Dale Yuzuki, M.A., M.Ed.
Most human beings have an almost infinite capacity for taking things for granted.
I knew that going back to graduate school after a few years of full-time work was going to be difficult, but I hadn’t thought it would be this tough. I faced the pressures of time, of opportunities lost, of finances, and of living like a hermit, not to mention the nature of a competitive program where I wanted to show myself as worthy of that degree.
It was another late night in the laboratory for me. It was 1989, and I was heading back downstairs into a well-lit basement with benches crowded with devices (power supplies, acrylic boxes of various sizes, and sleeves of clear plastic Petri dishes). No one else was there at 2:30 a.m.
I had bicycled back to the lab earlier in the evening, pulled a few plates from the fridge labeled Amp+ which I had prepared the day before, lit the Bunsen burner, and dipped the bent glass rod into a dish of alcohol to sterilize it. Spinning the plastic plate on a rotating device, tube after tube of transformed E. coli spread out, and then they went off to the 37°C incubator.
Bicycling the few miles back home in the late Spring San Francisco air was exhilarating. Way past midnight, I felt as though I was accelerating time as I figured out the next steps in my newest cloning project — imagining the restriction enzyme digest, separating the target DNA fragment on an agarose gel, purifying it afterwards, and then setting up the ligation reaction.
The following morning was bright and sunny. After four hours of sleep, I got up to teach an 8:00 a.m. undergraduate biology lab. Not a lot of prep work was necessary, as this session was the equivalent of teaching out of a cookbook, and the students were a lot of fun.
One older student had returned to school to work on additional undergraduate requirements. It was clear he was going places — he had a successful career in software development and had returned to San Francisco State to go to medical school. He was at SFSU for a few years to take the prerequisite biology, inorganic chemistry, biochemistry, and organic chemistry coursework, followed by the MCAT (Medical College Admissions Test).
Charlie was fun to teach — especially for me, a former high school Honors Chemistry and Biology teacher. The reason he was fun was his incisive questioning; at the college level, the edges and frontiers of science could be explored, investigated, and pushed a little further.
At the end of this class, Charlie asked, “How’s the research going?”
“I’m getting to the exciting part,” I replied. “If the ligation worked, it means I’ve got the fragment I’ve been trying to get at for the past six months.”
A ligation is where fragments of DNA are shuffled into the appropriate position and glued together, and I had been having difficulty getting this step to work the way it was supposed to. Calls into the company that sold the enzyme ligase had given me a few suggestions, and a few lab-mates had also given me their ideas on why it was not working. At that point, I had accumulated plenty of days where the ampicillin plates (those clear plastic dishes with transparent yellow agar growth medium) would be empty the following day. Another day’s work down the drain.
Ampicillin, an antibiotic, disallows any bacteria that does not have the engineered plasmid in it. There are places engineered into these microscopic loops of DNA where you can cut and paste, just like in any computer program. The ligase performs the final step in pasting the connecting pieces of DNA together. These plasmids have an ampicillin resistance gene and a properly working ligation reaction would confer that resistance to the specially designed and specifically treated bacteria I had plated onto the Amp+ plates only 6 hours before.
The moment of truth came right after the undergraduate lab session.
Opening up the 37°C incubator, I eagerly picked up the stack of ampicillin plates.
The plates were littered with dozens of blue and white dots, which were around a millimeter or two across, each about the size of a pinhead. I just suppressed yelling aloud. There were not many days working in the lab where you felt elated, so I treasured this one.
Any bacteria that survived on the Amp+ plate had the plasmid with the ampicillin resistance gene in it. Any bacteria that had turned blue showed that that particular colony (called a clone, this is a single bacteria that has multiplied many million-fold) contained a plasmid without any gene interrupting another gene called beta-galactosidase. In normal plasmids, the beta-galactosidase gene codes for a protein that metabolizes a dye called X-gal to turn blue. Blue colonies were not good, as they did not have the stretch of DNA that I wanted to capture. The white colonies were the good ones; the bacteria contained plasmids where the beta-galactosidase gene had been disrupted by the engineered piece inside it.
I would choose a few of the white ones for further analysis, scooping up the small dot of bacteria with a sterile wire loop, then stirring it in a test tube with around a half-inch of nutrient broth.
In one of those tubes was a group of bacteria containing what I was after: a snippet of DNA that would become a Master’s Thesis. One technique after another, I had learned how to cut DNA, how to stitch it together, how to sequence the DNA, and how to amplify it up from minuscule picograms.
It is these technologies that are fully brought to bear when tackling the problem of a pandemic: how to detect infected individuals, how to develop therapies and perhaps even cures for severe COVID-19, and how to bring a vaccine to market in record time.
At its essence, molecular biology is being able to manipulate DNA for a desired effect, and in this pandemic, all DNA technology is on full display. Detecting live virus is far too complicated and dangerous; therefore, sensitive DNA technologies such as the polymerase chain reaction (PCR) are utilized for detection of the SARS-CoV-2 nucleic acid. Antibodies are sequenced, characterized, and engineered as promising therapeutics to both treat and potentially prevent infection. No less than five distinct technical approaches are currently being utilized in a global effort, with over 200 vaccines and vaccine candidates in various stages of development.
We will take a closer look at these fundamental building blocks and how they directly relate to the diagnostics technology, the therapeutic development, and the vaccine development against COVID-19 in the coming chapters. But first, we must take a look at how science in general progresses.
Science is hard and, at its essence, science is humble. Hard-fought knowledge gained at the edges of scientific understanding will only extend knowledge in a tiny area. Fundamental breakthroughs and new realms of knowledge open up infrequently. Sometimes, a new opening will unfurl into a new geography, and other times, the discovery of new knowledge is only the window to a small, unique archipelago. All too often, it’s just a tiny little sandbar — an observation that doesn’t lead anywhere.
In science, there is something called “The Streetlight Effect.”
A person (typically a drunkard) is looking for their wallet under a lamp post. A passer-by asks, “Have you looked in any other places?” To which the person replies, “No, I’m looking here because the light is better.”
Scientists are world-class experts within the scope of their narrow area of expertise, working at the edges of their specialty to increase what is known. Steve Jobs once said that he wanted to make a ‘dent in the universe’ — and in that spirit, scientists make hard-won progress every day[i]. Some dents are bigger than others and sometimes small movements lead to much larger effects.
You may be familiar with the scientific method of a three-part cardboard poster from a grade school science fair, either as one who made one or as a parent of a child tasked with making one. The origin of the method comes from the Arab world of the 12th century, from a polymath named Ibn al-Haytham (Latinized name: Alhazen) who wrote,
A person who studies scientific books with a view of knowing the real facts ought to turn himself into an opponent of everything he studies; he should thoroughly assess its main as well as its margin parts, and oppose it from every point of view and all its aspects…If he takes this course, the real facts will be revealed to him.
— Shukūk ʿalā Baṭlamyūs (“Doubts About Ptolemy”)
While this may seem counterintuitive, you can see the pride of knowledge displayed in the current conflicting viewpoints of experts, public leaders, and informal discourse as we work through the coronavirus pandemic. Instead of making dents, we have witnessed the birth of a new, chattering class of instant experts bloviating on mass media. With social-media amplification, narrow-mindedness and biases are the norms. Ask yourself, when was the last time you heard someone say ‘I don’t know’ in a televised interview or video clip?
al-Haytham refers to “real facts.” At its heart, the scientific method finds its way through the darkness by expanding a circle of knowledge through hypotheses and testing, repeating the process over and over again with additional facts, data, and observation. This method is humble at its core. A quote familiar to many of you could speak directly to the process:
But he that is greatest among you shall be your servant. And whosoever shall exalt himself shall be abased; and he that shall humble himself shall be exalted.
— Matthew 23:11-12
While you will not find this humility in public discourse, it remains a fundamental attitude within the realm of science. A hypothesis is used to fit the data, in all of its incomplete forms, until a collection of hypotheses can be amalgamated into a model. Alternatively, conflicting data will change such a hypothesis in mid-stream, eventually forming an enlarged model. This model could be used to make predictions and/or changed as new data emerges. Often enough, a completely new model must be invented to account for what has been learned.
In this Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) pandemic, many edges of science are being expanded at the same time. Scientists are using their current models of complex systems and applying them to a real-world problem where there are millions of lives, trillions of dollars, and untold suffering at stake.
In broad strokes, you can look at the pandemic as an emergency, such as a brush fire in a large area traveling rapidly from home to home. Diagnostics — whether we speak of a chest X-ray with the tell-tale ‘ground-glass’ appearance or a nasal-cavity swab from a drive-through test site — amount to the fire alarm. This is the warning system telling us to take action.
The calling in of firefighters and their equipment equates to therapeutics, of which at present, only a handful exist — although more are on the way.
Vaccines are the final answer, a protective layer. In the brush fire scenario, imagine five days of constant rain preventing any new fires from starting.
As far as diagnostics go, the new SARS-CoV-2 tests are based on tried-and-true technologies. For example, think of the at-home plastic test strip pregnancy test with the “–” and “+” readouts. This is a Lateral Flow ImmunoAssay (LFIA), which relies on looking for the telltale signs of a particular hormone in the urine that comes from a growing fetus (this hormone called human chorionic gonadotropin or HCG for short). The change in color detects the presence of HCG with a specific antibody preloaded on the cassette. It costs less than a dollar to manufacture!
Today, molecular diagnostic testing for SARS-CoV-2 requires a much more complicated methodology and equipment (which I promise we will cover the details of in a later section). Yet innovation using molecular methods (either detecting the viral RNA or viral antigen) combined with convenient and cheap at-home methods is well underway by a number of companies that are using a variety of molecular tricks.
This method of pushing the edges of the known in new combinations is a common theme. Until now, we have not had the urgency of public health and economic emergencies driving us to move as quickly as possible. But the processes here were already underway before this pandemic, with other public health challenges in mind; the combination of molecular methods with the LFIA cassette has been worked on by several groups over the past three years or so. The pandemic has simply accelerated all of the timelines.
The same theme of extending the edges and boundaries of science is observed in the development of therapeutics and vaccines. With underlying knowledge and the infrastructure of monoclonal antibodies being put to use with urgency, new combinations of ideas are being tested and refined. The course of developing novel vaccines (there are five generally-used approaches, all of which are being tried at once with over 165 in pre-clinical development and 36 in Phase I to Phase III clinical trials as of the Fall of 2020) is being accelerated in creative ways never tried before.
Attitude is everything.
In the second century AD, Galen of Pergamon made observations of animals (mainly monkeys and pigs) and formulated theories on the circulatory, nervous, and muscle systems. Galen’s ideas then stood for over a thousand years. Even today, terms such as “sanguine” come from Galen’s belief that human moods are caused by an imbalance between four bodily humours. He carefully observed humans in their diseased state as a physician and conducted experiments and observations on animals; he thus dominated Western medical science for 1,300 years.
In the Renaissance, it was Michelangelo who snuck into a morgue to examine underlying anatomy. He acknowledged the existing limits of depicting the human form and sought a more realistic art, carefully observing and drawing the corpses. Ignorance of human anatomy, a stumbling block to progress in art, was slowly melting away.
A few hundred years later, Charles Darwin’s five-year voyage to the west coast of South America on the HMS Beagle revealed to him thousands of specimens that he would study for several decades. Observation coupled with careful thought led to the questions that Darwin asked: Why are there so many kinds of finches in the Galapagos? Where did they come from? What do they have in common? In what important ways do they differ? How could this change have come about?
These advances in science, whether coming from the ancient Greeks, the Renaissance or the 19th and 20th centuries, were fueled by our innate curiosity and a distinct awareness of our own ignorance.
There are known knowns. There are things we know we know. We also know there are known unknowns. That is to say, we know there are some things we do not know. But there are also unknown unknowns, the ones we don’t know we don’t know.
— Secretary of Defense Donald Rumsfeld, 2002 Department of Defense News Briefing[ii]
The literal translation of the word “science” is “to know” in Latin. The history of science is a history of mankind trying to reveal the known unknowns and on the way to such a revelation discovering unknown unknowns. In the translation of space between the two, there is no other attitude that can be taken than that of a student, a learner…one who does not know.
Science starts and continues in the state of unknowing. As a seeker and a learner, try to be skeptical as well as open, unsure, and humble. It is a large request, I know, and not easy to accomplish.
In the past ten years, knowledge of human origins and migrations has exploded, thanks to genomic analysis of ancient DNA coupled with archeological discovery. Neanderthals (Homo sapiens Neanderthalis) are known to have co-existed with Homo Sapiens (Homo sapiens Sapiens) for almost 20,000 years (47K to 65K years ago) and hence bred human-Neanderthal hybrids. Within the Denisova Cave in the Siberian mountains, the discovery of a single finger bone fragment led to the discovery of a third species of humans that co-existed with Neanderthals and Homo Sapiens for about 10,000 years (44,000 to 54,000 years ago). There is also additional evidence of “ghost” human-like species in our early history.
Where did these other ancient humans come from? Why did they become extinct? What were they like? These are all examples of unknown unknowns becoming known unknowns.
Several international missions to Mars were launched in the Summer of 2020. The U.S. mission is set to analyze a 3-billion-year-old delta for signs of life. It will prepare specimens for eventual retrieval to Earth perhaps as early as 2031. This $2.2 billion investment is a clear demonstration of our own curiosity, our awareness of our own unknowing, and the ability and resources to do something about it.
Scientists, by nature, are methodical and cautious in making bold statements. Yet the urgency of the moment is unleashing a flood of effort, data, and techniques as they dissect how best to get ahead of a disease that is wreaking havoc on the world.
In order to make sense of this, we will next look deeper into the biotechnology of the science.
[i] “Steve Jobs’ dent in the universe—the shocking truth revealed!”, SolveNext Blog, accessed October 20, 2020, https://solvenext.com/blog/steve-jobs-dent-in-the-universethe-shocking-truth-revealed
[ii] Michael Shermer, “Rumsfeld’s Wisdom,” Scientific American, September 1, 2005, https://www.scientificamerican.com/article/rumsfelds-wisdom/