Making Connections: How A Worm’s Nerves Regulate Their Networks with other Nerves

Written By: Kaitlyn Sadtler

Original Article: Noblett et al. JCB 2018

The Gist of It:

There are roughly 100 billion neurons in the human brain and roughly 86 billion neurons throughout the rest of our bodies. Each neuron can have more than 10,000 connections or synapses with other nerves to create the information superhighway that regulates our movement, our breathing, our digestion, and more. So how does the body maintain and regulate these different connections? That’s what researchers from the University of Ottawa have been trying to figure out. Now, the nervous system is a very complex thing, especially in mammals like us, so they chose to work with a small worm called Caenorhabditis elegans, or C. elegans. These animals have a total of 302 neurons which are all mapped out in this small, millimeter-long organism. Before this study, the authors identified a mutant worm that had an increase in the number of neurites (extensions from the nerve). By sequencing the genome of that worm, they found that the gene that was responsible for this increase in neurites was known as dip-2. When they deleted this gene, they saw an increase in neurite growth; if they looked at nerve regeneration (specifically that of axons – the long information highway part of the neuron), they saw that in worms that didn’t have DIP-2, there was an increase in regeneration. Through these studies, researchers were able to identify a specific protein that regulates how nerves make different extensions (which are responsible for connecting to other neurons) and whether the axons are able to regenerate. Understanding this balance will allow us to understand how the nervous system prevents connections that aren’t supposed to be there and helps build back neural connections that are missing

DIP-2 helps tell neurons to regulate how many neurites (sprouts from the cell body) to form. Knocking out DIP-2 increases neurite formation and axon regeneration.

The Nitty Gritty:

Previously Carr et al. described a genetic mutant screen with a mutant nde-5 that displayed increased neurite formation on the ventral cord motor neurons of C. elegans. Through whole genome sequencing Noblett et al. identified the gene responsible for this phenotype, disco-interacting protein 2 (dip-2). Knockout of dip-2 (both genetic and inducible) resulted in an increase in neurite formation. Compared to a vang-1 (Planar Cell Polarity; PCP) knockout that yields a similar phenotype, a dip-vang- double mutant has a heightened ectopic neurite phenotype, suggesting that these two pathways act independently of each other. As age is associated with increased neurite formation, the authors over-expressed dip-2 and saw a correlating decrease in ectopic neurite formation in aged animals. DIP-2 protein was detected cytoplasmically in neurons using a GFP tag. In axon regeneration, dip-2 knockout worms subjected to femtosecond laser axotomy had enhanced regrowth when compared to WT (dip-2) controls. This function of the protein was reliant upon its AFD (adenylate-forming domain) domains but not its DMAP1-binding (DNA methyltransferase-associated protein ]1) domain.

Original Research Article: Noblett, N, et al. “DIP-2 suppresses ectopic neurite sprouting and axonal regeneration in mature neurons.” J Cell Biol 218.1 (2019): 125-133.

Do you say Orgo or OChem? And what the heck is a Mr. Frosty…

If you walked into a biomedical research lab, just like when you’ve experienced any other cultures, you would find that there is a lot of slang and shortened names for things we work with daily. Just as a peek into the lab, here are a few of our favorites (from our labs in the USA). Have any favorites we should add to the list? Let us know!
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The evolving world of lab slang — have any of your favorite words for lab instruments or shorthand? This is a short list of a few we use all the time.

Lab Slang and Culture:

Eppie: One of the most common terms in biomedical sciences. Eppendorf tubes are small tubes that usually hold about 1.5 mL of liquid (half of a tenth of an ounce) and are used for a wide variety of things. Though Eppendorf is not the only company that makes these, most people have fallen into using the phrase “eppie” for the tiny tubes.
Mr Frosty: When you store mammalian cells (not plant or bacteria) in cryogenic storage, you have to slowly cool them down so that ice crystals do not form and damage the cells you are trying to save. To do this, you use a jar that has a rack for small tubes and is filled with isopropyl alcohol (rubbing alcohol) below the rack. The isopropyl alcohol cools the tubes slowly – at 1 degree Celsius per minute – to allow the cells to slowly cool down far below freezing and eventually be transferred into liquid nitrogen storage at −196 degrees Celsius (or −321 degrees Fahrenheit). There are several vendors that sell for these jars, but one of the favorite names is “Mr. Frosty.”
Fleas: To mix up solutions in beakers, we use stir bars that are spun by magnetic stir plates with a magnet inside them that spins around at a controlled speed. These bars come in all shapes and sizes – including super tiny itty bitty ones. Some labs have called these “fleas”!
Hood: When we work with sterile solutions or cells, in order to keep them clean we work inside an enclosed workspace called  a Biosafety Cabinet, sometimes called a Biosafety hood. Instead of calling it a BSC, a lot of scientists just call them hoods.
Sup: Pronounced “soup”, it’s an abbreviation for supernatant, which is the liquid in the tube after you’ve spun something down. What does “spun something down” mean? That’s when you put cells or other material that is in a liquid into a centrifuge to use centrifugal force to collect that material into a pellet at the bottom of the tube, leaving the liquid “sup” on the top.
Falcon Tubes: Similar to Eppendorf, but a bit bigger. Several companies make these larger tubes that hold 15 – 50 mL (half an ounce to just over 1.5 ounces), but a lot of people call them “Falcon Tubes” because of the main brand name that they are sold under.
Quals/Prelim: Beyond the lab, graduate students take coursework to further their education and fulfill the requirements of a PhD degree. At the end of the classes, they take comprehensive exams that are either written exams, oral exams, or both, and  determine whether or not they will continue on to finish the research portion of their PhD. Often, it’s a stressful time because you normally get 2 chances to pass the exam and if you don’t, you leave with a Master’s degree – eek! Hug a grad student that is studying for their quals! (note: some institutions are phasing these out in favor for different evaluations)
Orgo/OChem: There seems to be an East Coast vs West Coast debate on the short phrase for Organic Chemistry. East Coaster’s seem to prefer Orgo, while West Coaster’s prefer OChem –  if you’ve taken Orgo (I’m an East Coaster) do you see this difference?
Plating cells: Most of the time if you think of plating something, it’s food being plated at a restaurant, but for biomedical researchers it is something very different – we plate cells. Cell culture is growing cells outside of the body in a culture dish. Most people think of the little round petri dishes, but more often than not we use tissue culture flasks, which are more rectangular and have a lid with a filter that can prevent outside contaminants from entering but can still allow exchange of oxygen and carbon dioxide.
Pipettes & Lab Markers: Now, this isn’t slang or an abbreviation, but it is a common feature of lab culture – the ever-elusive pipettes and lab markers. Two things that always seem to walk away from your lab bench. Pipettes help measure out small amounts of liquid, and lab markers and just that, markers. For some odd reason, these are the two things that the lab fairy likes to steal and never return.

 

Can a drug mimic the effects of exercise?

Written by: Ken Estrellas

Original Article: Muise et al. PLOS One 2019
The Gist of It:
Obesity, diabetes, and cardiovascular disorders affect countless individuals worldwide, decreasing their quality of life and, in some cases, shortening life. The common mantra of “diet and exercise” has consequently been echoed in a number of different ways, ranging from various diet plans such as Atkins, keto, and paleo, to new types of physical activity such as CrossFit, SoulCycle, and Zumba. The benefits of physical activity are undeniable, but the underlying science is much more complex than simply burning fat and building muscle. Could the molecular mechanisms driving the positive effects of exercise be harnessed in the form of a drug, particularly for individuals who cannot exercise due to disability? In a recent study, Muise and colleagues sought to answer this question. They studied the role of signaling associated with the molecule 5′ adenosine monophosphate-activated protein kinase (AMPK), which is activated in response to exercise. The researchers made AMPK activators  and tested to see whether they could be given as a drug. Two of these compounds, LA1 (a long-acting AMPK activator) and SA1 (a short-acting AMPK activator), decreased blood glucose levels in healthy mice. Treatment with LA1 was also associated with significantly increased glycogen levels, a sign of proper glucose metabolism, and increased modification of a protein associated with AMPK signaling, pACC, in skeletal muscle. Another long-acting compound, LA2, was associated with elevations in pACC in mice to roughly the same level as having mice run 1300 meters on a treadmill. Treatment with LA1 and exercise were associated with similar glycogen levels in skeletal muscle and in the heart, and LA1 alone actually showed better reductions in blood glucose and free fatty acid levels compared to a single 800-meter run. Strikingly, this set of compounds reduced the effects of hyperglycemia and increased overall energy expenditure in mice and rats with diabetes, but body weight was not affected. Overall, although AMPK activators may not be the next miracle weight loss drug, their effects on metabolism might someday prove them to be an effective alternative for individuals who would not normally be able to reap the benefits of exercise.
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Small molecules that activate AMPK have effects on metabolism similar to those of exercise.

The Nitty Gritty:
In this study funded by Merck & Co., Inc., Muise et al. evaluated the effects of four small-molecule activators of 5′ adenosine monophosphate-activated protein kinase (AMPK) on metabolic parameters in healthy C57BL/6 and eDIO mice, obese db/db and B6.V-Lepob/J mice, and diabetic fZDF rats. Two long-acting compounds (LA1, LA2) and two short-acting compounds (SA1, SA2), each dosed at 30 mg/kg orally, demonstrated pharmacokinetic properties in line with the expected long- and short-acting properties. LA1 significantly decreased blood glucose levels compared to those in control mice at several timepoints over the course of a 90-minute assay performed 1 hour and 24 hours post-dose, but SA1 failed to do so. LA1 was also associated with increased phosphorylation of acetyl-CoA carboxylase (pACC, a marker of AMPK pathway activation) as measured by the pACC/ACC ratio in skeletal muscle and increased glycogen levels in heart and skeletal muscle at 1 and 24 hours post-dose when compared to treatment with the vehicle control and SA1. When comparing the effects of AMPK activation and exercise, LA2 was associated with significantly elevated pACC/ACC compared to treatment with the vehicle control, and LA2 produced a similar pACC/ACC ratio as was observed after strenuous exercise (cycles of 30 minutes treadmill running and 30 minutes rest, up to a total distance of 1300 meters). Probeset analysis revealed that six genes demonstrated elevated activation in heart, skeletal muscle, liver, brown adipose tissue, and white adipose tissue, including Fkbp5. Significantly lower levels of serum glucose and free fatty acids and increased levels of skeletal muscle and heart glycogen were observed with LA1 treatment or an 800-meter run + LA1 treatment when compared to exercise alone. In db/db diabetic mice, LA1 was associated with a significantly elevated pACC/ACC ratio at 2 and 7 hours post-dose, an increased heart rate, and increased heart glycogen levels compared to any of the short-acting therapies or vehicle. Fasting glucose was significantly improved in diabetic eDIO mice with SA2, and serum glucose levels at 24 hours after the last dose were significantly reduced in diabetic ob/ob mice and fZDF rats with SA2. Plasma levels of β-hydroxybutyrate, a marker of successful fatty acid oxidation, were significantly increased 1 hour post-dose in db/db mice with either LA1 or SA2 compared to vehicle, as were other markers of fatty acid oxidation. Despite these observed metabolic changes, no changes in body weight were observed; however, AMPK activation could still potentially serve as an option to ameliorate some symptoms of metabolic disease, particularly in individuals physically unable to exercise due to disability.
Original Research Article: Muise, E, et al. “Pharmacological AMPK activation induces transcriptional responses congruent to exercise in skeletal and cardiac muscle, adipose tissues and liver.PLoS One 14 (2019): e0211568.

Figuring out how to treat the plague: A new, rapid test to help select treatment for Yersinia pestis infection

Written by: Rebecca Tweedell

Original Article: Shifman et al. Frontiers in Microbiology 2019
The Gist of It:
The Black Death (aka the plague) caused devastating losses in the 1300s, wiping out about 60% of Europe’s population. In modern times, people do not often think about the pathogen responsible for this devastation, Yersinia pestis, especially since there are typically fewer than 20 infections in the United States each year. However, this bacterium still causes epidemics in Africa and is considered a serious bioterrorism threat due to its ease of transmission and lethality. While antibiotic treatment is highly effective in most cases, identifying the infection and picking the right antibiotic quickly remain challenging. Y. pestis is extremely deadly if treatment is not started within 18–24 hours. Unfortunately, most current tests to determine what antibiotics will work against the infection take much longer than that because Y. pestis grows very slowly in the lab. In a recent study at the Israel Institute for Biological Research, researchers developed a new test for Y. pestis that takes just over 7 hours to determine whether an antibiotic will work (much faster than the current standard test, which takes up to 72 hours). To develop the test, they first exposed bacteria to the antibiotic doxycycline and looked for genetic transcripts that changed in quantity. After looking through the big list of changes, they honed in on two transcripts, mgtB and bioD. They found that the ratio of the changes in these two transcripts after exposure to doxycycline had the strongest ability to predict the bacteria’s susceptibility to the antibiotic. They tested their new method on several strains of Y. pestis and found that they were able to successfully identify which strains would be killed with doxycycline treatment. The biggest excitement behind this new rapid method is that it can be used not only to improve the treatment of Y. pestis but also to develop more rapid tests for other dangerous pathogens that do not grow well in the lab, improving treatment and saving lives.
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The new rapid test for antibiotic susceptibility is lightning fast and only takes 7.5 hours, compared to the sluggish 72 hours that can be required by current tests.

The Nitty Gritty:
Shifman et al. exposed Y. pestis to a range of concentrations of doxycycline (0.125–8 μg/mL) for 2 hours and quantified mRNA expression levels using a DNA microarray. They identified 15 candidate transcripts by selecting those that had a fold change (FC) of ≥ 3 when transcript levels in bacteria treated at 1 × the minimal inhibitory concentration (MIC) were divided by transcript levels in unexposed bacteria, an FC ≥ 2.5 when transcript levels at 1 × MIC were divided by those at 0.5 × MIC, and an FC of ≥ 5 when transcript levels at 32 × MIC were divided by those at 0.5 × MIC. Based on additional in silico analyses, they further narrowed their list to include 2 upregulated (mgtB and lcrF) and 3 downregulated (irp7, bioD, and iucA) genes and developed quantitative real-time PCR (qRT-PCR) assays for them. By performing the qRT-PCR assays over a range of doxycycline concentrations, they found that the FC ratio of mgtB to bioD correlated well with the bacteria’s MIC and had the best predictive power. They then validated the qRT-PCR assay by testing various Y. pestis strains with different doxycycline MICs and found that their assay could successfully predict the MIC of the bacteria after using just 4 hours of culture and 2 hours of antibiotic exposure, a much quicker timeframe than the standard 72 hours required in current assays.
Original Research Article: Shifman, O., et al. “A rapid antimicrobial susceptibility test for determining Yersinia pestis susceptibility to doxycycline by RT-PCR quantification of RNA markers.Frontiers in Microbiology 10 (2019): 754. doi: 10.3389/fmicb.2019.00754.