In 2000, members of an armed Filipino Islamic separatist group arrived on Sipadan Island, taking twenty-one of the twenty-three vacationers and hosts hostage. They were transported to Jolo Island where they were held with several other hostages taken from other archipelago islands over subsequent months. They were eventually rescued and released through a series of negotiations, ransoms, and military actions. While still a popular site for day trips and diving, Sipadan is no longer a place one can stay. Eden cannot last forever.
A COLD DAY IN HELL
I HAD everything ready. All the background research done, a schedule, assignments, and documents for signatures. Assembly line organized, the students returned and Anna delivered the samples in person. Behind closed doors we told the story and I asked everyone for their commitment and silence. Joining us were Drs. Martenson and Pierce who would run the toxicology and endocrinology assays. Participation was unanimous.
We explored the ethics of sharing only those samples from the CRFS dogs. Everyone had an opinion. While Jamie didn’t think there was any use in sending non-CRFS samples to CDC, even if they were from dogs that eventually got CRFS, Anna felt that there was value in looking at these in the context of looking for differences in the same dogs pre- and post-infection. It was critical that we eventually share these samples. Martenson and Pierce saw legitimacy in her argument, but pointed out that since the CDC was not collecting these from other vets, there wasn’t an established pool of like samples and ours would not make up a statistically valid population in the context of the thousands of other samples in their bank. Megan and I both felt that we should submit the samples eventually, but Anna’s team—our team—should at least get first crack at them since she made the initial observation. We came to a consensus that we would conduct our research and if we didn’t find anything in our initial analysis, we would contact the CDC and see if they were interested in taking these samples. Anna would at least make a note in the files of submitted samples from Addison’s dogs with CRFS that they seemed to die more slowly than dogs that didn’t have Addison’s.
We were not alone in this. If we found something we would follow it closely, but we had to let others contribute their expertise. The thought that Regnum and thus Kendal would likely gain access to our work made me sick to my stomach, but I had to just deal with, or better, just ignore it. Even if we were inclined to keep the samples initially to ourselves, considering the endless possibilities and seeing the vision of all those dead dogs, there is no way we could expect to do this on our own. We needed to do the initial work and prepare for scientific collaboration beyond our team.
Messenger RNA (mRNA) extraction had to be done first since it is very unstable. When a gene is activated, short pieces of mRNA are created from the gene’s template, which is then read by the ribosomes to make proteins. Changes in the amount of mRNA for a particular gene suggests that the organism has a reason to make more or less of that specific protein.
Proteins were next. They are fragile and degrade quickly if samples are not kept cold. There’s always a fear that an elusive protein wasn’t found not because it wasn’t there, but because the sample had degraded. Again, changes in levels or structure of a protein can point to how the dogs’ bodies are reacting to CRFS. Evidence of a foreign protein could point to the agent of CRFS itself.
Lastly, DNA. It is made up of a complex of molecules involving some of the strongest bonds in chemistry, and it’s sturdy as hell. Its covalent bonds are simple but strong, and the scaffold made by the double-stranded helical structure further physically protects the bonds, making it difficult to break them. By contrast, single-stranded mRNA leaves bonds exposed; thus they are oxidized easily and the strand degrades quickly. DNA is so stable that it has been pulled from such paleontological finds as bone marrow of 38,000-year-old saber-toothed cats permeated with tar from the La Brea tar pits of California, a femur and teeth of a 430,000-year-old pre-human found in Spain, and bones of a 700,000-year-old horse from the Canadian permafrost.
Extracted DNA can be kept indefinitely. It can be frozen, dried, stored in alcohol, or left in a vial on a benchtop. Just don’t heat it above 94C or the complementary strands that make up DNA will break apart. Capitalizing on this feature, a controlled denaturing of DNA is the initial step in one of the most commonly performed DNA analyses, the polymerase chain reaction (PCR). In this procedure, DNA is heated until the two strands fall apart; the tube containing the denatured DNA also contains the building blocks for more DNA along with other required enzymes and primers. As the temperature is subsequently lowered, new strands are assembled off the template of the original single strands of DNA, making a second copy. Successive cycles of this result in hundreds of thousands of copies of the targeted piece of DNA.
It took the best of two weeks to set up protocols, process all the samples, and begin the analysis phase. With mRNA, proteins, and DNA in hand, we set out to see if we could detect any differences between normal dogs with and without CRFS, and Addison’s dogs with and without CRFS.
We spent the next four weeks in a factory-like process to conduct every possible type of laboratory procedure on the samples, and meticulous data entry and analysis of every correlation we could think of. Everyone was working full-time on their part of the assembly line, to the detriment of their own research. The regimented process was punctuated by thrilling moments when there seemed to be an intriguing result, followed by a reality check when it proved to not be statistically valid.
Throughout February came a blizzard of dogs lost to CRFS. While the outbreak was still largely isolated to the East Coast, no population of dogs was left unaffected. Purebreds and mutts, pampered city dogs and working dogs in rural communities all fell victim with scattered losses that still seemed to show no pattern. There were mass losses at shelters, many of which temporarily stopped taking in dogs. Curiously, dog rescue centers were still largely untouched, even as they took in those dogs that would have gone to the shelters.
Two weeks before spring break and still nothing significant. The sun had set, closing another work week, but no one bothered to turn on the lights. Thinking that I might go home early for once and catch the girls before they engaged in their weekend plans, I headed out through the lab to find Megan and Jamie, huddled in the luminosity of computers and equipment monitors, hovering over output from the DNA sequencer. The energy was palpable.
I crept up behind them. “What are you two conspiring over?” I whispered. Megan yelped. “Whoa, I was only kidding. You find something?”
“Maybe.” Jamie motioned me over with a rapidly waving hand. He looked strangely serious, even for him.
Megan looked up, as excited as Sofie finding a mole hole. “If it is what we think, then we have something very interesting.”
I wedged in between them. The three of us focused on the computer screen covered with rows and rows of thin lines making hundreds of little colored peaks, each indicating which nucleotide base was at what position in the sequence. Running underneath each row was the resulting sequence of A’s, T’s, C’s, and G’s. On a separate monitor were statistics of the peaks telling us the concentration of the nucleotide at each position. Jamie searched the screen for the pattern of interest, and pointed out the peaks and corresponding statistics on the intensity. “See? Right here.”
“Whoa, sorry guys, my eyes are too old for this,” I said, walking to the wall and flipping on the lights.
Blinking fast to adjust to the light he focused back to the sequences he had highlighted. “We’ve been looking at a particular mRNA that appears to increase in amount in all the dogs with CRFS. See here how the intensity indicates a large number of copies?” I’d hardly taken it in when he switched the sheet for another screen with rows and rows of the same sequence.
“See here, the first few rows? These are the normal dogs that don’t have Addison’s. We’ve matched these to the next few samples which are the same dogs after they got CRFS.
Then below these are the Addis
on’s dogs, again the first few samples before they got CRFS, the next are the same dogs after getting CRFS.
“We of course also have control dogs that are healthy and did not get CRFS. And see? Every dog has this same short sequence.” He pointed to rows and rows of the same series of letters, and grinned broadly.
Not in on the secret yet, I said, “So, every dog has this sequence. Then I would say that it’s just a dog sequence, but I’m sure you’re setting me up.” He shifted to his sneaky boyish, wait-for-it look.
“Oh!” I said. “You said you see an increase in the number of copies of the sequence in the dogs with CRFS.”
“Correct, they all have the sequence, but when we look at the intensity statistics, there’s an increase in the amount in the CRFS dogs, though the increase is very slight. Megan has the quantitative data.” He beamed at his teammate. “Show her.”
She rapidly swapped out his spreadsheets for hers, trading lines of letters for numbers representing the concentrations of mRNA sequence. “See here? These are the matched sets of healthy dogs to those with CRFS. Both the normal and one’s with Addison’s show an increase in the mRNA level when they have CRFS.”
“Hold on. You said you have matched sets for normal dogs. The same normal dog pre- and post CRFS infection?”
“Uh-huh.” Megan smiled.
“I thought all we got from normal dogs were samples after they got the disease. How did you get matched sets for these dogs?”
“Anna gave them to us. She’s been collecting blood from any patient that came in, with the owner’s permission of course, telling them that she wants it just in case, and that it may help find the cause. She said no one had refused. So even though we don’t have matching sets for every normal dog that died of CRFS, she has a fairly decent number of samples. See these, here? There are eighteen samples from normal dogs that eventually got CRFS.”
Anna was amazing. She’d thought of everything. I plopped down on the stool, shaking my head as I slowly processed the implications of the data. “Do you know what the sequence codes for?”
“Not yet. Jamie started picking this up yesterday and we’ve spent the day repeating the analysis, running every sample through a second time to be sure.” Megan said.
“We’ll have to run comparisons through the NIH NCBI databases tonight and tomorrow, but we have a theory. Want to venture a guess?” Jamie teased. They looked like two little kids who put the frog in the teacher’s desk.
“Is the amount of mRNA the same in dogs that you say are healthy, or without CRFS, regardless of whether they’re normal or have Addison’s?”
“Yes!” Megan pounced. Obviously she and Jamie thought they had the answer. I thought I did too, but it was worth carefully considering each detail to make sure every angle was covered.
“Okay, since Anna says the dogs are first affected by adrenal failure, her theory is that CRFS messes up something in the overall hypothalamic–pituitary–adrenal system—the HPA.”
“Correct,” Megan said.
“And, since the Addison’s dogs don’t have functioning adrenal glands, they get medication to replace the adrenal hormones. So even if CRFS were to directly affect the adrenals, Addison’s dogs would probably be protected.”
“Correct.” Jamie nodded rapidly.
I continued, “But since you did see a difference in Addison’s dogs that get CRFS, and since they obviously do get it, CRFS can’t be directly acting on the adrenal glands, but probably somewhere else in the HPA other than the adrenals.
“That’s what we’re thinking,” Megan agreed. “But there is definitely a difference between how normal and Addison’s dogs respond.”
“How so?”
“Well, see the numbers here? Even though both types of sick dogs show an increase in gene expression, the dogs with Addison’s show a slightly lower level.”
“So you also validated Anna’s clinical observations with mRNA evidence.” The slow trickle of thought started churning into whitewater. “This should be relatively easy to identify. Why didn’t you start running it through NCBI’s database when you first suspected something!?”
“Because we wanted to be sure!” Megan said defensively.
“We’ve been checking and rechecking all day to make sure that we have all the samples correctly categorized, and all the sequences match, and that all the levels of mRNA are confirmed, since the difference is very small,” Jamie added. “We were actually holding off on searching the databases to see what it was, sort of as a reward to ourselves once we had it all confirmed. We were just about to come tell—”
“You didn’t have to, I could feel something was up from across the room!”
We looked at one another in stunned silence, the excitement hovering between elation and realization of the implications of their finding. We could have a major clue to the process, or even be close to finding the cause. Each step from here would have to be carefully calculated so we didn’t waste a lot of time chasing phantom links.
NO WHINING ON THE YACHT
TRUMPING STORIES of war and falling economies, when a well-to-do American systematically killed Cecil the Lion for the sole purpose of having a wall trophy, the clamoring for the offender’s head was deafening. For a brief time, Cecil’s story drowned out those of starvation in Ethiopia and the Central African Republic, and mass migration of refugees from the Sudan, even though all of these stories originated from the same continent half a world away. To be honest, those who questioned our collective sympathetic priorities did have a point. Those of us who love our dogs and toss out phrases like “they are my family,” “they are like my children” speak from the privilege of first-world problems and the luxury of isolation from the impact of world events that don’t directly touch our lives, unless we choose to post a related picture on Facebook to show how in touch we are.
It is estimated that 40% of American households own a collective seventy-five million dogs, spending money on everything from the purchase of their pet, to food, vet care, toys, accessories, and services like boarding and grooming. Americans spend over $60 billion a year on our pets—more than the gross domestic product of most countries in the world. Our pets have become more than companions; they have become a reflection of our capitalist indulgences, proudly signifying that their owners have achieved the American dream. Simple food and shelter have been replaced with multi-level cat apartments, doggy daycare, and an assortment of outfits and costumes to celebrate every conceivable occasion.
When a dog is dying you will know it is time when it stops eating. But often we extend their lives with veterinary interventions that may call into question who the care really benefits. We love our dogs, but sometimes love is blind.
The slowly creeping downstream economic impact of CRFS first and most significantly hit the dog show world, with groomers and handlers and breed clubs practically shut down and out of work. Then business dropped precipitously for national pet store chains, as the fear of contaminated products from overseas grew, while cottage industries surfaced, including many who locally produced raw dog food. All of this happened without the slightest link to the cause.
While it was clear that CRFS was a problem of an economically advanced nation, the economic impact was felt worldwide, as American owners hunkered down and stopped buying everything from collars from china and toys from India. But though sympathies abounded for those who mourned the loss of their pets, they did not extend to those who were affected downstream, and whose lives depended on a meager percentage of industry revenues.
At least at first. There was a slow-growing social media movement afoot to highlight the absurdities of our obsession with our pets, juxtapositioning images of owners agonizing over their dying pets with those of mothers agonizing over their starving children. The movement’s position, in some ways valid, was that there should be no whining on the yacht.
Another text, my fourth of the day. Can you pick Tess up after rehearsal?
Of course.
&
nbsp; I could feel the irritated force of Chris’ typing. It had been more than two months and his patience was wearing thin, especially since until now we hadn’t made any breakthroughs. But he did not know yet about the hope.
I’m sorry, again. We’re on to something that is looking pretty interesting and I can’t just walk out in the middle our discussion, so I don’t know what time I will be home.
My phone sounded Moondance, Chris’ ringtone.
“Hi, I’m sorry,” I said. “I just can’t stop. I completely forgot she had rehearsal. Can you get her?”
“It’s okay,” he said without emotion. “She told me this morning and we worked it out.” There was a numbness to his voice.
“She did? But I told her that I would take care of it.”
“Well apparently she is learning not to depend on you either.” I deserved that, but it still pissed me off.
“That’s not fair. It’s a critical time.”
“It’s always a critical time for you.”
His point was not lost on me. Each step, each node of progress, whether forward or backward, was critical. No matter how small. So it was always a critical time, to me at least.
“I know I keep saying that, I just—Anna’s samples are showing something really interesting and we need to determine what to do next, quickly. We’re just about out of time.”
“What are you seeing?”
“I’ll tell you later when I know something more definitive.”
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