proteins at unprecedented yields, with a freedom of design that is not possible in living organisms. The consequence of this innovative strategy is enormous.”

Jewett, associate professor of chemical and biological engineering at Northwestern’s McCormick School of Engineering, and his team worked with Yale colleagues led by Jesse Rinehart. Jewett and Rinehart are co-corresponding authors of the study.

As a synthetic biologist, Jewett uses cell-free systems to create new therapies, chemicals and novel materials to impact public health and the environment.

“This work addresses the broader question of how can we repurpose the protein synthesis machinery of the cell for synthetic biology,” Jewett said. “Here we are finding new ways to leverage this machinery to understand fundamental biological questions, specifically protein phosphorylation.”

Jewett and his colleagues combined state-of-the-art genome engineering tools and engineered biological “parts” into a “plug-and-play” protein expression platform that is cell-free. Cell-free systems activate complex biological systems without using living intact cells. Crude cell lysates, or extracts, are employed instead.

Specifically, the researchers prepared cell lysates of genomically recoded bacteria that incorporate amino acids not found in nature. This allowed them to harness the cell’s engineered machinery and turn it into a factory, capable of on-demand biomanufacturing new classes of proteins.

“This manufacturing technology will enable scientists to decrypt the phosphorylation ‘code’ that exists in the human proteome,” said Javin P. Oza, the lead author of the study and a postdoctoral fellow in Jewett’s lab.

To demonstrate their cell-free platform technology, the researchers produced a human kinase that is involved in tumour cell proliferation and showed that it was functional and active. Kinase is an enzyme (a protein acting as a catalyst) that transfers a phosphate group onto a protein. Through this process, kinases activate the function of proteins within the cell. Kinases are implicated in many diseases and, therefore, of particular interest.

“The ability to produce kinases for study should be useful in learning how these proteins function and in developing new types of drugs,” Jewett said.

The National Institutes of Health (grants NIDDK-K01DK089006 and P01DK01743341), the Defense Advanced Research Projects Agency (grant N66001-12-C-4211) and the David and Lucille Packard Foundation Fellowship supported the research.

The title of the paper is “Robust production of recombinant phosphoproteins using cell-free protein synthesis.” The other co-first author is Hans R. Aerni, of Yale.

Northwestern University

Cancer patient receives 3-D printed ribs

After being diagnosed with a chest wall sarcoma, a 54-year-old Spanish man's surgical team made the decision to remove his sternum and a portion of his rib cage and replace it with an implant.

The implant was designed and manufactured by medical device company, Anatomics, who utilised the CSIRO's 3D printing facility, Lab 22 in Melbourne, Australia.

The surgical team, Dr José Aranda, Dr Marcelo Jimene and Dr Gonzalo Varela from Salamanca University Hospital, knew the surgery would be difficult due to the complicated geometries involved in the chest cavity.

The procedure has been described in the European Journal of Cardio-Thoracic Surgery.

"We thought, maybe we could create a new type of implant that we could fully customise to replicate the intricate structures of the sternum and ribs," Dr Aranda said.

"We wanted to provide a safer option for our patient, and improve their recovery post-surgery."

That's when the surgeons turned to Anatomics.

After assessing the complexity of the requirements, Anatomics CEO Andrew Batty said the solution lay in metallic 3D printing.

"We wanted to 3D print the implant from titanium because of its complex geometry and design," Mr Batty said.

"While titanium implants have previously been used in chest surgery, designs have not considered the issues surrounding long term fixation.

"Flat and plate implants rely on screws for rigid fixation that may come loose over time. This can increase the risk of complications and the possibility of reoperation."

Through high resolution CT data, the Anatomics team was able to create a 3D reconstruction of the chest wall and tumour, allowing the surgeons to plan and accurately define resection margins.

"From this, we were able to design an implant with a rigid sternal core and semi-flexible titanium rods to act as prosthetic ribs attached to the sternum," Mr Batty said.

Working with experts at CSIRO's 3D printing facility Lab 22, the team then manufactured the implant out of surgical grade titanium alloy.

"We built the implant using our $1.3 million Arcam printer," Alex Kingsbury from CSIRO's manufacturing team said. "The printer works by directing an electron beam at a bed of titanium powder in order to melt it. This process is then repeated, building the product up layer-by-layer until you have a complete implant. "3D printing has significant advantages over traditional manufacturing methods, particularly for biomedical applications. "As well as being customizable, it also allows for rapid prototyping - which can make a big difference if a patient is waiting for surgery." Once the prosthesis was complete it was couriered to Spain and implanted into the patient. "The operation was very successful," Dr Aranda said. "Thanks to 3D printing technology and a unique resection template, we were able to create a body part that was fully customized and fitted like a glove." Discovery of a highly efficient catalyst eases way to hydrogen economy Hydrogen could be the ideal fuel: Whether used to make electricity in a fuel cell or burned to make heat, the only byproduct is water; there is no climate-altering carbon dioxide. Like gasoline, hydrogen could also be used to store energy. Hydrogen is usually produced by separating water with electrical power. And although the water supply is essentially limitless, a major roadblock to a future "hydrogen economy" is the need for platinum or other expensive noble metals in the water-splitting devices. Noble metals resist oxidation and include many of the precious metals, such as platinum, palladium, iridium and gold. "In the hydrogen evolution reaction, the whole game is coming up with inexpensive alternatives to platinum and the other noble metals," says Song Jin, a professor of chemistry at the University of Wisconsin-Madison. In an online edition of Nature Materials, Jin's research team reports a hydrogen-making catalyst containing phosphorus and sulfur — both common elements — and cobalt, a metal that is 1,000 times cheaper than platinum. Catalysts reduce the energy needed to start a chemical reaction. The new catalyst is almost as efficient as platinum and likely shows the highest catalytic performance among the non-noble metal catalysts reported so far, Jin reports. The advance emerges from a long line of research in Jin's lab that has focused on the use of iron pyrite (fool's gold) and other inexpensive, abundant materials for energy transformation. Jin and his students Miguel Cabán-Acevedo and Michael Stone discovered the new high-performance catalyst by replacing iron to make cobalt pyrite, and then added phosphorus. Although electricity is the usual energy source for splitting water into hydrogen and oxygen, "there is a lot of interest in using sunlight to split water directly," Jin says. The new catalyst can also work with the energy from sunlight, Jin says. "We have demonstrated a proof-of-concept device for using this cobalt catalyst and solar energy to drive hydrogen generation, which also has the best reported efficiency for systems that rely only on inexpensive catalysts and materials to convert directly from sunlight to hydrogen." Many researchers are looking to find a cheaper replacement for platinum, Jin says. "Because this new catalyst is so much better and so close to the performance of platinum, we immediately asked WARF (the Wisconsin Alumni Research Foundation) to file a provisional patent, which they did in just two weeks." Many questions remain about a catalyst that has only been tested in the lab, Jin says. "One needs to consider the cost of the catalyst compared to the whole system. There's always a tradeoff: If you want to build the best electrolyzer, you still want to use platinum. If you are able to sacrifice a bit of performance and are more concerned about the cost and scalability, you may use this new cobalt catalyst." Strategies to replace a significant portion of fossil fuels with renewable solar energy must be carried out on a huge scale if they are to affect the climate crisis, Jin says. "If you want to make a dent in the global warming problem, you have to think big. Whether we imagine making hydrogen from electricity, or directly from sunlight, we need square miles of devices to evolve that much hydrogen. And there might not be enough platinum to do that." The collaborative team included Professor J.R. Schmidt, a theoretical chemist at UW-Madison, and electrical engineering Professor Jr-Hau He and his students from King Abdullah University of Science and Technology in Saudi Arabia. The U.S. Department of Energy provided major funding for the study. University of Wisconsin-Madison ‘Lab-on-a-Chip’ technology cuts costs of lab tests for HIV, Lyme disease, other diseases Rutgers engineers have developed a breakthrough device that can significantly reduce the cost of sophisticated lab tests for medical disorders and diseases, such as HIV, Lyme disease and syphilis. The new device uses miniaturized channels and valves to replace “benchtop” assays – tests that require large samples of blood or other fluids and expensive chemicals that lab technicians manually mix in trays of tubes or plastic plates with cup-like depressions. “The main advantage is cost – these assays are done in labs and clinics everywhere,” said Mehdi Ghodbane, who earned his doctorate in biomedical engineering at Rutgers and now works in biopharmaceutical research and development at GlaxoSmithKline. Ghodbane and six Rutgers researchers recently published their results in the Royal Society of Chemistry’s journal, Lab on a Chip. The lab-on-chip device, which employs microfluidics technology, along with making tests more affordable for patients and researchers, opens doors for new research because of its capability to perform complex analyses using 90 percent less sample fluid than needed in conventional tests. “A great deal of research has been hindered because in many cases one is not able to extract enough fluid,” Ghodbane said. The Rutgers breakthrough also requires one-tenth of the chemicals used in a conventional multiplex immunoassay, which can cost as much as$1500. Additionally, the device automates much of the skilled labor involved in performing tests. “The results are as sensitive and accurate as the standard benchtop assay,’’ said Martin Yarmush, the Paul and Mary Monroe Chair and Distinguished Professor of biomedical engineering at Rutgers and Ghodbane’s adviser.

Until now, animal research on central nervous system disorders, such as spinal cord injury and Parkinson’s disease, has been limited because researchers could not extract sufficient cerebrospinal fluid to perform conventional assays. “With our technology, researchers will be able to perform large-scale controlled studies with comparable accuracy to conventional assays,” Yarmush said.

The discovery could also lead to more comprehensive research on autoimmune joint diseases such as rheumatoid arthritis through animal studies. As with spinal fluid, the amount of joint fluid, or synovial fluid, researchers are able to collect from lab animals is minuscule. The Rutgers team has combined several capabilities for the first time in the device they’ve dubbed “ELISA-on-a-chip” (for enzyme-linked immunosorbent assay). A single device analyzes 32 samples at once and can measure widely varying concentrations of as many as six proteins in a sample. The researchers are exploring the commercial potential of their technology. Other members of the research team are Elizabeth Stucky, a doctoral student in the Department of Chemical and Biochemical Engineering, and assistant research professor Tim Maguire, associate research professor Rene Schloss, professor David Shreiber and associate professor Jeffrey Zahn, all in the Department of Biomedical Engineering. The National Institutes of Health, the National Science Foundation, the New Jersey Commission on Brain Injury Research and Corning, Inc. provided funding for the research.

Rutgers University

New drug approach could offer relief to patients, hospitals fighting antibiotic resistance

Virginia Tech researchers have discovered a new group of antibiotics that may provide relief to some of the more than 2 million people in the United States affected by antibiotic resistance.

The new antibiotics target the bacteria Staphylococcus aureus, or staph, and the antibiotic resistant strains commonly known as MRSA, short for methicillin-resistant Staphylococcus aureus.

In 2013, invasive MRSA infections were responsible for an estimated 9,937 deaths in the U.S. Although current infection rates are declining, the majority of these deaths, about 8,150, were associated with inpatient stays in health care facilities, according to the Active Bacterial Core surveillance report by the Centers for Disease Control and Prevention.

The discovery, published in Medicinal Chemistry Communications, shows that the potential new antibiotics are unlike contemporary antibiotics because they co-ntain iridium, a silvery-white transition metal. New transition metal complexes do not easily breakdown, which is important for delivery of antibiotics to where they are needed to fight infections in the body.

Even though these compounds contain iridium, further testing by the researchers shows that they are nontoxic to animals and animal cells. Thus, they are likely safe for use in humans, according to the researchers.

"So far our findings show that these compounds are safer than other compounds made from transition metals," said Joseph Merola, a professor of chemistry in the College of Science, a Fralin Life Science Institute affiliate, and a corresponding author of the study. "One of the reasons for this is that the compounds in this paper that target MRSA are very specific, meaning that a specific structure-function relationship must be met in order to kill the bacteria."

Researchers showed the antibiotics effectively kill the bacteria without inhibiting mammalian cells. A version of the antibiotic was tested for toxicity in mice with no ill effects.

"We are still at the beginning of developing and testing these antibiotics but, so far, our preliminary results show a new group of antibiotics that are effective and safe," said Joseph Falkinham, a professor of microbiology in the College of Science and an affiliate of the Virginia Tech Center for Drug Discovery. "Within the next few years, we hope to identify various characteristics of these antibiotics, such as their stability, their distribution and concentration in animal tissue, their penetration into white blood cells, and their metabolism in animals."

The team is currently testing the compounds in human cell lines and, so far, the cells have remained normal and healthy.

This discovery comes at a time when antibiotic resistance is a significant health concern all over the world, for people and for livestock.

Last September, the U.S. federal government issued an executive order to combat the rise of antibiotic-resistant bacteria, stating that it “represents a serious threat to public health and the economy.” In March, a National Action Plan outlined critical next steps for key federal agencies and departments.

According to estimates cited by the Centers for Disease Control and Prevention, antibiotic resistance is a problem that adds around \$20 billion annually to health care costs in the U.S.

"The biggest question scientists have to ask to tackle antibiotic resistance is, how can we stay on top of the bacteria? Fortunately, these new organometallic antibiotics are coming at a time when bacteria have not evolved to resist them," said Merola, who is also an affiliate of the Virginia Tech Center for Drug Discovery.

In both the U.S. and Europe, the spread of MRSA is a major threat to people in hospitals and other health care facilities.

When people contract the bacteria in a hospital setting, the infection can be life-threatening and cause pneumonia and infections in the bloodstream and in surgical wounds, according to the CDC.

Staphylococcus aureus is a bacterium commonly found on the skin and nose, which is how it spreads into hospitals and other medical facilities.

"Before you go into the hospital for surgery, many hospitals will do a nasal swab, and if you have staph, they will treat you before surgery because it could be transferred into your body and cause serious infection," Falkinham said.

A university-level Research Institute of Virginia Tech, the Fralin Life Science Institute enables and enhances collaborative efforts in research, education, and outreach within the Virginia Tech life science community through strategic investments that are often allied with colleges, departments, and other institutes.

Virginia Tech news

FDA approves the first 3D printed drug product

Aprecia introduces its first product using the ZipDose® Formulation Platform for the treatment of epilepsy

Aprecia Pharmaceuticals Company has announced that the U.S. Food and Drug Administration (FDA) has approved SPRITAM® levetiracetam for oral use as a prescription adjunctive therapy in the treatment of partial onset seizures, myoclonic seizures and primary generalized tonic-clonic seizures in adults and children with epilepsy (1). SPRITAM utilizes Aprecia’s proprietary ZipDose® Technology platform, a groundbreaking advance that uses three-dimensional printing (3DP) to produce a porous formulation that rapidly disintegrates with a sip of liquid (1). While 3DP has been used previously to manufacture medical devices, this approval marks the first time a drug product manufactured with this technology has been approved by the FDA.

“By combining 3DP technology with a highly-prescribed epilepsy treatment (2), SPRITAM is designed to fill a need for patients who struggle with their current medication experience,” said Don Wetherhold, Chief Executive Officer of Aprecia. “This is the first in a line of central nervous system products Aprecia plans to introduce as part of our commitment to transform the way patients experience taking medication.”

ZipDose Technology enables the delivery of a high drug load, up to 1,000 mg in a single dose (2). As a result, SPRITAM enhances the patient experience - administration of even the largest strengths of levetiracetam with just a sip of liquid. In addition, with SPRITAM there is no measuring required as each dose is individually packaged, making it easy to carry this treatment on the go. SPRITAM is expected to be available in the first quarter of 2016.

“In my experience, patients and caregivers often have difficulty following a treatment regimen. Whether they are dealing with a swallowing disorder or the daily struggle of getting a child to take his or her medication, adherence can be a challenge,” said Marvin H. Rorick III, M.D., neurologist at Riverhills Neuroscience in Cincinnati, Ohio. “Especially for children and seniors, having an option for patients to take their medication as prescribed is important to managing this disease.”

Nearly three million people in the United States have been diagnosed with active epilepsy, with an estimated 460,000 of those cases occurring in children (3). Additionally, in a recent survey of people age 65 and older living in an independent living facility, 15 percent reported difficulty swallowing (4). Other chronic conditions can impair the ability to swallow, further exacerbating the problem (5).

While there are many reasons, including swallowing difficulties, for which patients may not take their medication as prescribed, missed doses of medication can undermine treatment outcomes for conditions like epilepsy (6, 7). Patients with poor adherence to epilepsy drugs are more likely to have a breakthrough seizure (6). In one survey completed by patients, 71 percent acknowledged having forgotten, missed or skipped a dose of seizure medication at some time, and almost half reported having had a seizure after a missed dose at some time during treatment.

References

1.      SPRITAM [package insert]. East Windsor, N.J. Aprecia Pharmaceuticals Company; 2015.

2.     Data on file. Aprecia Pharmaceuticals Company.

3.     Centers for Disease Control and Prevention. Epilepsy Fast Facts. March 12, 2015. Available: http://www.cdc.gov/epilepsy/basics/fast-facts.htm. Accessed July 29, 2015.

4.     Chen PH, et al. Prevalence of Perceived Dysphagia and Quality-of-Life Impairment in a Geriatric Population. Dysphagia. 2009;24(1):1-6.

5.     Ekberg O. et al. Social and psychological burden of dysphagia; its impact on diagnosis and treatment. Dysphagia. 2002;17:139-146.

6.     Davis KL, Candrilli SD, Edin HM. Prevalence and Cost of Nonadherence with Antiepileptic Drugs in an Adult Managed Care Population. Epilepsia. 2008. 49.

7.     Stegemann S, Gosch M, Breitkreutz J. Swallowing dysfunction and dysphagia is an unrecognized challenge for oral drug therapy. International Journal of Pharmaceutics. 430 (2012) 197-206.

8.     Cramer JA, Glassman M, Rienzi V. The relationship between poor medication compliance and seizures. Epilepsy and Behavior. 08/2002; 3(4):338-342.

Making pharmaceuticals that degrade before they can contaminate drinking water

In recent years, researchers have realized that many products, including pharmaceuticals, have ended up where they're not supposed to be -- in our drinking water. But now scientists have developed a way to make drugs that break down into harmless compounds before they contaminate our taps. Their report appears in ACS' journal Environmental Science & Technology.

A wide range of active ingredients originating from pesticides, shampoos, lotions, cosmetics, disinfectants and drugs get washed into sewage systems or rivers and streams, ending up in our tap water. Scientists don't have a complete picture yet of what effects these substances have on wildlife and human health, but they are a major concern. Researchers have detected them in low levels in streams and rivers across the United States and in other countries. To address the specific problem of medications in the environment, Klaus Kümmerer and colleagues made tweaks to pharmaceuticals so they degrade after they've passed through both the body and sewage treatment systems, which aren't capable of scrubbing wastewater of all contaminants.

The researchers chose to work with a commonly used drug called propranolol - a beta blocker prescribed to treat high blood pressure and to prevent heart problems. It is very stable and has been found in sewage.

They made a small molecular change in its structure that didn't affect its beta blocking activity but allowed it to break down more easily than the original form. Further studies are needed, but initial testing showed that the altered drug and its byproducts are likely not toxic. The researchers suggest that a similar approach could be used to re-design other classes of drugs and chemicals to make them more environmentally friendly, too.

The authors acknowledge funding from the German Ministry of Education and Research.

American Chemical Society