Repairing Our Inner Clock with a Two-Inch Zebrafish

A model organism is a non-human animal used in research.  Zebrafish have gained much popularity as a research organism for a variety of disciplines such as genetics, pharmacology and biological research.  This is due to a number of reasons including their high fecundity, production of transparent embryos, cost-effectiveness, similarities to humans, as well as an abundance of available data. In addition, the larvae and embryos of Zebrafish develop externally and at a rapid pace compared to some other model organisms. This allows for manipulation and observation of early life stages to be less difficult. The embryos of Zebrafish are able to absorb additives from the water, with a high tolerance to chemical mutagens, allowing for easier administration of chemicals at greater dosages.

Posted by Biomechanism 

Circadian rhythms — the natural cycle that dictates our biological processes over a 24-hour day — does more than tell us when to sleep or wake. Disruptions in the cycle are also associated with depression, problems with weight control, jet lag and more. Now Prof. Yoav Gothilf of Tel Aviv University’s Department of Neurobiology at the George S. Wise Faculty of Life Sciences is looking to the common zebrafish to learn more about how the human circadian system functions.

Prof. Gothilf and his Ph.D. student Gad Vatine, in collaboration with Prof. Nicholas Foulkes of the Karlsruhe Institute for Technology in Germany and Dr. David Klein of the National Institute of Health in Maryland, has discovered that a mechanism that regulates the circadian system in zebrafish also has a hand in running its human counterpart.

The zebrafish discovery provides an excellent model for research that may help to develop new treatments for human ailments such as mental illness, metabolic diseases or sleep disorders. The research appears in the journals PLoS Biology and FEBS Letters.

A miniature model

Zebrafish may be small, but their circadian system is similar to those of human beings. And as test subjects, says Prof. Gothilf, zebrafish also have several distinct advantages: their embryos are transparent, allowing researchers to watch as they develop; their genetics can be easily manipulated, and their development is quick — eggs hatch in two days and the fish become sexually mature at three months old.

Previous research on zebrafish revealed that a gene called Period2, also present in humans, is associated with the fish’s circadian system and is activated by light. “When we knocked down the gene in our zebrafish models,” says Prof. Gothilf, “the circadian system was lost.” This identified the importance of the gene to the system, but the researchers had yet to discover how light-triggered gene activity.

The team subsequently identified a region called LRM (Light Responsive Model) within Period2 that explains the phenomenon. Within this region, there are short genetic sequences called Ebox, which mediate clock activity, and Dbox, which confer light-driven expression — the interplay between the two sequences is responsible for light activation. Based on this information, they identified the proteins which bind the Ebox and Dbox and trigger the light-induction of the Period2 gene, a process that is important for synchronization of the circadian system.

To determine whether a similar mechanism may exist in humans, Prof. Gothilf and his fellow researchers isolated and tested the human LRM and inserted it into zebrafish cells. In these fish cells, the human LRM behaved in exactly the same way, activating Period2 when exposed to light — and unveiling a fascinating connection between humans and the two-inch-long fish.

Shedding new light on circadian systems and the brain

Zebrafish and humans could have much more in common, Prof. Gothilf says, leading to breakthroughs in human medicine. Unlike rats and mice but like human beings, zebrafish are diurnal — awake during the day and asleep at night — and they have circadian systems that are active as early as two days after fertilization. This provides an opportunity to manipulate the circadian clock, testing different therapies and medications to advance our understanding of the circadian system and how disruptions, whether caused by biology or lifestyle, can best be treated.

Prof. Gothilf believes this model has further application to brain and biomedical research. Researchers can already manipulate the genetic makeup of zebrafish, for example, to make specific neurons and their synapses (the junctions between neurons in the brain) fluorescent — easy to see and track. “Synapses can be actually counted. This kind of accessible model can be used in research into degenerative brain disorders,” he notes, adding that several additional research groups at TAU are now using zebrafish to advance their work.

Bacterial Attack Strategy Uses Special Delivery of Toxic Proteins

Posted by Biomechanism 

(“Biomechanism.com“) — When competing for food and resources, bacteria employ elaborate strategies to keep rival cells at bay. Scientists have now identified a pathway that allows disease-causing bacteria to attack other bacterial cells by breaking down their cell wall.

Alistair Russell, NSF Graduate Research Fellow in the laboratory of Joseph Mougous at the University of Washington, studies how Pseudomonas aeruginosa successfully outcompetes other types of bacteria. Credit: Leila Gray/UW

Pseudomonas aeruginosa is a type of bacteria readily found in everyday environments. It easily forms colonies in a wide variety of settings, including medical devices, body organs and skin wounds. This allows it to cause disease and act as a major pathogen, particularly in hospitals.

Research led by Joseph Mougous, assistant professor of microbiology at the University of Washington in Seattle, provides new insight into how P. aeruginosa acts as a master colonizer and maintains a competitive advantage over other bacteria. The study, which will be published in the July 21 issue of the journalNature, describes how P. aeruginosa uses a specialized secretion system–the type VI secretion system–to deliver toxic proteins to recipient opponent bacteria. These proteins break down a structural protein known as peptoglycan, which the authors describe as “an Achilles heel of bacteria.” Degrading this target in turn causes the breakdown of the protective cell wall of competitor bacteria. Furthermore, P. aeruginosa protects itself from this attack by using specific, strategically localized immunity proteins that counteract the effects of the toxic proteins it secretes.

This discovery not only sheds light on P. aeruginosa‘s pathogenic success, but is also interesting from an evolutionary perspective as it highlights the functional similarity between the bacterial type VI secretion system and viruses that infect bacteria, or bacteriophages.

View a video with NSF graduate research fellow Alistair Russell of the University of Washington below: 

{Video Description: Research led by Joseph Mougous, assistant professor of microbiology at the University of Washington in Seattle, provides new insight into how the disease-causing bacteria Pseudomonas aeruginosa acts as a master colonizer and maintains a competitive advantage over other bacteria. Alistair Russell, NSF Graduate Research Fellow in the Mougous lab, discusses this research and describes how it helps our understanding of Pseudomonas as well as other pathogenic bacteria.}

________________

Fool’s gold gives scientists priceless insight into Earth’s evolution

Posted by Biomechanism 

(“Biomechanism.com“) — Fool’s gold is providing scientists with valuable insights into a turning point in the Earth’s evolution, which took place billions of years ago.

Scientists are recreating ancient forms of the mineral pyrite – dubbed fool’s gold for its metallic lustre – that reveal details of past geological events.

Pyrite is a shiny yellow mineral which is often called "fool’s gold" because many prospectors were fooled into thinking they had found gold. Pyrite gets its name from the Greek word for fire, because it can give off sparks when it is struck. It has been used to light fires for thousands of years. Pyrite is commercially used for the production of sulphur dioxide and in the manufacture of sulphuric acid.

Detailed analysis of the mineral is giving fresh insight into the Earth before the Great Oxygenation Event, which took place 2.4 billion years ago. This was a time when oxygen released by early forms of bacteria gave rise to new forms of plant and animal life, transforming the Earth’s oceans and atmosphere.

Studying the composition of pyrite enables a geological snapshot of events at the time when it was formed. Studying the composition of different forms of iron in fool’s gold gives scientists clues as to how conditions such as atmospheric oxygen influenced the processes forming the compound.

The latest research shows that bacteria – which would have been an abundant life form at the time – did not influence the early composition of pyrite. This result, which contrasts with previous thinking, gives scientists a much clearer picture of the process.

More extensively, their discovery enables better understanding of geological conditions at the time, which informs how the oceans and atmosphere evolved.

The research, funded by the Natural Environment Research Council and the Edinburgh Collaborative of Subsurface Science and Engineering, was published in Science.

Dr Ian Butler, who led the research, said: “Technology allows us to trace scientific processes that we can’t see from examining the mineral composition alone, to understand how compounds were formed. This new information about pyrite gives us a much sharper tool with which to analyse the early evolution of the Earth, telling us more about how our planet was formed.”

Dr Romain Guilbaud, investigator on the study, said: “Our discovery enables a better understanding of how information on the Earth’s evolution, recorded in ancient minerals, can be interpreted.”

Harvard bioengineers identify the cellular mechanisms of traumatic brain injury

Posted by Biomechanism 

Findings offer new hope for treatment of TBI in veterans wounded by explosions.

(“Biomechanism.com“) — Bioengineers at Harvard have identified, for the very first time, the mechanism for diffuse axonal injury and explained why cerebral vasospasm is more common in blast-induced brain injuries than in brain injuries typically suffered by civilians.

The research addresses two major aspects of traumatic brain injury (TBI), with significant implications for the medical treatment of soldiers wounded by explosions.

Caption: Left: A healthy neuron, with its dendrites and axon intact. Right: The damaged neuron has retracted its arms, breaking essential connections with its neighbors. Credit: Photo courtesy of Matthew Hemphill, Borna Dabiri, and Sylvain Gabriele.

Two papers, published in the journals Proceedings of the National Academy of Sciences (PNAS) and PLoS One, provide the most comprehensive explanation to date of how mechanical forces can be translated into subtly disastrous physiological changes within the brain’s neurons and vasculature.

“These results have been a long time coming,” says principal investigator Kevin Kit Parker, a Professor of Bioengineering at Harvard’s School of Engineering and Applied Sciences (SEAS) and a major in the U.S. Army. “So many young men and women are returning from military service with brain injuries, and we just don’t know how to help them.”

When the brain encounters a jarring force, such as an exploding roadside bomb, the delicate tissue slams against the skull. The result, if the patient survives, can be a temporary concussion, a more dangerous hemorrhage, or long-term TBI, which can even lead to the early onset of Parkinson’s or Alzheimer’s diseases.

Inspired by Parker’s own military experience, the Disease Biophysics Group (based at SEAS and at the Wyss Institute for Biologically Inspired Engineering at Harvard) has taken up the cause. Using cutting-edge tissue engineering techniques—essentially creating a living brain on a chip—biologists, physicists, engineers, and materials scientists collaborate to study brain injury and potential targets for treatment.

Now, researchers in his group have identified the cellular mechanism that initiates diffuse axonal injury, offering urgently needed direction for research in therapeutic treatments.

Their studies show that integrins, receptor proteins embedded in the cell membrane, provide the crucial link between external forces and internal physiological changes.

Integrins connect the structural components within the cell (such as actin and other cytoskeletal proteins) with the extracellular matrix that binds cells together into tissue. Collectively, this network of structural and signaling components is referred to as the focal adhesion complex.

Parker’s research has demonstrated that the forces unleashed by an explosion physically disrupt the structure of the focal adhesion complex, setting off a chain reaction of destructive molecular signals within the nerve cells of the brain.

Inside the neuron, integrins normally mediate the activation of the proteins RhoA and Rho kinase (ROCK). When the focal adhesion complex is disturbed, the Rho-ROCK signaling pathway goes haywire: it directs the motor protein actin to retract the cell’s arm-like axons, disconnecting the neurons from each other and collapsing the cellular networks that constitute the brain.

Caption: Bioengineers at Harvard have identified the mechanism for diffuse axonal injury and explained why cerebral vasospasm is common after an IED explosion. (NOTE: PI Kit Parker is NOT in this photo.) Photo: U.S. Department of Defense.

“Our research has shown that abrupt mechanical forces, such as those from a blast wave and transduced by integrins, can result in neural injury,” says Matthew A. Hemphill, who with Borna Dabiri (S.B. ’07) and Sylvain Gabriele, is a lead author of the paper in PLoS One. Dabiri and Hemphill are currently graduate students at SEAS, and Gabriele is a former postdoctoral fellow in Parker’s lab.

Adds Dabiri: “Encouragingly, we also found that treating the neural tissue with HA-1077, which is a ROCK inhibitor, within the first 10 minutes of injury, reduced the number of focal swellings. We think that further study of ROCK inhibition could lead to viable treatments within the near future.”

A second direction of research in Parker’s lab has solved another mystery in TBI, explaining why cerebral vasospasm, a dangerous remodeling of the brain’s blood vessels, occurs more commonly in TBI caused by explosions than in other types of brain trauma.

“Until now, other researchers looking at TBI focused on ion channels and membrane poration, and it was generally accepted that cerebralvasospasm was only caused by hemorrhaging. It turns out that it’s much morecomplicated than that,” says Patrick W. Alford, a former postdoctoral fellow in Parker’s lab and lead author of the paper inPNAS. “Integrins and Rho-ROCK signaling appear to be players in both diffuse axonal injury and cerebral vasospasm.”

As reported in PNAS, the forces exerted on arteries are different during an explosive blast than during blunt force trauma. Subarachnoid hemorrhage, which can occur in very severe head injuries, is known to cause vasospasm, but Parker’s new research shows that the unique force of an explosion can also cause vasospasm by itself.

The blast from an explosion creates a surge in blood pressure, which stretches the walls of the blood vessels in the brain. To study this, Parker’s team of bioengineers built artificial arteries, made of living vascular cells, and used a specialized machine to rapidly stretch them, simulating an explosion. While this stretching did not overtly damage the cellular structure, it did cause an immediate hypersensitivity to the protein endothelin-1.

Endothelin-1 is known to stimulate vascular cells to absorb calcium ions, which affect actin—the same protein involved in the retraction of axons.

In the 24 hours following the simulated blast, the vascular tissues hypercontract and undergo a complete phenotypic switch, disrupting the overall function of the tissue. Both of these behaviors are characteristic of cerebral vasospasm.

Most importantly, as in the neural tissue, the Rho-ROCK signaling pathway plays an important role in the behavior of actin and the cells’ contraction. Parker’s team found that inhibition of Rho soon after the injury can mitigate the harmful effects of the blast on the brain’s vascular system.

“We have established a toe-hold as we try to climb up on top of this problem,” says Parker. “In many ways, this work is just the beginning.”

_______________________

Parker’s coauthors on the paper in PLoS One are Hemphill, currently at the University of Mons in Belgium; Dabiri, who beganworking in Parker’s lab as an undergraduate; Gabriele, who is now at the University of Mons; Lucas Kerscher, a visiting student; Christian Franck, formerly a postdoctoral fellow at SEAS and now at Brown University; Josue A. Goss, a staff engineer at SEAS; and Alford, who is now at the University ofMinnesota.

Parker’s coauthors on the paper in PNAS are Alford; Dabiri; Goss; Hemphill; and Mark D. Brigham, a graduate student at SEAS.

The Disease Biophysics Group received financial support from the Defense Advanced Research Projects Agency (DARPA) Preventing Violent Explosive Neurologic Trauma (PREVENT) Program, the Department of Defense, and the Harvard School of Engineering and Applied Sciences (SEAS).

The researchers also gratefully acknowledge the use of facilities at the Harvard Center for Nanoscale Systems, a member of the National Nanotechnology Infrastructure Network (NNIN), which is funded by the National Science Foundation (NSF).