Hi, my name is Jack. I'm a biology instructor at the University of Kansas in the southern metro area of Kansas City where I've been teaching since 2016. When I started it was a struggle to get back to basics and remember all the general material that I'd forgotten long ago, but in time I've come to really love being in front of a class and helping people get to know a subject that I love.
My background is science - I've been working in laboratories since I was an undergraduate at the University of Delaware. My primary interest has been immunology and how the immune system and its components can be of use in fighting cancer and infectious diseases. I received my PhD in 2010 and have since been working as an educator bringing life science awareness to the largest audience possible.
Over the years, I have commonly found that about 10-20% of my students have difficulty isolating DNA from their buccal (Cheek) samples. This typically occurs when centrifugation of these isolates (even at very low speed) intended to pellet cells prior to lysis and DNA isolation, results in the immediate lysis of cells and release of DNA into the saline solution. Sometimes this goes without the student noticing or appreciating what is happening in the tube and leads to a failure to obtain any detectable (by UV) or amplifiable (by PCR) DNA in their final tube.
Just today, Dr. Jerry Coyne (Faculty at the University of Chicago, author of ‘Why Evolution is True‘ and host of a blog of the same name), reported that the buccal sample he sent to 23andMe had a similar outcome.
They write to him, “Our laboratory tried to extract DNA from your sample, but unfortunately the concentration wasn’t high enough to meet our standards. While it is uncommon, it does happen occasionally due to biological variability between people.”
I still don’t know why some individuals’ cells appear fragile in this method, but perhaps it’s not entirely unique.
This week in our Topics in Biotechnology course, we discussed a paper from the laboratory of Karen Vousden of the Francis Crick Institute, TIGAR, a p53-inducible regulator of glycolysis and apoptosis. I wanted to take some time to summarize the conclusions of this paper here in order to prevent any misunderstanding that might have arisen following our in-class discussion of the place of TIGAR with respect to glucose metabolism.
The body is extraordinary in its ability to maintain itself. It keeps a temperature of 37 degrees. It keeps bad stuff out and good stuff in. It has an immune system that attacks and destroys invasive viruses, bacteria, fungi, and other parasites. Cells maintain a constant pH and balance the concentrations of salts, proteins, and nutrients. All these regulatory mechanisms work together to keep us healthy and functioning properly.
One of the ways our body does this is by having our cells monitor their own health and make difficult choices when they are unhealthy. When our cells suffer damage, they work hard to repair the damage, but they also balance this against the greater good of the body as a whole. If they can repair damage, they do so, but when they can’t, cells eliminate themselves by a process called apoptosis. A central protein that controls many of these processes is the ‘guardian of the genome,’ p53.
This paper explores the role of TIGAR (Tp53-induced glycolysis and apoptosis regulator) in modulating the pro-apoptotic effects of p53 and in reducing free radicals. Specifically, TIGAR exerts its effects by rebalancing the normal metabolism of glucose during glycolysis. This prevents further damage and also allows time for repair to occur before making a decision to terminate the cell if repair is unsuccessful.
Under normal conditions, cells take up and process glucose as a fuel for making ATP, which is used directly to power enzymes and carry out all the processes that keep us alive. As part of this process, glucose is broken down stepwise during glycolysis. Some fraction of the products of this reaction gets diverted by an enzyme called PFK-2, which makes Fructose-2,6,-bisphosphate. This sugar goes on to bind to, and activate PFK-1, which keeps the pathway flowing.
Under conditions following DNA damage, p53 will become activated and the cell will arrest glycolysis as well as any cell division while it initiates DNA repair mechanisms and acts to remediate the radical oxygen species (ROSs) that are often associated with this sort of damage.
Among the many genes that are turned on to carry out these operations is TIGAR. Bensaad et al. show that the gene for TIGAR is transcribed and translated into protein, and that protein goes on to act as an enzyme to regulate metabolism.
Specifically, TIGAR functions as an enzyme with a high degree of homology to FBPase-2, which converts Fructose-2,6,-bisphosphate to Fructose-6-phosphate. This has an important regulatory function because, as stated above, Fructose-2,6,-bisphosphate is required to activate the enzyme PFK-1, which is required for glycolysis. In the absence of Fructose-2,6,-bisphosphate, PFK-1 shuts down and the products of glycolysis start backing up.
At first, this results in a backlog of Fructose-6-phosphate. As this accumulates, it will result in the accumulation of Glucose-6-phophate. With nowhere also to go, this will be processed to 6-phosphogluconolactone, making NADPH. NADPH will then oxidize glutathione, which will break down the ROS, H2O2 to water.
With the reduction in the number of ROSs, DNA damage will cease and repair can take place, thus diverting the cell away from a pathway leading to apoptosis.
This paper represents an amazing amount of work and is nearly bulletproof in its findings. I highly recommend it to anyone interested in how DNA damage and p53 interact with metabolic pathways and how this interaction directly leads to a more complete understanding of how p53 does its job.
Neutrophils are the first responding immune cells following exposure to pathogens.
This is due to several characteristics of these cells. Primarily, these cells are very populous in the blood. In fact, they are the most common type of circulating leukocyte, so no matter where in the body there is an insult/injury, there will be neutrophils close by to respond. Secondly, these cells are primed to respond to chemoattractants elicited by complement and from pathogens themselves.
One immediate response that will occur following an injury is the cleavage of complement molecules as they react with invading pathogens. Complement may be activated in three ways, spontaneously (i.e., the alternative pathway), by pathogen-associated carbohydrates (i.e., the lectin pathway), and by antibody (i.e., the classical pathway). In all three pathways, C3 convertase is a common element where the complement protein, C3, is cleaved into C3b, which precipitates onto the membrane of the invading cell, and C3a, which diffuses away and acts as a powerful anaphylotoxin.
C3a has a number of effects, notably activating vasculature to dilate (thus slowing blood flow in the area), upregulating adhesion molecules for cells to attach to, and promoting permeability, making it easy to Neutrophils to extravasate (leave circulation and enter the tissue). Neutrophils are quick to respond and to all of these cues and will further be attracted to the C3a gradients themselves.
Once in the vicinity of an infection, neutrophils will become more attracted by more specific attractants, such as those secreted by the pathogens themselves. One beautiful example of this kind of chemoattraction can be seen in the video of a neutrophil pursuing a bacterium in vitro:
Consider the following:
If you were given any (antibody or other) reagents you wished, how might you determine whether the neutrophil pursuing the bacterium was following a trail of fMLF? (what is fMLF? Why might this particular chemoattractant be relevant to this example? How do you determine specificity?)
Dick Hallorann lends his grandmother’s words to describe people like himself and Danny Torrance. She called it “Shining.” The Overlook Hotel is one of those places that shines too.
Here are a few random thoughts, sounds, and images from the many lives of The Shining.
Room 217 of the Stanley Hotel
Before there was an Overlook, there was the Stanley Hotel. Stephen King once stayed in the Presidential Suite of the Stanley, where he was visited by bad dreams, hallucinations, and was inspired to sketch out an outline for his best novel.
But The Shining is not just King’s best novel. It also served as the basis for the film of the same name, as written and directed by Stanley Kubrick. Like the hotel, this Stanley would also haunt King as he took his novel and stretched and twisted it into one of the most influential films ever made. So different from the book, King has said that it is Kubrick’s film, not his.
A reviewer calls the film, “a brilliant, ambitious attempt to shoot a horror film without the Gothic trappings of shadows and cobwebs so often associated with the genre.”
One of the most striking things, aside from masterful performances by Jack Nicholson and Shelley Duvall (heresy!? Yes, I think her performance was nearly as perfect as Nicholson’s – just drawn more from Kubrick’s haranguing rather than her innate talent.) is the spellbinding score and incidental music. From the first intonations of Dies Irae to the frenetic energy of the final chase scene, the music shapes your emotions and pulls you into the haunted world of the Overlook.
Rolling Stone, on the music accompaniment to Stanley Kubrick’s The Shining…
“Kubrick and music editor Gordon Stainforth cherry-picked maximally queasy passages from works by a clutch of Eastern European mavericks: “Lontano” by György Ligeti, the Hungarian genius whose music gave 2001: A Space Odyssey its unearthly atmosphere, and even more importantly Krzysztof Penderecki, the Polish radical whose strangulated strings, barbaric brass yawp, clatter-bone rhythms and hissing choruses in “Utrejna,” “De Natura Sonoris,” “The Awakening of Jacob” and more provided the Overlook Hotel and its denizens with an appropriately unhinged environment.”
To address the differences between the novel and the film, King sought to make a new version of the film that tracked more with the original storyline. In 1997, this version was released as a miniseries staring Steven Weber and Rebecca De Morney as the Torrances and using the Stanley Hotel as the Overlook. This version eschewed the hedge maze that Kubrick had created in place of the technically difficult hedge lions and, most notably stayed faithful to the novel’s end with Jack dying in the fiery explosion sparked by the Overlook’s overpressurized boiler.
Three decades after the original novel, King released Doctor Sleep, following the troubled life of Danny in the years after his ordeal at the Overlook. Fans of the Shining particularly enjoyed the last act of Doctor Sleep which brought us back to the remains of the Overlook, a place that exists only in the world of the original Shining film.
Radiolab did a good piece on Challenge trials for the COVID-19 vaccine which you can find here.
Challenge trials are an expedient, but fraught way to generate data on a vaccine’s efficacy quickly. Briefly, they involve taking a (relatively) small group of people, say 100, and dividing them into two groups of 50 each. One group gets the vaccine and the other group gets the placebo. Then, after waiting some period of time for the immune system to generate a protective response, you expose all 100 people to live virus.
This is essentially the same thing as what is done in the more traditional trial, with the exception of directly exposing subjects.
In the end, it’s a simple matter to calculate the efficacy (or not) of the vaccine by comparing the number of people who got sick in each group. If these numbers are equal, the vaccine is ineffective, etc. Even if all 100 people got sick from this challenge, it would still be less than the number of people who got sick (170) during the Pfizer trial that involved 43,000+ total participants (reported here).
Further, a challenge trial can be completed in as little as two months, while more traditional trials are ongoing over two years.
In the Radiolab piece, trial participants were asked why they participated. Answers ranged from appeals to mathematics (one participant said exposure to COVID did not significantly increase his risk of dying in any given year) to a need to make their experience meaningful (if they caught covid in the trial, at least it would provide important evidence toward approving a drug, whereas if they caught it any other time, it would just be a waste.)
However, the one reason I did not hear was one of financial inducement. Although I could not find actual data on this, challenge trials may pay up to $4000 for the 3-6 weeks of legal quarantine. There are a number of issues associated with these payments – the most obvious being that it may induce those without means to participate because they feel the money if too good to pass up. Jennifer Blumenthal-Barby and Peter Ubel write that, in fact, the payment for these trials may not be enough as it fails to meet the proposed minimum living wage of $15/hr.
I recommend that you listen to the Radiolab piece (it’s less than 30 minutes long) and take a look at the Blumenthal-Barby and Ubel, which is an interesting read.