Student Research Results in Recent JIB Paper

Images from research paper from Pochapsky and Lovett labsBy Thomas Pochapsky, Professor of Chemistry & Biochemistry

We don’t usually consider PineSol, Vick’s VapoRub and Lemon Pledge as food, but it is a good thing that some bacteria can.  The active components of those products are terpenes, small organic molecules that are produced by evergreens to repel insects, promote wound healing and prevent infection.  The bacteria that can use terpenes as food are a critical part of the forest ecosystem:  Without them, the soil would rapidly become saturated with toxic terpenes.  Members of the Pochapsky and Lovett laboratories in Chemistry and Biology are curious about what enzymes are involved in terpene metabolism.  In particular, why would one bacterial strain feast on a particular terpene (camphor, for example) while ignoring others?

The first step in terpene breakdown by bacteria is often the addition of an oxygen atom at a particular place in the terpene molecule, providing a “handle” for subsequent enzymes in the breakdown pathway.  The enzymes that catalyze these oxygenation reactions are called cytochromes P450.  P450 enzymes perform important reactions in humans, including steroid hormone biosynthesis and drug metabolism and activation.  Human P450s are targets for cancer chemotherapy and treatment of fungal infections.  A specific inhibitor of P450 is a component of the AIDS “cocktail” treatment, slowing the breakdown of the other cocktail components so the drugs do not have to be taken as often.

Despite the importance and wide scope of the P450 enzyme family, we don’t know much about how a particular P450 goes about choosing a molecule to work on (the substrate) or where it will put the oxygen (the product).  This is what the Brandeis labs are interested in finding out.  What particular sequence of amino acids gives rise to the substrate/product combination of a given P450? Answers to this question will aid in drug design and bio-engineering projects.

The project employs multiple scientific techniques in order to get at the answers to these questions, including bacterial genome sequencing, messenger RNA transcription, enzyme isolation, activity assays, mass spectrometry and enzyme structure determination.  As complicated as it sounds, though, the project lends itself nicely to undergraduate research:  Three of the authors on this paper are undergraduates, Phillix Esquea ‘18, Hannah Lloyd ’20 and Yihao Zhuang ’18.  Phillix was a Brandeis Science Posse recruit, and is now working with a Wall Street investment bank in NYC.  Yihao is enrolled in graduate school at the University of Michigan School of Pharmacy, and Hannah Lloyd is still at Brandeis, continuing her work on the project.  Even high school students got in on the act:  Teddy Pochapsky and Jeffrey Matthews are both seniors at Malden Catholic High School, and collected soil samples used for isolation of terpene-eating bacterial strains.  (One of the newly isolated bacterial strains is named in their honor, Pseudomonas strain TPJM).

“A new approach to understanding structure-function relationships in cytochromes P450 by targeting terpene metabolism in the wild.” Nathan R.Wong, Xinyue Liu, Hannah Lloyd, Allison M. Colthart, Alexander E. Ferrazzoli, Deani L. Cooper, Yihao Zhuang, Phillix Esquea, Jeffrey Futcher, Theodore M. Pochapsky, Jeffrey M. Matthews, Thomas C. Pochapsky.  Journal of Inorganic Biochemistry. Volume 188, November 2018, Pages 96-101.  https://doi.org/10.1016/j.jinorgbio.2018.08.006.

Encoding taste and place in the hippocampus

The ambience of a good meal can sometimes be as memorable as the taste of the food itself. A new study from Shantanu Jadhav and Donald Katz’s labs, published in the February 18 edition of The Journal of Neuroscience, may help explain why. This research identified a subset of neurons in the hippocampus of rats that respond to both places and tastes.

The hippocampus is a brain region that has long been implicated in learning and memory, especially in the spatial domain. Neurons in this area called “place cells” respond to specific locations as animals explore their environments. The hippocampus is also connected to the taste system and active during taste learning. However, little is known about taste processing in the hippocampus. Can place cells help demarcate the locations of food?

To test this hypothesis, Neuroscience PhD student Linnea Herzog, together with staff member Leila May Pascual and Brandeis undergraduates Seneca Scott and Elon Mathieson, recorded from neurons in the hippocampus of rats as the rats explored a chamber. At the same time, different tastes were delivered directly onto the rats’ tongues.

Analyzing how place cells responded to tastes delivered inside or outside of their place field

The researchers found that about 20% of hippocampal neurons responded to tastes, and could discriminate between tastes based on palatability. Of these taste-responsive neurons, place cells only responded to tastes that were consumed within that cell’s preferred location. These results suggest that taste responses are overlaid onto existing mental maps. These place- and taste-responsive cells form a cognitive “taste map” that may help animals remember the locations of food.

Read more:  So close, rats can almost taste it

Leading Science: Magnifying the Mind

Brandeis Magnify the Mind

Written by Zosia Busé, B.A. ’20

Joshua Trachtenberg, graduated from Brandeis in 1990 and is a leader in studying the living brain in action using advanced imaging technology. After establishing his research laboratory at UCLA, he founded a company – Neurolabware – in order to build the sophisticated custom research microscopes that are needed to perform groundbreaking work in understanding how the brain develops and how diseases and injuries interrupt its normal functioning. His company is created by scientists and for scientists, and is unique in creating high quality microscopes that are easy to use but also have the flexibility to be used in creative ways in innovative experiments, and in combination with a variety of other devices.

Brandeis University is now seeking to acquire one of these advanced microscopes that can observe fundamental processes inside the living brains of animals engaged in advanced behaviors. The resonant scanning two-photon microscope from Neurolabware allows researchers to understand and image large networks of neurons in order to visualize which cells and networks are involved with specific memories or how these processes go awry in disease. “This approach is unparalleled. There is no other technique around that could possibly touch this,” Trachtenberg says.

Previous two-photon technologies permitted only very slow imaging, allowing scientists to take a picture about every two seconds, but the resonant two-photon technology is a major breakthrough that allows scientists to take pictures at about 30 frames per second. This speed increase is a major game changer. Not only can one observe activity in the brain at a higher speed, but it is possible to take pictures at a speed that is faster than the movement artifacts that must be accounted for, such as breathing or heart beats. Because one can see the movement, it can be corrected, allowing high resolution functional imaging of structures as small as single synaptic spines in the living brain. Further, advances in laser technology and fluorescent labels now allow scientists to see deeper into the brain than ever before, compounding the recent advantages of increased speed.

[Read more…]

How do batteries work?

How do batteries really work? A convincing simple yet quantitative answer to this question has remained elusive. Textbooks and on-line sources have provided only descriptions but not explanations of basic electrochemistry. All calculations in electrochemistry are based on measured voltages, not atomic or molecular properties. Made-up explanations of batteries in terms of different “electron affinities” of different metals are widely believed but easily disproved, e.g. by concentration cells using the same metal for both electrodes.

A paper in the Journal of Chemical Education by Klaus Schmidt-Rohr (Chemistry) explains how batteries store and release energy, in quite simple terms but based on quantitative data. In the classical Zn/Cu galvanic cell, it is the difference in the lattice cohesive energies of Zn and Cu metals, without and with d-electron bonding, respectively, that is released as electrical energy. Zinc is also the high-energy material in a 1.5-V alkaline household battery. In the lead–acid car battery, intriguingly the energy is stored in split water (two protons and an oxide ion). Atom transfer into or out of bulk metals or molecules plays as big a role as electron transfer in driving the processes in batteries.

How Batteries Store and Release Energy: Explaining Basic Electrochemistry, Klaus Schmidt-Rohr, Journal of Chemical Education, 2018, 95 (10), pp 1801–1810.

Paradis and Van Hooser labs collaborate on eLife paper

Figure 3 from research paper

Figure 3. Rem2 is required for late-phase critical period ocular dominance plasticity.

“Rem2 stabilizes intrinsic excitability and spontaneous firing in visual circuits.” Anna R Moore, Sarah E Richards, Katelyn Kenny, Leandro Royer, Urann Chan, Kelly Flavahan, Stephen D Van Hooser, Suzanne Paradis. eLife 2018;7:e33092.

Throughout our waking hours, we experience an ever-changing stream of input from our senses. The brain responds to this varying input by adjusting its own activity levels and even its own structure. It does this by changing the strength of the connections between neurons, or the properties of the neurons themselves. Known as plasticity, this process of continuous change enables the brain to develop, learn and to recover from injury.

The visual systems of mammals are particularly well suited to studying how sensory experience alters the brain. Studies in animals show that lack of sensory input to one or both eyes during a critical period in development causes long-lasting changes in the brain’s visual circuits. Similarly, children with the condition amblyopia or ‘lazy eye’ – in which one eye has impaired vision and the brain ignores input from that eye – can end up with permanent deficits in their vision if the condition is not treated during childhood. Changes in sensory input are thought to trigger plasticity in the brain by altering the activity of specific genes. But exactly how this process works is unclear.

Anna Moore, Sarah Richards et al. now show that a gene called Rem2 has an important role in regulating visual plasticity. In the key experiments, young mice had their vision in one eye blocked for a few days. Analysis of their brains showed that mice that had been genetically modified to lack the Rem2 gene responded differently to this change in their environment (i.e. the loss of input to one eye) than their normal counterparts. Further experiments suggest that Rem2 regulates the excitability of individual neurons: that is, how much the neurons respond to any given input. In the absence of Rem2, neurons in visual areas of the brain become hyperactive. This prevents them from adjusting their activity levels in response to changes in sensory input, which in turn leads to impaired plasticity.

Being able to harness the brain’s visual plasticity mechanisms on demand, for example by regulating Rem2 activity, could benefit individuals with disorders such as amblyopia. Rem2 is also active in many other parts of the brain besides those that support vision. This suggests that manipulating this gene could affect numerous forms of plasticity. However, various barriers must be overcome before we could use this approach to treat brain disorders. These include obtaining a more in depth understanding of the role of the Rem2 gene in the human brain.

Advanced spectroscopy reveals mechanism of vectorial action in a membrane pump

Judith Herzfeld research imageSome proteins in cell membranes are responsible for actively pumping desired molecules in or unwanted molecules out. Since their discovery, it has been expected that their vectorial action involves the existence of two protein conformations, one in which the active site has a low affinity for substrate and is open to the discharge side of the membrane and the other in which the active site has a high affinity for substrate and is open to the uptake side of the membrane. The driver of the pump is a source of energy that converts the pump from the lower energy state to the higher energy state, from which it can relax back and begin the cycle anew.

However, this model has never fit the longest-studied pump, the light-driven ion pump bacteriorhodopsin. At rest, the active site has a high proton affinity but is open to the discharge side of the membrane. Disruption of the active site by light reduces the proton affinity, but it has been a decades-long mystery how this occurs while maintaining access to the discharge side of the membrane. This mystery has now been solved through advanced spectroscopic studies of photocycle intermediates trapped at low temperatures. Obtained collaboratively by Judith Herzfeld’s group at Brandeis and Robert Griffin’s group at MIT, the spectra trace the establishment of an essential U-shaped pathway to the discharge side of the membrane. The results also explain how this pathway is broken as soon as the proton is released, thereby preventing back flow and enforcing the vectorial action of the pump.

“Primary transfer step in the light-driven ion pump bacteriorhodopsin: an irreversible U-turn revealed by DNP-enhanced MAS NMR.” Qing Zhe Ni, Thach Van Can, Eugenio Daviso, Marina Belenky, Robert G. Griffin, and Judith Herzfeld. J. Am. Chem. Soc., DOI: 10.1021/jacs.8b00022. Publication Date (Web): February 28, 2018

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