New method reveals nano-scale drug molecules in cells

               

 Researchers at Chalmers University of Technology and partners within the Chemical Imaging Infrastructure have produced a method whereby it is possible to see at the nano level where a medicinal drug ends up in the cells and how much of it is needed for optimum treatment. The technique enables the development of new pharmaceuticals and tailored treatments for diseases that have not previously been treatable.

‘Without needing to add anything to affect the cell, we can produce unique precision at the nano level. That is not possible with comparable methods that are currently in use,’ says Per Malmberg, Director of the infrastructure and senior researcher in the Department of Chemistry and Chemical Engineering at Chalmers University of Technology.

Thanks to the comprehensive knowledge of the human genome, researchers can design more effective drugs that work by engaging specific targets in the interior of the cell. This advance also makes it necessary for drug designers to consider how their molecules behave inside the cell.

The new method, developed by the partners within the Chemical Imaging Infrastructure, is described in a recently released whitepaper. It involves enhanced cutting-edge technology and knowledge to analyse and quantify biological medicines, such as peptides and oligonucleotides, in human cells with considerable reliability.

The new method is based on the NanoSIMS (nanoscale secondary ion mass spectrometry) instrument developed by CAMECA, which can measure and image molecules at high resolution on the nanoscale and has been available at the chemical imaging infrastructure since 2015. The instrument has been widely adopted by the scientific community for research, but it has not yet been applied to the development of medicinal drugs.

‘Compared with similar techniques, the NanoSIMS methodology provides much faster and more accurate answers. With our technique, a drug project can receive an answer within about four weeks, and there are good opportunities to reduce the time even further, says Per Malmberg.

Significance for unmet medical needs
So far, researchers have worked with cultured cells, but the technique can also be used to examine tissue. In the long term, it could also be used to investigate what happens in individual cells in an organ where the drug is expected to act. This could provide a key to a deeper understanding of, for example, neurodegenerative diseases, such as ALS or Parkinson’s disease, and cancer.

The pharmaceutical industry has a significant need to develop and apply methodologies for nanoscale quantification of drug molecules and their distribution at the sub-cellular level. 

‘I am extremely pleased that we have succeeded in imaging medicinal drugs in cells. There are many things that can happen to a drug once it enters the cell. Now that we can make observations at this level for the first time, we can obtain critical information that will help us design drugs for diseases that have not previously been treatable,’ says Michael Kurczy, Associate Principal Scientist at AstraZeneca.

Collaboration key to new results
Researchers at Chalmers University of Technology and the University of Gothenburg are responsible for the development, in collaboration with AstraZeneca, AstraZeneca’s BioVentureHub and the company CAMECA. When the infrastructure partners’ collective knowledge and expertise in terms of the preparation and measurement of samples were combined, results were achieved that would not have been possible without such collaboration.

‘It is a great opportunity for researchers, especially young ones, to work at the interface of academia, industry and engineering. The synergy between the developers’ direct insight into the industry’s needs and problems, and the researchers’ expertise and ideas on how they could be resolved, has been crucial in enabling us to present new, valuable tools, which will lead to a significant improvement in drug development processes and therefore the quality of people’s lives,’ says Thi Ngoc Nhu Phan, Assistant Professor at the University of Gothenburg.

Read the full white paper about the new method

Aston University launches ground-breaking project to replicate brain’s neural networks through 3D nanoprinting

Aston University has launched MESO-BRAIN, a major stem cell research project which it hopes will develop three-dimensional (3D) nanoprinting techniques that can be used to replicate the brain’s neural networks.

The cornerstone of the MESO-BRAIN project will be its use of pluripotent stem cells generated from adult human cells that have been turned into brain cells, which will form neural networks with specific biological architectures. Advance imaging and detection technologies developed in the project will be used to report on the activity of these networks in real time.

Such technology would mark a new era of medical and neuroscience research which would see screening and testing conducted using physiologically relevant 3D living human neural networks. In the future, this could potentially be used to generate networks capable of replacing damaged areas in the brains of those suffering from Parkinson’s disease, dementia or other brain trauma.

The MESO-BRAIN initiative, which will span three years, received €3.3million of funding from the European Commission as part of its prestigious Future and Emerging Technology (FET) scheme. Aston University is leading the project, with partners from industry and higher education across Europe: Axol Bioscience Ltd, Laser Zentrum Hannover, The Institute of Photonic Sciences, University of Barcelona and Kite Innovations. This unique partnership brings together stem cell biologists, neuroscientists, photonics experts and physicists.

Head of the MESO-BRAIN project, Professor Edik Rafailov, said: “What we’re hoping to achieve with this project has, until recently, been the stuff of science-fiction.

“If we can use 3D nanoprinting to improve the connection of neurons in an area of the brain which has been damaged, we will be in a position to develop much more effective ways to treat those with dementia or brain injuries.

“To date, attempts to replicate and reproduce cells in this way have only ever delivered 2D tissues or poorly defined 3D tissues that do not resemble structures found within the human body. The new form of printing we are aiming to develop promises to change this. The MESO-BRAIN project could improve hundreds of thousands of lives.”

Dr Eric Hill, Programme Director for MSc Stem cells and Regenerative Medicine at Aston University, commented: “This research carries the potential to enable us to recreate brain structures in a dish. This will allow us to understand how brain networks form during development and provide tools that will help us understand how these networks are affected in diseases such as Alzheimer’s disease.”

  

Distinct Brain Cells Recognize Novel Sights

The brain's ability to learn to recognize objects plays out in the inferior temporal cortex. A new study offers a possible explanation of how two classes of neurons play distinct roles to help that happen.

No matter what novel objects we come to behold, our brains effortlessly take us from an initial "What's that?" to "Oh, that old thing" after a few casual encounters. In research that helps shed light on the malleability of this recognition process, Brown University neuroscientists have teased apart the potentially different roles that two distinct cell types may play.
In a study published online in advance in the journal Neuron, the researchers document that this kind of learning is based in the inferior temporal cortex (ITC), a brain area buried deep in the skull. Scientists already knew the area was important for visual recognition of familiar items, but they hadn't figured out the steps required to move from novelty to familiarity, a process they refer to as "plasticity."
"We know little about that because of the level at which this plasticity is taking place," said senior author David Sheinberg, professor of neuroscience and a member of the Brown Institute for Brain Science. "The inner workings made up of individual neurons make it very hard to actually track what's going on at that level."
Working with two monkeys, in whom they monitored single neuron activity using tiny microelectrodes, Sheinberg and graduate student Luke Woloszyn tracked the firing patterns of individual neurons in the ITC while monkeys viewed 125 objects they had been trained to recognize and 125 others that they had never seen before.
The scientists found that the two major classes of cells found in the brain, excitatory and inhibitory, responded differently depending on what the monkeys saw. Excitatory neurons were especially active when the monkeys saw a preferred familiar object -- the familiar image, out of the 125 such images, that the cell "liked" best. Although the particular preferred familiar image varied across the sample of neurons, almost every excitatory cell had at least one familiar image to which it responded more robustly than its preferred novel image, Sheinberg said. Inhibitory neurons, meanwhile, were much more active when the monkeys saw any novel image, independent of the object's actual identity.
Woloszyn and Sheinberg were able to distinguish between the neuron classes by the shapes of the voltage changes picked up by the microelectrodes. Excitatory neurons had characteristically broad spikes, while inhibitory neurons had narrower spikes.
Not only did the researchers see differences in what made the neurons respond, but also in when they did so. Excitatory neurons peaked in activity within 100 milliseconds to their preferred familiar objects, for example, while inhibitory neurons responded to a broad set of novel objects over a wider timeframe of up to 325 milliseconds.
Sheinberg speculated that the different roles of the cells and the specific timing of their responses might be explained by the following interplay: When the monkey sees something familiar (banana!), excitatory neurons fire to signal recognition, sending that signal to other parts of the brain to drive the appropriate behavioral response. But when the monkey sees something unfamiliar (stapler?), the excitatory response is more diffuse, permitting the inhibitory neurons to maintain their elevated activity, which in turn signals a learning event.
"When a familiar object has been recognized, that's a positive signal and that can cause the system to move on," Sheinberg said. "In the absence of that signal, that means the object isn't familiar. What we think is going on is that the ongoing inhibitory activity actually promotes a learning process. It can be a signal to learn."
Another finding is the possible manifestation of that learning. Sheinberg and Woloszyn found considerable significance in their observation that individual excitatory neurons would modulate their firing rate only in response to a few of the images the monkeys saw. This "sparseness" of firing is a measure of the neuron's specialized attention to just a few images.
"What really drove me and Luke along the way was this question of whether ... through repeated exposure, neurons really do specialize in a marked way," Sheinberg said. "The effect of the learning was surprising to me."
They further speculate that the specialization is originally driven by the inhibitory cells.
"We thus propose that the increased activity of our putative inhibitory cells is the neurochemical trigger for the robust selectivity changes within the excitatory population," Sheinberg and Woloszyn wrote in the journal.
The study ties into a separate effort at Brown, Stanford, and other institutions in which a team of scientists is striving to lay the basic science groundwork for ultimately treating people who have suffered traumatic brain injury.
"There is a huge diversity [of neurons] and this diversity could be very important because in the case where you need to repair circuits -- say you've had a stroke, and you want to retrain that area -- it may be that certain cell classes need to be functional in order to support that plasticity," he said. "We're only beginning to appreciate the interplay between these cell types that might support learning and reorganization."
The study was supported by the National Institutes of Health and the National Science Foundation.

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David Sheinberg. Scientists already knew the inferior temporal cortex was important for visual recognition of familiar items, but they hadn’t figured out the steps required to move from novelty to familiarity — “plasticity.” (Credit: Mike Cohea/Brown University)

**Published in "SCIENCE DAILY"

Stem Cells from Pelvic Bone May Preserve Heart Function

 Stem cells from the pelvic bone may help hearts beat stronger. Doctors and other clinicians at the Orlando Health Heart Institute are researching the use of stem cells from pelvic bone marrow to restore tissue and improve heart function after muscle damage from heart attacks.

"The thought is the body may use itself to heal itself," said Vijaykumar S. Kasi, MD, PhD, an interventional cardiologist, director, Cardiovascular Research, and principal investigator for the clinical trial at ORMC. "Because stem cells are immature cells they have the potential to develop into new blood vessels and preserve cardiac muscle cells. By infusing certain stem cells into the area of the heart muscle that has been damaged from a heart attack, tissue can be preserved and heart function restored."
The PreSERVE-AMI Study, sponsored by Amorcyte, LLC, a NeoStem, Inc. company (NYSE Amex: NBS), is for patients who have received a stent to open the blocked artery after a specific heart attack history (in part a ST-Segment Elevation Myocardial Infarction, or STEMI, a critical type of heart attack caused by a prolonged period of blocked blood supply, affecting a large area of the heart muscle and causing changes in the blood levels of key chemical markers). The study evaluates the effectiveness and safety of infusing stem cells collected from a patient's bone marrow into the artery in the heart that may have caused the heart attack. About 160 patients will participate in this national study at approximately 34 sites.
The infusion procedure begins with a catheter inserted through an incision in the groin. An X-ray camera is used to guide doctors in positioning the catheter in the heart artery where the stent was placed. A balloon is inflated within the stent and the infusion takes place in the area impacted by the heart attack. Because the study is randomized, double blinded and placebo controlled, patients are infused with either AMR-001, a cell therapy product composed of stem cells taken from one's own bone marrow, or a placebo (inactive substance).
Prior to the infusion, patients are screened using various assessments including an electrocardiogram, a cardiac MRI (magnetic resonance image) and a cardiac nuclear test. After the necessary screenings, patients have a mini-bone marrow procedure where the stem cells are "harvested" (removed) from the bone marrow in their pelvic bone, using a special needle. The stem cells are processed at Progenitor Cell Therapy, another NeoStem, Inc. company, in preparation for infusion. Patients who are randomized to placebo will have their bone marrow frozen and stored and available to them for clinical use, should they require bone marrow for any reason.
"We are excited to participate in innovative clinical trials as part of our continued efforts to play a vital role in future solutions to improve patient outcomes," said Dr. Kasi. "Heart disease remains the No.1 killer of men and women in our country." Effective treatment options are part of the medical journey to more heart healthy communities locally and globally.
"Severe heart failure, often the end result of large or multiple heart attacks, is a major health care challenge, impacting more than five million people in the United States and costing more than $35 billion annually," said Dr. Kasi. "Stem cell therapy is part of the movement from treatment to cure and has the potential to overcome limitations and expenses of heart transplants and offers hope for patients who are desperately praying for another chance at life."

**Published in "SCIENCE DAILY"

Newly Found Protein Helps Cells Build Tissues

Brown University biologists have found a new molecule in fruit flies that is key to the information exchange needed to build wings properly. They have also uncovered evidence that an analogous protein may exist in people and may be associated with problems such as cleft lip, or premature ovarian failure.

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As they work together to form body parts, cells in developing organisms communicate like workers at a construction site. The discovery of a new signaling molecule in flies by Brown University biologists not only helps explain how cells send many long-haul messages, but also provides new clues for researchers who study how human development goes awry, for instance in cases of cleft lip and palate.
For all the diversity of life, animal cells employ only a small set of proteins to send those jobsite signals that coordinate construction. For that reason, said Kristi Wharton, associate professor of molecular biology, cell biology and biochemistry, studying these proteins and pathways in fruit flies can allow biologists and physicians to explain how development and other cellular processes occur in a wide variety of creatures and tissues.
"We are interested in how the pattern of a hand forms or how the pattern of a wing forms," Wharton said. "How do cells know their position in a developing tissue?"
In humans a key family of the signaling molecules that convey such messages are bone morphogenic proteins (BMPs). In fruit flies the directly analagous proteins carry the name "glass-bottom boat" (Gbb), because a mutant form makes larvae appear clear instead of milky white. To date, the conventional wisdom has been that signaling comes from a fly form of BMP known as Gbb15.
"The thought for the longest time is that this smaller protein is the only product that is formed and important for signaling," Wharton said. "But we found another form of this signaling molecule that was not previously known."
Wharton and former postdoctoral fellow Takuya Akiyama introduce the new molecule, Gbb38, in the April 3 edition of the journal Science Signaling. Experiments showed that in tissues where it was abundant, particularly parts of the wing, Gbb38 proved responsible for more signaling activity than Gbb15, and appeared especially important for carrying long-haul signals.
Possible links to humans
In addition to the findings in flies, Akiyama found that mutations in the genes for making BMPs in humans that directly mirror the genetic code for making Gbb38 in flies, occur in people with cleft lip (with or without cleft palate), and the reproductive disorders premature ovarian failure and persistent Mullerian duct syndrome. In other words, a mutation that interrupts Gbb38 production in flies, is analogous to the mutations associated with developmental disorders in different tissues in people.
The genetic analysis doesn't prove that mutations that hinder the production of an analogous signaling protein in humans would be the cause of those diseases, Wharton said. In fact, a longer-form BMP like Gbb38 has yet to be discovered in people. But the new discovery at least suggests the need for research to investigate that link, perhaps first in mice, she said.
Another potential benefit of the finding, she said, is that finding a Gbb38 analogue in humans could improve the current use of BMPs as therapeutics for bone repair, spinal fusions, and reconstruction of maxillofacial bone defects.
"If large forms of human BMPs are indeed present, which is suggested by the three human mutations, then they could be a very useful alternatives to the short BMPs because the large forms are more active in terms of signaling and have different properties in vivo," Wharton said.
Discovery on the wing
In the new paper, aided by an antibody provided by second author Guillermo Marques of the University of Alabama, Akiyama and Wharton were able to discover Gbb38 because they first asked what happened when they interrupted the creation of Gbb15. When they did that, by mutating the genetic instructions that tell enzymes where to cut Gbb15 out of a longer protein, they noticed that signaling activity was only mildly reduced instead of completely gone as conventional wisdom would have predicted.
Further research showed that there was another place where enzymes could cut to make a protein. Cutting at that spot yielded the longer Gbb38 protein. When they interrupted that cleavage in flies, the researchers found that signaling was significantly hindered. A total reduction in signaling came from interrupting both Gbb15 and Gbb38.
In local areas of wing tissue, meanwhile, Akiyama found that interrupting Gbb15 had consequences for signaling only among neighboring cells. Interrupting Gbb38, meanwhile, left local signaling intact, but created problems significantly farther away.
"The small protein doesn't move very far across the tissue," Wharton said. "But we found the large protein has a very long range. That may provide one answer to the long-standing question about what regulates the range of these signaling molecules."
The view for developmental biologists, therefore, may indeed be clearer in a larger glass-bottom boat.
The National Institute of General Medical Sciences funded the research.

**Published in "SCIENCE DAiLY"

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Communication on the tissue construction site Kristi Wharton studies “glass-bottom boat” proteins, which allow organisms to shape tissue into wings, hands, organs, and everything else. (Credit: Mike Cohea/Brown University)