Scientists develop futuristic brain probe allowing for wireless control of neurons

Researchers at the Washington University School of Medicine, St. Louis, and University of Illinois, Urbana-Champaign, created a remote controlled, nextgeneration tissue implant that allows neuroscientists to inject drugs and shine lights on neurons deep inside the brains of mice. The revolutionary device is described online in the journal Cell.

“It unplugs a world of possibilities for scientists to learn how brain circuits work in a more natural setting,” said Michael R. Bruchas, Ph.D., associate professor of anesthesiology and neurobiology at Washington University School of Medicine and a senior author of the study.

The Bruchas lab studies circuits that control a variety of disorders including stress, depression, addiction, and pain. Typically, scientists who study these circuits have to choose between injecting drugs through bulky metal tubes and delivering lights through fibre optic cables. Both options require surgery that can damage parts of the brain and introduce experimental conditions that hinder animals’ natural movements.

To address these issues, Jae-Woong Jeong, Ph.D., a bioengineer formerly at the University of Illinois at Urbana- Champaign, worked with Jordan G. McCall, Ph.D., a graduate student in the Bruchas lab, to construct a remote controlled, optofluidic implant. The device is made out of soft materials that are a tenth the diameter of a human hair and can simultaneously deliver drugs and lights.

“We used powerful nanomanufacturing strategies to fabricate an implant that lets us penetrate deep inside the brain with minimal damage,” said John A. Rogers, Ph.D., professor of materials science and engineering, University of Illinois at Urbana-Champaign and a senior author. “Ultra-miniaturized devices like this have tremendous potential for science and medicine.”

With a thickness of 80 micrometres and a width of 500 micrometres, the optofluidic implant is thinner than the metal tubes, or cannulas, scientists typically use to inject drugs. When the scientists compared the implant with a typical cannula they found that the implant damaged and displaced much less brain tissue.

The scientists tested the device’s drug delivery potential by surgically placing it into the brains of mice. In some experiments, they showed that they could precisely map circuits by using the implant to inject viruses that label cells with genetic dyes. In other experiments, they made mice walk in circles by injecting a drug that mimics morphine into the ventral tegmental area (VTA), a region that controls motivation and addiction.

The researchers also tested the device’s combined light and drug delivery potential when they made mice that have lightsensitive VTA neurons stay on one side of a cage by commanding the implant to shine laser pulses on the cells. The mice lost the preference when the scientists directed the device to simultaneously inject a drug that blocks neuronal communication. In all of the experiments, the mice were about three feet away from the command antenna.

“This is the kind of revolutionary tool development that neuroscientists need to map out brain circuit activity,” said James Gnadt, Ph.D., program director at the NIH’s National Institute of Neurological Disorders and Stroke (NINDS). “It’s in line with the goals of the NIH’s BRAIN Initiative.”

The researchers fabricated the implant using semi-conductor computer chip manufacturing techniques. It has room for up to four drugs and has four microscale inorganic light-emitting diodes. They installed an expandable material at the bottom of the drug reservoirs to control delivery. When the temperature on an electric heater beneath the reservoir rose then the bottom rapidly expanded and pushed the drug out into the brain.

l doi: 10.1016/j.cell.2015.06.058

Findings on cell differentiation controls can advance understanding of stem cells

Singapore scientists from A*STAR’s Genome Institute of Singapore (GIS) have, for the first time, found further evidence of how the differentiation of pluripotent cells is tied to and controlled by the cell cycle clock. This deeper understanding of how cells become differentiated is extremely important when considering therapeutic potentials.

Embryonic stem cells (ESCs) are cells that have not differentiated into specific cell types, and are said to be in a pluripotent state. The cell cycle is divided into four phases: G1, S, G2 and M[1]. Previous studies have shown that cell differentiation of ESCs is initiated only in the G1 phase, attributed to G1-specific properties that contribute to lineage specification. The absence of these properties in the other three phases was believed to passively hinder differentiation.

This study, using high-throughput screening, provides the first evidence that during the S and G2 phases, the ESCs are more potent towards maintaining its stemness; that is, they actively resist differentiation. Additionally, the scientists found that in instances of DNA damage, ESCs do not differentiate, so as to prevent the formation of specialised (differentiated) cells with compromised genomic integrity. Findings from the study were published July 2015 in Cell.

“Many studies have been devoted to looking at what keeps the ESCs in their undifferentiated state. Hence, to address a gap in the understanding of cell differentiation, our team at the GIS decided to focus on what regulates the ESCs’ exit from their pluripotent state,” said lead author of the research, Dr Kevin Gonzales, Post Doctoral Fellow at the Stem Cell and Regenerative Biology at GIS. “Moreover, most functional screens are carried out in mouse ESCs. The only functional screen on human ESCs was published in 2010 from our laboratory at the GIS. This latest study was also performed on human ESCs, making it more clinically relevant than studies using mouse ESCs.”

Co-lead author Research Fellow Dr Liang Hongqing at GIS’ Stem Cell and Regenerative Biology added, “Our research has shifted the current paradigm from a G1-phase centric view in stem cell regulation to a balanced view that different cell cycle phases perform different roles to orchestrate the stem cell fate.”

GIS Executive Director Prof Ng Huck Hui said, “Knowing that the S and G2 phases employ active pathways to prevent differentiation of ESCs, we can propose that, conversely, the absence of these pathways contributes to G1 phase amenability towards differentiation. This is truly an exciting and huge step forward in the study of cell pluripotency to advance fundamental understanding of human stem cells.”

[1] Cell cycle stages: (a) G1, first gap phase in preparation for DNA replication (G1); (b) S, DNA replication phase; (c) G2, second gap phase in preparation for cell division; (d) M, mitosis [cell division] phase.

l doi: 10.1016/j.cell.2015.07.001

One in four patients with defibrillators experiences boost in heart function over time

In a study which highlights the dynamic nature of cardiac disease and need for ongoing risk assessment, Johns Hopkins-led research of outcomes among 1,200 people with implanted defibrillators shows that within a few years of implantation, one in four experienced improvements in heart function substantial enough to put them over the clinical threshold that qualified them to get a defibrillator in the first place.

A report on the study, published in the 4 August 2015 issue of the Journal of the American College of Cardiology, reveals these patients had markedly lower risk of dying and were far less likely to suffer arrhythmia- terminating device shocks, suggesting their hearts had grown less prone to developing lethal rhythms.

All of the patients in the study had received defibrillators because of declining heart function – a condition that puts them at high risk for lethal rhythm disturbances – but none of them had experienced an actual cardiac arrest. Such preemptive defibrillator placement is known as primary prevention and is distinct from implantation of devices in cardiac arrest survivors with a history of dangerous rhythm anomalies, an approach known as secondary prevention.

The investigators attribute the improvement in heart function primarily to the concurrent use of heart failure medications that enhance the heart’s ability to pump and, in a small portion of patients, to the concurrent use of a special pacemaker that synchronizes the contraction of the heart’s chambers. The team says the real surprise was not the fact that patients got better, but rather how many did.

Because the number of rhythm-restoring device shocks never reached zero among those with improved heart function, researchers say arrhythmia risk was not completely eliminated and patients may continue to derive at least some protection from defibrillators even as their hearts become less susceptible to fatal rhythms. But because defibrillators can also cause serious complications, the risk-benefit ratio does shift substantially in people whose heart function improves dramatically, the researchers say.

“Our results highlight an urgent need to refine the risk-benefit assessment in people repeatedly, over the course of their treatment, and not just at the time of device implantation,” says senior investigator Alan Cheng, M.D., a cardiac electrophysiologist and an associate professor of medicine at the Johns Hopkins University School of Medicine.

“Determining if patients with defibrillators whose hearts get better over time may be better off without the device is just as important as determining who needs a defibrillator in the first place,” he says.

Implanted defibrillators detect and correct arrhythmias that cause cardiac arrest, but they can also misfire, delivering startling, painful, unnecessary and, at times, dangerous shocks. And because placing the device inside the chest is an invasive and complex procedure, the investigators say, there is risk of blood vessel damage and dangerous heart valve infections. Predicting which patients are most at risk for a cardiac arrest and stand to benefit most from a defibrillator is often tricky and invariably involves some guesswork, researchers add.

Long-standing clinical guidelines from the American College of Cardiology and the American Heart Association call for all people with heart failure whose hearts’ ejection fraction is below 35% to receive a device if they are able to undergo the implantation.

“Heart function below 35 percent is the current standard guiding device placement. But our study shows in one-quarter of people with defibrillators, heart function, over time, goes above that threshold, suggesting that a person’s risk for arrhythmia is not static. Monitoring such fluctuations is essential to optimize the clinical management of these patients,” Cheng says.

For the study, researchers enrolled and followed nearly 1,200 people, ages 18 to 80, who received defibrillators at four heart centres in the United States between 2003 and 2013 and who were followed for an average of five years. A portion of the group, 538 (45%), underwent at least one heart function re-assessment after initial device placement.

The patients’ heart function was evaluated by measuring how well their left ventricles propelled blood to the rest of the body. Of the 538 patients, 40% experienced increased left ventricular ejection fraction. In one-quarter of patients, left ventricular performance increased above 35%. These patients had a 33% lower risk of dying and a 30% lower risk of appropriate device shocks, compared with patients whose heart function remained unchanged.

The investigators say the fact that fewer than half of all patients in the study had their heart function re-assessed prior to their regularly scheduled device replacement points to the need for better ongoing monitoring.

l doi: 10.1016/j.jacc.2015.05.057

Implanted neurons become part of the brain

Scientists at the Luxembourg Centre for Systems Biomedicine (LCSB) of the University of Luxembourg have grafted neurons reprogrammed from skin cells into the brains of mice for the first time with long-term stability. Six months after implantation, the neurons had become fully functionally integrated into the brain. This successful, because lastingly stable, implantation of neurons raises hope for future therapies that will replace sick neurons with healthy ones in the brains of Parkinson’s disease patients, for example. The Luxembourg researchers published their results in the August 2015 issue of Stem Cell Reports.

The LCSB research group around Professor Dr Jens Schwamborn and Kathrin Hemmer is working continuously to bring cell replacement therapy to maturity as a treatment for neurodegenerative diseases. Sick and dead neurons in the brain can be replaced with new cells. This could one day cure disorders such as Parkinson’s disease. The path towards successful therapy in humans, however, is long. “Successes in human therapy are still a long way off, but I am sure successful cell replacement therapies will exist in future. Our research results have taken us a step further in this direction,” says stem cell researcher Prof Schwamborn, who heads a group of 15 scientists at LCSB.

In their latest tests, the research group and colleagues from the Max Planck Institute and the University Hospital Münster and the University of Bielefeld succeeded in creating stable nerve tissue in the brain from neurons that had been reprogrammed from skin cells. The stem cell researchers’ technique of producing neurons, or more specifically induced neuronal stem cells (iNSC), in a petri dish from the host’s own skin cells considerably improves the compatibility of the implanted cells. The treated mice showed no adverse side effects even six months after implantation into the hippocampus and cortex regions of the brain. In fact it was quite the opposite – the implanted neurons were fully integrated into the complex network of the brain. The neurons exhibited normal activity and were connected to the original brain cells via newly formed synapses.

The tests demonstrate that the scientists are continually gaining a better understanding of how to treat such cells in order to successfully replace damaged or dead tissue.

“Building upon the current insights, we will now be looking specifically at the type of neurons that die off in the brain of Parkinson’s patients – namely the dopamine-producing neurons,” Prof Schwamborn says. In future, implanted neurons could produce the lacking dopamine directly in the patient’s brain and transport it to the appropriate sites. This could result in an actual cure, as has so far been impossible. The first trials in mice are in progress at the LCSB laboratories on the university campus Belval.

l doi: 10.1016/j.stemcr.2014.06.017

Endoscopes still contaminated after cleaning, study shows

Potentially harmful bacteria can survive on endoscopes used to examine the interior of the digestive tract, despite a multistep cleaning and disinfecting process, according to a study published in the August issue of the American Journal of Infection Control, the official publication of the Association for Professionals in Infection Control and Epidemiology (APIC).

Though endoscopes were cleaned in accordance with multi-society guidelines, viable microbes and residual contamination remained on surfaces after each stage of cleaning, according to study findings. Researchers from Ofstead & Associates in Saint Paul, Minnesota and Mayo Clinic in Rochester, Minnesota tested samples collected from 60 encounters with 15 colonoscopes and gastroscopes used for gastrointestinal procedures after each reprocessing step to assess contamination levels. Investigators observed all reprocessing activities, using a checklist to ensure that cleaning protocols were performed in accordance with published guidelines.

Reprocessing consisted of: bedside cleaning, manual cleaning in dedicated reprocessing rooms, and automated endoscope reprocessing with a high-level disinfectant. Disinfected endoscopes were stored vertically after drying with isopropyl alcohol and forced air. When contamination levels exceeded pre-determined benchmarks for each cleaning step, technicians went beyond guidelines and repeated cleaning procedures, retesting after each attempt to reduce contamination.

Researchers performed microbial cultures and various rapid tests to detect viable organisms and organic residue that remained after each step of cleaning. Viable organisms were detected on 92% of devices after bedside cleaning; 46% after manual cleaning; 64% after high-level disinfection, and 9% after overnight storage. Rapid indicator tests detected contamination above benchmarks on 100% of devices after bedside cleaning; 92% after manual cleaning; 73% after high-level disinfection, and 82% after overnight storage.

“This study demonstrates that colonoscopes and gastroscopes can harbour residual organic material, including viable microbes, even when adherence with recommended reprocessing guidelines is verified,” said the study authors. “More research is needed to identify processes that can ensure all flexible endoscopes are free of residual contamination and viable microbes prior to patient use, including the potential use of routine monitoring with rapid indicators and microbiologic cultures. Results from this study suggest that current standards and practices may not be sufficient for detecting and removing residual contamination.”

The authors list several potential limitations of the study including that it is a single- site study and may not be generalizable. In addition, reprocessing technicians were aware of the researchers’ use of a checklist to ensure guideline compliance and therefore may have devoted more time and effort to reprocessing. Another caveat is that technicians were immediately informed about contamination that exceeded benchmarks and repeated cleaning steps – actions that are not generally part of standard practice.

Colonoscopes and gastroscopes are endoscopic devices with thin tubes, channels, and ports that are used to examine the interior of the colon and the stomach. Recent reports of multidrug-resistant infections related to contaminated duodenoscopes, which have intricate elevator mechanisms and channels that are especially difficult to clean, have raised awareness about the necessity for meticulous reprocessing of all types of endoscopes to prevent the transmission of pathogens to patients.

l doi: 10.1016/j.ajic.2015.03.003

Researchers show how single genetic mutation increases autism risk

Last December, researchers identified more than 1,000 gene mutations in individuals with autism, but how these mutations increased risk for autism was unclear. Now, University of North Carolina (UNC) School of Medicine researchers are the first to show how one of these mutations disables a molecular switch in one of these genes and causes autism.

Published 6 August 2015 in the journal Cell, the research shows that an enzyme called UBE3A can be switched off when a phosphate molecule is tacked onto UBE3A. In neurons and during normal brain development, this switch can be turned off and on, leading to tight regulation of UBE3A.

But a research team led by Mark Zylka, PhD, associate professor of cell biology and physiology, found that an autism-linked mutation destroys this regulatory switch. Destruction of the switch creates an enzyme that cannot be turned off. As a result, UBE3A becomes hyperactive and drives abnormal brain development and autism.

“Genetic studies are showing that there will be about 1,000 genes linked to autism. This means you could mutate any one of them and get the disorder. We found how one of these mutations works,” said Zylka, senior author of the Cell paper and member of the UNC Neuroscience Center. The work was done in human cell lines, as well as mouse models.

Because this one autism-linked UBE3A mutation was part of the Simons Simplex Collection – and Zylka previously had been funded through a Simons Foundation grant – he had access to the cells that were used to find this one mutation. When Jason Yi, PhD, a postdoctoral fellow in Zylka’s lab, sequenced the genes from the cell samples – including cells from the child’s parents – he found that the parents had no hyperactive UBE3A but the child did.

The child’s regulatory switch was broken, causing UBE3A to be perpetually switched on. “When this child’s mutation was introduced into an animal model, we saw all these dendritic spines form on the neurons,” said, Zylka, who is also a member of the Carolina Institute for Developmental Disabilities. “We thought this was a big deal because too many dendritic spines have been linked to autism.”

Their findings thus pointed to hyperactivation of UBE3A as the likely cause of this child’s autism. It was previously thought that too much UBE3A might cause autism because duplication of the 15q chromosome region – which encompasses UBE3A and several other genes – is one of the most commonly seen genetic alterations in people with autism. This is called Dup15q Syndrome.

As part of their study, Zylka and Yi found that protein kinase A (PKA) is the enzyme that tacks the phosphate group onto UBE3A. This finding has therapeutic implications, particularly since drugs exist to control PKA.

“We think it may be possible to tamp down UBE3A in Dup15q patients to restore normal levels of enzyme activity in the brain,” Zylka said. “In fact, we tested known compounds and showed that two of them substantially reduced UBE3A activity in neurons.”

One of the drugs, rolipram, previously had been tested in clinical trials to treat depression but was discontinued due to side effects. One of the symptoms associated with Dup15q syndrome is sudden unexpected death in epilepsy. In light of these life-threatening seizures, Zylka pointed out that it may be worth examining whether lower doses of rolipram, or other drugs that increase PKA activity, provide some symptom relief in Dup15q individuals. “The benefits might outweigh the risks,” he said.

l doi: 10.1016/j.cell.2015.06.045

Researchers discover endogenous process that controls reproduction of cardiac muscle cells

Heart failure is the most common cause of death worldwide. The main reason for this is that damage to the human heart causes cardiac muscle cells to die, which in turn leads to reduced heart function and death. However, this is not the case for zebrafish or amphibians. If their hearts become damaged and cardiac muscle cells die, their remaining cardiac muscle cells can reproduce, allowing the heart to regenerate. Researchers at Florida Atlantic University (FAU) have now found a possible explanation as to why this does not happen in humans. The results of their research have been published in the journal eLife.

The ability of most cardiac muscle cells to reproduce disappears in humans and all other mammals shortly after birth. What remains unclear, however, is how this happens and whether it is possible to restore this ability and therefore to regenerate the heart.

FAU researchers Dr David Zebrowski and Prof. Dr. Felix B. Engel from the Department of Nephropathology at Universitätsklinikum Erlangen’s Institute of Pathology and their colleagues have now found a possible explanation for this phenomenon. “In our study we discovered that the centrosome in cardiac muscle cells undergoes a process of disassembly which is completed shortly after birth,” Prof. Engel explains. “This disassembly process proceeds by some proteins leaving the centrosome and relocating to the membrane of the cell nucleus in which the DNA is stored. This process causes the centrosome to break down into the two centrioles of which it is composed, and this causes the cell to lose its ability to reproduce.”

The centrosome is an organelle found in almost every cell. In recent years, experiments have shown that if the centrosome is not intact, the cell can no longer reproduce. This raised the key question to what extent centrosome integrity could be manipulated – such as in cancer where cells reproduce at an uncontrolled rate.

The FAU researchers have now investigated whether the state of centrosome integrity is regulated naturally in the animal kingdom in order to control the reproduction of certain cells. “We were incredibly surprised to discover that the centrosome in the cardiac muscle cells of zebrafish and amphibians remains intact into adulthood,” says Dr David Zebrowski, who has been studying centrosomes for five years. “For the first time, we have discovered a significant difference between the cardiac muscle cells of mammals and those of zebrafish and amphibians that presents a possible explanation as to why the human heart cannot regenerate.”

The discovery that there is a natural process that regulates centrosome integrity in the cardiac muscle cells of mammals opens up a range of possibilities for future research. Firstly, this observation provides a new starting point for attempts to stimulate the reproduction of cardiac muscle cells in humans to regenerate the heart. At the same time, centrosome integrity can be examined in order to find adult cardiac muscle cells that may have retained their ability to reproduce, which may enable new forms of medical treatment. Finally, a detailed understanding of the mechanism could also help researchers to develop methods of inhibiting the uncontrolled growth of cancer cells.

l doi: 10.7554/eLife.05563

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