Genetic Flip Helped Organisms Go From One Cell to Many

Clockwise from top left: microscopic views of glands in frog skin, a sheep’s hoof, a tamarin’s skin and fish scales. 

Narwhals and newts, eagles and eagle rays — the diversity of animal forms never ceases to amaze. At the root of this spectacular diversity is the fact that all animals are made up of many cells — in our case, about 37 trillion of them. As an animal develops from a fertilized egg, its cells may diversify into a seemingly limitless range of types and tissues, from tusks to feathers to brains.

The transition from our single-celled ancestors to the first multicellular animals occurred about 800 million years ago, but scientists aren’t sure how it happened. In a study published in the journal eLife, a team of researchers tackles this mystery in a new way.

The researchers resurrected ancient molecules that once helped single-celled organisms thrive, then recreated the mutations that helped them build multicellular bodies.

The authors of the new study focused on a single molecule called GK-PID, which animals depend on for growing different kinds of tissues. Without GK-PID, cells don’t develop into coherent structures, instead growing into a disorganized mess and sometimes even turning cancerous.

GK-PID’s job, scientists have found, is to link proteins so cells can divide properly. “I think of it as a molecular carabiner,” said Joseph W. Thornton, an evolutionary biologist at the University of Chicago and a co-author of the new study

When a cell divides, it first has to make an extra copy of its chromosomes, and then each set of chromosomes must be moved into the two new cells. GK-PID latches onto proteins that drag the chromosomes, then attaches to anchor proteins on the inner wall of the cell membrane. Once those proteins are joined by GK-PID, the dragging proteins pull the chromosomes in the correct directions.

Bad things happen if the chromosomes head the wrong way. Skin cells, for example, form a stack of horizontal layers. New cells needs to grow in the same direction so skin can continue to act as a barrier. If GK-PID doesn’t ensure that the chromosomes move horizontally, the cells end up in a jumble, like bricks randomly set at different angles.

Previous studies have offered clues to how this important molecule might have evolved in the ancestors of animals. All animals (ourselves included) carry a gene sequence that’s very similar to the one producing GK-PID. But that gene encodes a different molecule with a different job: an enzyme that helps build DNA. The enzyme can be found even in other organisms, like fungi to bacteria.

Dr. Thornton and his colleagues wondered whether that enzyme and its cousin GK-PID shared some kind of evolutionary history.

First, they made a careful study of the different forms of GK-PID and the DNA-building enzyme in about 200 species. Then they worked out how the genes for these molecules must have mutated over the millenniums.

That analysis allowed the scientists to figure out the DNA sequence for GK-PID in the single-celled ancestors of animals — a gene that hasn’t been seen in hundreds of millions of years. Then Dr. Thornton and his colleagues did something even more amazing: They recreated those ancient molecules to see how they once functioned.

The ancestral version of GK-PID wasn’t a carabiner, the scientists found. Instead, it behaved like a DNA-building enzyme. That finding suggests that in the ancestors of animals, the gene for the enzyme was accidentally duplicated. Later on, mutations in one copy of the gene turned it into a carabiner.

But how many mutations did it take to transform the molecule? That’s the most remarkable part of the new study. The scientists altered the gene for the ancestral enzyme with the earliest mutations that evolved in it. They found it took a single mutation to flip GK-PID from an enzyme to a carabiner.

“Genetically, it was much easier than we thought possible,” Dr. Thornton said. “You don’t need some elaborate series of thousands of mutations in just the right order.”

The evolution of a molecular carabiner did not by itself give rise to the animal kingdom, of course. Other adaptations were needed to grow multicellular bodies. Dr. Thornton said that it might be possible to resurrect other ancestral molecules to figure out how those adaptations evolved, as well.

And if GK-PID is any guide, Dr. Thornton said, their evolution may have been surprisingly simple. A single mutation might have been enough to switch a molecule from one job to another.

Antonis Rokas, an evolutionary biologist at Vanderbilt University who was not involved in the study, agreed. “One of evolution’s most striking major innovations may be the end-product of a series of many minor innovations,” he said.


Researchers See New Importance in Y Chromosome

The male, or Y, chromosome in humans, right, is much smaller than the X, left. 

There is new reason to respect the diminutive male Y chromosome.

Besides its long-known role of reversing the default state of being female, the Y chromosome includes genes required for the general operation of the genome, according to two new surveys of its evolutionary history. These genes may represent a fundamental difference in how the cells in men’s and women’s bodies read off the information in their genomes.

When researchers were first able to analyze the genetic content of the Y chromosome, they found it had shed hundreds of genes over time, explaining why it was so much shorter than its partner, the X chromosome. All cells in a man’s body have an X and a Y chromosome; women’s have two X chromosomes.

The finding created considerable consternation. The Y had so few genes left that it seemed the loss of a few more could tip it into extinction.

But an analysis in 2012 showed that the rhesus monkey’s Y chromosome had essentially the same number of genes as the human Y. This suggested that the Y had stabilized and ceased to lose genes for the last 25 million years, the interval since the two species diverged from a common ancestor.

Two new surveys have now reconstructed the full history of the Y chromosome back to its evolutionary origin. One research group was led by Daniel W. Bellott and David C. Page of the Whitehead Institute in Cambridge, Mass., and the other by Diego Cortez and Henrik Kaessmann of the University of Lausanne in Switzerland. Their findings were reported on Wednesday in the journal Nature.

In the past 12 years, with the help of the genome sequencing centers at Washington University in St. Louis and the Baylor College of Medicine in Houston, Dr. Page’s group has decoded the DNA sequence of the Y chromosome of eight mammals, including the rhesus monkey and humans. The Y chromosome is so hard to decode that many early versions of the human genome sequence just omitted it. Dr. Kaessmann’s group, on the other hand, devised a quick method of fishing out Y chromosome genes by simply comparing the X and Y DNA of various species and assuming that any genetic sequences that did not match to the X must come from the Y.

Dr. Kaessmann calculates that the Y chromosome originated 181 million years ago, after the duck-billed platypus split off from other mammals but before the marsupials did so.

In some reptiles, sex is determined by the temperature at which the egg incubates. Genetic control over sex probably began when a gene on one of the X chromosomes called SOX3 became converted to SRY, the gene that determines maleness, and thus the Y chromosome came into being.

Until this time, the predecessors of the X and the Y had been an equal pair of chromosomes just like any of the others. Humans have 23 pairs of chromosomes, with one member of every pair being inherited from each parent. People with an XX pair among their 23 are female; those with an XY pair are male.

Before generating eggs and sperm, the 23 pairs of chromosomes line up and each chromosome exchanges chunks of DNA with its partner, a process known as recombination. But recombination between the X and Y had to be banned, except at their very tips, lest the male-determining SRY gene slip across to the X and wreak havoc.

Recombination creates novel arrays of DNA that keep genes adapted to the environment; without recombination they decay and are shed from the genome.

The reconstructions by the Page and Kaessmann groups show that most such genes were shed almost immediately and that the few genes remaining on the Y have been stable for millions of years.

One of these genes is SRY, and others are involved in sperm production. A third category of genes is unusual in being switched on not just in the testis but in tissues all over the body. These active genes, of which there are 12 in humans, all have high-level roles in controlling the state of the genome and the activation of other genes.

The 12 regulatory genes have counterpart genes on the X with which they used to recombine millions of years ago. They escaped the usual decay caused by lack of recombination, presumably being kept functional by purifying selection, a geneticists’ term meaning that any mutations were lethal to the owner. They have, however, become somewhat different from their 12 counterpart genes on the X.

This means that female, or XX, cells have a slightly different set of these powerful genes from male or XY cells, since the X and Y genes are producing slightly different proteins. In females, usually one X chromosome is inactivated in each cell, but the 12 genes are so important that they escape inactivation, and XX cells, like XY cells, receive a double dose of the gene’s products.

“Throughout human bodies, the cells of males and females are biochemically different,” Dr. Page said. The genome may be controlled slightly differently because of this variation in the 12 regulatory genes, which he thinks could contribute to the differing incidence of many diseases in men and women.

Differences between male and female tissues are often attributed to the powerful influence of sex hormones. But now that the 12 regulatory genes are known to be active throughout the body, there is clearly an intrinsic difference in male and female cells even before the sex hormones are brought into play.

“We are only beginning to understand the full extent of the differences in molecular biology of males and females,” Andrew Clark, a geneticist at Cornell University, wrote in a commentary in Nature on the two reports.

A Gene Mystery: How Are Rats With No Y Chromosome Born Male?

An Amami spiny rat. Cells of the rat, which is from Japan, are sexually flexible and capable of adapting to either ovaries or testes. 

In most mammals, us included, biological sex is determined by a lottery between two letters: X and Y, the sex chromosomes. Inherit one X each from mom and dad, and develop ovaries, a womb and a vagina. Inherit an X from mom and a Y from dad, and develop testes and a penis.

But there are rare, mysterious exceptions. A small number of rodents have no Y chromosomes, yet are born as either females or males, not hermaphrodites. Now, scientists may be one step closer to figuring out how sex determination works in one of these rodents.

In a study published in Science Advances on Friday, Japanese scientists suggested that cells of the endangered Amami spiny rat, from Japan, are sexually flexible and capable of adapting to either ovaries or testes. When the researchers injected stem cells derived from a female rat into male embryos of laboratory mice, the cells developed into and survived as sperm precursors in adult males. The result was surprising since scientists have never been able to generate mature sperm from female stem cells, largely because sperm production normally requires the Y chromosome.

Found only in the subtropical forests of an island in Japan called Amami Oshima, Amami spiny rats are threatened by habitat destruction, competition with black rats not native to the island and predation by mongooses and feral cats and dogs. Their range has been reduced to less than 300 square miles, an area smaller than New York City.

Both female and male Amami spiny rats have only one X chromosome, an arrangement only known to occur in a handful of rodents among mammals. Arata Honda, associate professor at the University of Miyazaki and the lead author of the paper, said in an email that he was partly motivated to study Amami spiny rats in the hope that learning about them might reduce their risk of extinction.

No one knows how or why, but at some point the rats lost their Y chromosome and, along with it, an important gene called SRY that’s considered the “master switch” of male anatomical development in most mammals.

A chimera containing genes from an Amami spiny rat and a mouse. 

It’s possible that a new gene that wasn’t linked to the Y chromosome took over the role of SRY in these rats, said Monika Ward, a professor and expert on the Y chromosome at the University of Hawaii in Honolulu who was not involved in this study. In addition, research has shown that other genes involved in male sexual differentiation were not lost, but rather transferred from the Y chromosome to other parts of the rat’s genome, including to the X chromosome.

Because the rats are endangered, scientists cannot directly do experiments on them. To get around this, Dr. Honda and his colleagues converted skin cells from the tail tip of a female Amami spiny rat into special stem cells called induced pluripotent stem cells (also called iPS cells), which can multiply indefinitely and become any other cell type in the body. The scientists injected the stem cells into mice embryos and transplanted the embryos into female mice, which birthed 13 so-called chimeras.

After the chimeras reached adulthood, the researchers located the spiny rat iPS cells within their bodies. They were surprised to find some iPS cells appeared in the ovary as immature egg cells, and others in the testis as immature sperm cells.

This study shows that the spiny rat’s sex cells have “astounding” fluidity, said Diana Laird, an associate professor in reproductive sciences at the University of California, San Francisco, who was not involved in the study. The cells were able to sense whether they were in an ovary or testis, and “not only hear but obey the signals” coming from that foreign environment, she added.

Dr. Ward emphasized that these results are not universal — if you were to take iPS cells from a normal female mouse and put them in a male embryo, the few cells that became sperm precursors would die very quickly. The female stem cells in this study were able to approach mature sperm development because of the Amami spiny rat’s unique biology, she said.

The study is also significant because the researchers managed to create chimeras from an endangered species, said Marisa Korody, a postdoctoral associate at the San Diego Zoo Institute for Conservation Research who researches how iPS cells might be used to protect endangered animals.

“One of the lofty goals we have for using stem cells,” she said, is “to differentiate them into egg and sperm and hopefully create embryos that can be transplanted into a surrogate.”

But there are limits to the findings because the researchers have not yet shown that the spiny rat’s stem cells can fully develop into mature eggs and sperm. “That’s the million dollar question,” Dr. Laird said.

Somehow, This Fish Fathered a Near Clone of Itself

Squalius alburnoides and Squalius pyrenaicus inside an artificial pond where researchers studied them.

A female and male get together. One thing leads to another, and they have sex. His sperm fuses with her egg, half of his DNA combining with half of her DNA to form an embryo.

As humans, this is how we tend to think of reproduction.

But there are many other bizarre ways reproduction can take place. For instance, scientists have discovered a fish carrying genes only from its father in the nucleus of its cells. Found in a type of fish called Squalius alburnoides, which normally inhabits rivers in Portugal or Spain, this is the first documented instance in vertebrates of a father producing a near clone of itself through sexual reproduction — a rare phenomenon called androgenesis — the researchers reported in the journal Royal Society Open Science on Wednesday.

The possibility of androgenesis is just one of many mysteries about Squalius alburnoides. It’s not a species in the usual sense, but rather something called a hybrid complex, a group of organisms with multiple parental combinations that can mate with one another.

The group is thought to have arisen from hybridization between females of one species, Squalius pyrenaicus, and males of another species, now extinct, that belonged to a group of fish called Anaecypris. To sustain its population, Squalius alburnoides mates with several other closely related species belonging to the Squalius lineage.

That it can reproduce at all is unusual enough. Most hybrids, like mules, are sterile because the chromosomes from their parents of different species have trouble combining, swapping DNA and dividing — steps required for egg or sperm production.

Squalius alburnoides males circumvent this problem by producing sperm cells that do not divide, and therefore contain more than one chromosome set. This is important because most animals, Squalius alburnoides included, need at least two chromosome sets to survive.

Mostly, animals have sex cells containing only one chromosome set, which means the egg and sperm are both required for reproduction. But in Squalius alburnoides, sperm with multiple chromosome sets can provide all the nuclear genetic material needed for a viable offspring — paving the way for androgenesis.

A Squalius alburnoides fish, which researchers discovered had reproduced through androgenesis. 

The details in Squalius alburnoides are still unknown, but in general androgenesis is thought to occur in a couple of ways, said Miguel Morgado-Santos, a graduate student at the University of Lisbon and an author of the study: Sperm could fertilize an egg that contains no chromosomes, or it could destroy the genetic content from the nucleus of the egg after fertilization.

Mr. Morgado-Santos’s group found this instance of androgenesis by accident, while studying the mating patterns of Squalius alburnoides. The researchers put male and female Squalius alburnoides with males and females of another Squalius species in an artificial pond, let the fish reproduce and then genetically analyzed 100 randomly selected offspring. One of these offspring had only paternal chromosomes.

“We weren’t expecting to find that,” Mr. Morgado-Santos said, adding that at first he and his collaborators thought they had made a mistake.

Though he acknowledges one in 100 fish is a rare occurrence, Mr. Morgado-Santos thinks this instance of androgenesis could represent a “snapshot” of a population moving toward becoming its own species. Put another way, androgenesis may help this fish become independent from the other Squalius species it relies on to reproduce.

If androgenesis turns out to be a regular feature in this population, Mr. Morgado-Santos’s group might be catching the ”very early stages” of a new reproductive mode for the fish, which would be exciting, said Tanja Schwander, a professor of ecology and evolution at the University of Lausanne in Switzerland who did not participate in this research.

But for now, she added, the researchers cannot rule out the possibility that this one instance is a random exception (perhaps the fish’s mother accidentally produced an egg that contained no chromosomes, for instance).

Accident or not, it happened, and shows that reproduction can vary in all sorts of “weird and wonderful” ways across the natural world, said Benjamin Oldroyd, a professor of genetics at the University of Sydney who was not involved in this study.

“It may not add up to a hill of beans other than realizing that the world is much more complicated than we assume,” he said. “But it’s part of what life is, as a curious human, to understand these things.”

This Mutant Crayfish Clones Itself, and It’s Taking Over Europe

The marbled crayfish is a mutant species that clones itself, scientists report. The population is exploding in Europe, but the species appears to have originated only about 25 years ago. 

Frank Lyko, a biologist at the German Cancer Research Center, studies the six-inch-long marbled crayfish. Finding specimens is easy: Dr. Lyko can buy the crayfish at pet stores in Germany, or he can head with colleagues to a nearby lake.

Wait till dark, switch on head lamps, and wander into the shallows. The marbled crayfish will emerge from hiding and begin swarming around your ankles.

“It’s extremely impressive,” said Dr. Lyko. “Three of us once caught 150 animals within one hour, just with our hands.”

Over the past five years, Dr. Lyko and his colleagues have sequenced the genomes of marbled crayfish. In a study published on Monday, the researchers demonstrate that the marble crayfish, while common, is one of the most remarkable species known to science.

Before about 25 years ago, the species simply did not exist. A single drastic mutation in a single crayfish produced the marbled crayfish in an instant

The mutation made it possible for the creature to clone itself, and now it has spread across much of Europe and gained a toehold on other continents. In Madagascar, where it arrived about 2007, it now numbers in the millions and threatens native crayfish.

“We may never have caught the genome of a species so soon after it became a species,” said Zen Faulkes, a biologist at the University of Texas Rio Grande Valley, who was not involved in the new study.

The marbled crayfish became popular among German aquarium hobbyists in the late 1990s. The earliest report of the creature comes from a hobbyist who told Dr. Lyko he bought what were described to him as “Texas crayfish” in 1995.

The hobbyist — whom Dr. Lyko declined to identify — was struck by the large size of the crayfish and its enormous batches of eggs. A single marbled crayfish can produce hundreds of eggs at a time.

Soon the hobbyist was giving away the crayfish to his friends. And not long afterward, so-called marmorkrebs were showing up in pet stores in Germany and beyond.

As marmorkrebs became more popular, owners grew increasingly puzzled. The crayfish seemed to be laying eggs without mating. The progeny were all female, and each one grew up ready to reproduce.

In 2003, scientists confirmed that the marbled crayfish were indeed making clones of themselves. They sequenced small bits of DNA from the animals, which bore a striking similarity to a group of crayfish species called Procambarus, native to North America and Central America.

Ten years later, Dr. Lyko and his colleagues set out to determine the entire genome of the marbled crayfish. By then, it was no longer just an aquarium oddity.

For nearly two decades, marbled crayfish have been multiplying like Tribbles on the legendary “Star Trek” episode. “People would start out with a single animal, and a year later they would have a couple hundred,” said Dr. Lyko.

Many owners apparently drove to nearby lakes and dumped their marmorkrebs. And it turned out that the marbled crayfish didn’t need to be pampered to thrive. Marmorkrebs established growing populations in the wild, sometimes walking hundreds of yards to reach new lakes and streams. Feral populations started turning up in the Czech Republic, Hungary, Croatia and Ukraine in Europe, and later in Japan and Madagascar.

Sequencing the genome of this animal was not easy: No one had sequenced the genome of a crayfish. In fact, no one had ever sequenced any close relative of crayfish.

Dr. Lyko and his colleagues struggled for years to piece together fragments of DNA into a single map of its genome. Once they succeeded, they sequenced the genomes of 15 other specimens, including marbled crayfish living in German lakes and those belonging to other species.

The rich genetic detail gave the scientists a much clearer look at the freakish origins of the marbled crayfish.

It apparently evolved from a species known as the slough crayfish, Procambarus fallax, which lives only in the tributaries of the Satilla River in Florida and Georgia.

The scientists concluded that the new species got its start when two slough crayfish mated. One of them had a mutation in a sex cell — whether it was an egg or sperm, the scientists can’t tell.

Normal sex cells contain a single copy of each chromosome. But the mutant crayfish sex cell had two.

Somehow the two sex cells fused and produced a female crayfish embryo with three copies of each chromosome instead of the normal two. Somehow, too, the new crayfish didn’t suffer any deformities as a result of all that extra DNA.

It grew and thrived. But instead of reproducing sexually, the first marbled crayfish was able to induce her own eggs to start dividing into embryos. The offspring, all females, inherited identical copies of her three sets of chromosomes. They were clones.

Now that their chromosomes were mismatched with those of slough crayfish, they could no longer produce viable offspring. Male slough crayfish will readily mate with the marbled crayfish, but they never father any of the offspring.

In December, Dr. Lyko and his colleagues officially declared the marbled crayfish to be a species of its own, which they named Procambarus virginalis. The scientists can’t say for sure where the species began. There are no wild populations of marble crayfish in the United States, so it’s conceivable that the new species arose in a German aquarium.

[READ: A Gene Mystery: How Are Rats With No Y Chromosome Born Male?]

All the marbled crayfish Dr. Lyko’s team studied were almost genetically identical to one another. Yet that single genome has allowed the clones to thrive in all manner of habitats — from abandoned coal fields in Germany to rice paddies in Madagascar.

In their new study, published in the journal Nature Ecology and Evolution, the researchers show that the marbled crayfish has spread across Madagascar at an astonishing pace, across an area the size of Indiana in about a decade.

Thanks to the young age of the species, marbled crayfish could shed light on one of the big mysteries about the animal kingdom: why so many animals have sex.

Only about 1 in 10,000 species comprise cloning females. Many studies suggest that sex-free species are rare because they don’t last long.

In one such study, Abraham E. Tucker of Southern Arkansas University and his colleagues studied 11 asexual species of water fleas, a tiny kind of invertebrate. Their DNA indicates that the species only evolved about 1,250 years ago.

There are a lot of clear advantages to being a clone. Marbled crayfish produce nothing but fertile offspring, allowing their populations to explode. “Asexuality is a fantastic short-term strategy,” said Dr. Tucker.

In the long term, however, there are benefits to sex. Sexually reproducing animals may be better at fighting off diseases, for example.

If a pathogen evolves a way to attack one clone, its strategy will succeed on every clone. Sexually reproducing species mix their genes together into new combinations, increasing their odds of developing a defense.

The marbled crayfish offers scientists a chance to watch this drama play out practically from the beginning. In its first couple decades, it’s doing extremely well. But sooner or later, the marbled crayfish’s fortunes may well turn.

“Maybe they just survive for 100,000 years,” Dr. Lyko speculated. “That would be a long time for me personally, but in evolution it would just be a blip on the radar.”

Mozilla announces an open gateway for the internet of things

Apple, Google, Amazon and Samsung have all been working hard to create their own standard to control all the connected devices around your home. Mozilla just announced that anybody can now create an open gateway to control the internet of things. The organization also confirmed that it is still working on a set of frameworks and open standards so that we don’t end up with an internet of things controlled by big tech companies.

Connected devices are great, until you realize that your connected thermostat only works with the Amazon Echo and your connected lightbulbs only work with Siri and the Home app.

Accessory makers also don’t necessarily want a handful of big tech companies to control the internet of things. Tech giants could end up charging expensive licensing fees to work with their ecosystem. And customers end up having to make tough decisions.

Mozilla is a big proponent of the open web. So it seems natural that the not-for-profit organization has plans for connected devices. Project Things encompasses multiple projects, so let’s look at Mozilla’s work.

First, Mozilla wants to create an open standard with the W3C around the Web of Things. The idea is that accessory makers and service providers should use the same standard to make devices talk to each other. The specifications rely on JSON, and a REST and WebSockets API. Those are standard data and API models on the web, and they should work perfectly fine for connected devices.

Second, Mozilla is working on a Web of Things Gateway so that you can replace your Amazon Echo, Philips Hue hub, Apple TV and Google Home with an open device. You can already create a gateway using a Raspberry Pi 3, and ZigBee and Z-Wave USB dongles.

Eventually, manufacturers could leverage this work to create their own gateways. Maybe Netgear could embed a Web of Things gateway into their next router — your router is connected to the internet and runs 24/7 after all. Developers could also create bridges between the HomeKit API or Amazon’s Smart Home Skill API so that all devices work with your Amazon Echo, Google Home or iPhone without too much effort. Web of Things could become the common language between those proprietary APIs.

Finally, Mozilla is creating the interface to control your connected devices. You can add Mozilla’s progressive web app to your smartphone home screen and control your home. For instance, you can use your voice to turn on the lights, create IFTTT-style rules to automate your house, add a floor-plan to lay out your devices and more.

Mozilla has designed an add-on system so that you can add support for new devices and protocols by installing plugins. It’s important to note that all of this runs on your own gateway in your house. Google or Amazon can’t see when you turn on the light using your voice.

Eventually, I could also see app developers leveraging the Web of Things protocol to create native apps to control your house. But it’s clear that Mozilla wants to attack this issue from all angles. And developers can already start playing around with Project Things and contribute to development.

Science Says This Is the Simplest Way to Remember More of What You Read

Whether it’s Facebook content, Bill Gates’s favorite book, or the latest critical business report, most of us enjoy reading or have to do quite a bit of it through the day. But in the rush to do everything in less time, you might be missing a crazily simple way to commit more content to memory:

Just go back and give yourself a little time to reflect on what you just read.

Now, when I say “reflect,” I don’t mean sit there pondering for an hour. I mean sitting just long enough to

  • Mentally identify the main points or concepts
  • Jot down some notes (you can’t write everything, so this forces your brain to choose what’s most important)
  • Consider the ramifications or implications of the content
  • Think about how the content connects to your personal preferences, personality, and experiences

Why it works.

As Allison Preston, associate professor of psychology and neuroscience at the University of Texas at Austin, explains in this 2014 research study release,

We think replaying memories during rest makes those earlier memories stronger, not just impacting the original content, but impacting the memories to come. […] Nothing happens in isolation. When you are learning something new, you bring to mind all of the things you know that are related to that information. In doing so, you embed the new information into your existing knowledge.

With this in mind, when you give yourself a few minutes to rest and think about what you just ingested from the page, you’re allowing your brain to better connect the new information to what you’ve already done or understand. And because the brain is wired to respond to emotions quickly and efficiently, connecting them to memory formation and the interpretation of facts and rational thought, if you can allow yourself to really acknowledge and respond to what you feel during your reading reflections, you stand a better chance of the new memories being more powerful and easier to retrieve.

The myth of lost time.

I can hear you protesting from here.

“I barely have time to use the restroom! How am I supposed to take time to reflect on what I read?”

I get it. But when you can remember information from your content better, you actually can end up saving time. You don’t have to go back and look up as many facts or ideas, and whether it’s rubbing elbows with some big shots at a conference or explaining your rationale for a new process to your team, you can apply the information on the fly better. From this standpoint, reading reflection is an efficiency booster and worth the few brief minutes it takes.

More ways to level up.

To really get the most out of your reading and reading reflection, there are a few other add-on tricks you can try. You might want to

  • Read some of the content aloud or draw images for the main ideas. The brain doesn’t process the different types of sensory information in isolation from each other, so honing in on auditory or visual information might help you process the content.
  • Read when you are more rested. Fatigue can negatively influence your ability to focus, so pick a reading time where you feel energized.
  • Eliminate distractions. While turning off phone alerts or shutting your door are obvious distraction points, don’t forget about other factors, such as room temperature, hunger, and your position in your chair.
  • Be clear about your goal. Knowing the purpose behind what you’re reading can make it easier to feel motivated and engaged with the content.
  • Go for a hard copy. Researchers from the University of Oregon found that online content is harder to recall. One theory is that the disappear-reappear nature of online content is distracting, but the loss of tactile information, such as the feeling of the page, might contribute, too.

No matter how long your reflection time might happen to last, just read. Read anything. It’s by far one of the easiest things you can do to boost your intelligence and stay on top of your game.

Urothelial Carcinoma


Digital Object ThumbnailUrothelial Carcinoma (00:08)

A 69-year-old woman presented to the emergency department with new-onset gross hematuria. Her medical history was notable for 20 pack-years of smoking. Results of a physical examination, complete blood count, and metabolic panel were normal. Urinalysis showed more than 100 red cells per high-power field and 5 to 10 white cells per high-power field. A urine culture was negative, and results of urine cytologic testing showed no malignant cells. A computed tomographic urogram showed a filling defect in the right ureter. Examination of the bladder with a rigid cystoscope revealed a papillary mass that protruded through the right ureteral orifice during ureteral peristalsis (see video). In a ureteroscopic examination, it was determined that the mass was 4 cm in length and had a cylindrical stalk that was 5 mm in diameter; numerous smaller distal ureteral masses were also revealed. Biopsy was performed, and pathological examination confirmed the diagnosis of papillary urothelial carcinoma. Smoking and other chemical exposures are risk factors for urothelial carcinoma. After discussion of the treatment options, the patient elected to undergo robot-assisted laparoscopic nephroureterectomy with excision of a bladder cuff. The final pathological evaluation showed high-grade, multifocal urothelial cancer along the ureter, with negative surgical margins. Three months after the procedure, the patient was well and had no further hematuria, and surveillance cystoscopy showed no evidence of disease recurrence.

Risk of acute kidney injury associated with the use of fluoroquinolones


Background: Case reports indicate that the use of fluoroquinolones may lead to acute kidney injury. We studied the association between the use of oral fluoroquinolones and acute kidney injury, and we examined interaction with renin–angiotensin-system blockers.

Methods: We formed a nested cohort of men aged 40–85 enrolled in the United States IMS LifeLink Health Plan Claims Database between 2001 and 2011. We defined cases as men admitted to hospital for acute kidney injury, and controls were admitted to hospital with a different presenting diagnosis. Using risk-set sampling, we matched 10 controls to each case based on hospital admission, calendar time (within 6 wk), cohort entrance (within 6 wk) and age (within 5 yr). We used conditional logistic regression to assess the rate ratio (RR) for acute kidney injury with current, recent and past use of fluoroquinolones, adjusted by potential confounding variables. We repeated this analysis with amoxicillin and azithromycin as controls. We used a case-time–control design for our secondary analysis.

Results: We identified 1292 cases and 12 651 matched controls. Current fluoroquinolone use had a 2.18-fold (95% confidence interval [CI] 1.74–2.73) higher adjusted RR of acute kidney injury compared with no use. There was no association between acute kidney injury and recent (adjusted RR 0.87, 95% CI 0.66–1.16) or past (RR 0.86, 95% CI 0.66–1.12) use. The absolute increase in acute kidney injury was 6.5 events per 10 000 person-years. We observed 1 additional case per 1529 patients given fluoroquinolones or per 3287 prescriptions dispensed. The dual use of fluoroquinolones and renin–angiotensin-system blockers had an RR of 4.46 (95% CI 2.84–6.99) for acute kidney injury. Our case-time–control analysis confirmed an increased risk of acute kidney injury with fluoroquinolone use (RR 2.16, 95% CI 1.52–3.18). The use of amoxicillin or azithromycin was not associated with acute kidney injury.

Interpretation: We found a small, but significant, increased risk of acute kidney injury among men with the use of oral fluoroquinolones, as well as a significant interaction between the concomitant use of fluoroquinolones and renin–angiotensin-system blockers.

Fluoroquinolones are commonly prescribed broad-spectrum antibiotics.1 Although highly effective, they are known to cause cardiac arrhythmia, hypersensitivity reactions and central nervous system effects including agitation and insomnia.2,3 Recent reports of tendon rupture4 and retinal detachment5 suggest that these drugs may damage collagen and connective tissue. Case reports of acute kidney injury with the use of fluoroquinolones have been published,6 and the product label includes renal failure in a list of potential, but uncommon, adverse reactions.2 In clinical practice, when oral fluoroquinolones are prescribed, the potential for acute kidney injury is generally not a clinical consideration. We aimed to quantify the risk of acute kidney injury with the use of oral fluoroquinolones among men. This study population was limited to men because the cohort we studied was formed to investigate health issues that affect older men.


Data source

The IMS LifeLink Health Plan Claims Database contains paid claims from US health care plans. Compared with the US Census, the database captures 17% of men aged 45–54 years, 13% of men aged 55–64 years and 8% of men aged over 65 years. Data for men over 65 years are captured through Medicare Advantage programs. These privatized health care plans combine medical and prescription services, providing more inclusive health care data.7

The IMS LifeLink database contains fully adjudicated medical and pharmacy claims for over 68 million patients, including inpatient and outpatient diagnoses (via International Classification of Diseases, 9th revision, clinical modification [ICD-9-CM], codes) in addition to retail and mail-order prescriptions. The data are representative of US residents with private health care in terms of geography, age and sex. The IMS LifeLink database is subject to quality checks to ensure data quality and minimize errors,7 and it has been used in previous pharmacoepidemiologic studies.810

This study was approved by the University of Florida’s Institutional Review Board. All coding used in this study can be found in Appendix 1 (available at

Cohort formation

We used a nested case–control design for our primary analysis. Our cohort was formed to study health issues that affect older men. This population is at the greatest risk of acute kidney injury and is commonly prescribed fluoroquinolones. We extracted data for 2 million men from the IMS LifeLink database who had both prescription and medical coverage. We included men aged 40–85 years who met the inclusion criteria between Jan. 1, 2001, and June 30, 2011, and who had 365 days of enrolment with no acute kidney injury. We excluded men with a history of chronic kidney disease or dialysis because these men may be more prone to acute kidney injury. Censoring was performed at a study outcome, the end of enrollment and end of the study. The cohort was nested within inpatient hospital records, which were used to select cases and controls.

Cases and controls

Multiple studies have validated algorithms to determine acute kidney injury using ICD-9-CM coding. Several were not applicable because they were published only in abstract form,11 included ICD-10-CM coding,12 did not define acute kidney injury at hospital admission,13 included cases before 1990,14 assessed acute kidney injury that occurred after admission to hospital15 or included unspecified (nonacute) renal failure (ICD-9-CM 586.x).16 Two studies validated ICD-9-CM coding against a reference standard that required doubling of serum creatinine and found poor positive predictive values; however, this algorithm does not account for differences in baseline serum creatinine levels.17,18 A second algorithm was developed that identified acute kidney injury based on baseline serum creatinine level: acute kidney injury was defined by a change in serum creatinine of 0.5 mg/dL (44.2 μmol/L) for a nadir serum creatinine of 1.0 mg/dL (88.4 μmol/L) or lower, a change in serum creatinine of 1.0 mg/dL for a nadir serum creatinine between 2.0–4.9 mg/dL (176.8–433.2 μmol/L), or a change in serum creatinine of 1.5 mg/dL (132.6 μmol/L) for a nadir serum creatinine of 5.0 mg/dl (44.2 μmol/L).19 Two studies validated acute kidney injury using ICD-9-CM coding for all hospital discharges against this reference, finding positive predictive values of 80.2%17 and 87.6%.20

We defined acute kidney injury as ICD-9-CM 584.0 (acute renal failure, unspecified), 584.5 (acute tubular necrosis), 584.6 (cortical acute renal failure), 584.7 (medullary acute renal failure), 584.8 (acute renal failure with other specified pathologic lesion) and 584.9 (acute renal failure, not otherwise specified). We further restricted cases to the primary hospital discharge diagnosis, a diagnostic code that identifies the main reason for hospital admission. This is known to increase the positive predictive values and identify the primary reason for admission. We excluded cases if they had been admitted to hospital during the 6 months before the admission for acute kidney injury. Previous hospital admissions could indicate a greater degree of morbidity (confounding by disease severity) and prevent us from measuring prescription use (immeasurable time bias).21 We did not differentiate between subtypes of acute kidney injury because ICD-9-CM coding has not been validated to show this distinction.

We considered men who were admitted to hospital with a diagnosis other than acute kidney injury and who had not been admitted to hospital in the previous 6 months to be eligible for the control group. We used risk-set sampling to select the controls, whereby for each case, a pool of potential controls was formed that met the following criteria: were eligible for matching only on the day of hospital admission; were admitted to hospital within 6 weeks (calendar-time matching); entered the nested cohort no more than 6 weeks apart; and were within 5 years of age. From this risk set, 10 controls, who were still eligible to have an acute kidney injury, were randomly selected and matched for each case. This allows formation of an odds ratio equivalent to the rate ratio (RR).22 Matching on hospital admissions (a strong proxy for health status) was done to provide controls of more similar comorbidity and to reduce residual confounding.

Drug exposure

We included exposure to oral fluoroquinolones: ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin and norfloxacin. We excluded ophthalmic and topical fluoroquinolones because they have minimal systemic absorption. We excluded intravenous fluoroquinolones because our focus was on outpatient-dispensed preparations. We excluded prescriptions dispensed on the day of hospital admission to prevent reverse causality bias.

We defined a current user as someone who had an active supply of fluoroquinolone at hospital admission or had stopped taking a fluoroquinolone (prescription termination; final day of drug supply) in the 1–7 days before admission. Recent users were those who had a prescription termination 8–60 days before admission and had no active supply within the 7 days before admission. We defined past users as those who had a prescription termination 61–180 days before admission and who had no active prescriptions during days 0–60.

We selected 2 common oral antibiotics (amoxicillin and azithromycin) as control drugs. Although both have been implicated in rare cases of interstitial nephritis,2326 we hypothesized that the burden of acute kidney injury with these drugs would be insufficient to produce a positive association.

Statistical analysis

Primary analysis: nested case–control

We used conditional logistic regression to determine the RR for acute kidney injury with fluoroquinolone use. The model was adjusted by fluoroquinolone indication (genitourinary, respiratory or gastrointestinal tract infection; skin infection; and joint or bone infection in the past 6 mo), diseases associated with acute kidney injury (cancer, chronic obstructive pulmonary disease, congestive heart failure, diabetes mellitus, HIV and hypertension in the past year), potentially nephrotoxic drugs with high use (loop diuretics, nonsteroidal anti-inflammatory drugs and renin–angiotensin-system blockers at hospital admission) and markers of health care use (number of medications, billing codes and physician visits in the past 6 mo). We stratified the subsequent analyses by fluoroquinolone product (ciprofloxacin, levofloxacin and moxifloxacin).

We examined drug–drug interactions between fluoroquinolones (current use) and renin–angiotensin-system blockers (at admission) through the addition of an interaction term to our fully adjusted model. We defined renin–angiotensin-system blockers as angiotensin-converting-enzyme inhibitors and angiotensin-receptor blockers. We did not include aldosterone antagonists based on low use and concern for confounding based on the many indications for these medications. Although we hypothesized drug–drug interactions between fluoroquinolones and loop diuretics or nonsteroidal anti-inflammatory drugs, we did not have sufficient power for these analyses. We computed a number needed to harm (absolute risk increase × 100) in which the absolute risk increase equaled the estimated incidence among users (RR × incidence among nonusers) minus the incidence among nonusers.

Secondary analysis: case-time–control

A case-crossover design allows patients to serve as their own controls, using within-patient comparisons of drug exposure to assess the RR for the study outcome.27 This technique has the advantage of having no residual confounding from time-invariant covariates. Two cardinal requirements for a case-crossover study are an acute outcome and a transient exposure. Acute kidney injury is an acute outcome, and fluoroquinolones are typically prescribed for 7–14 days,2 meeting the assumption of transient exposure. Because most fluoroquinolone prescriptions are for 14 or fewer days, we chose the 14 days immediately before admission to hospital as the case-time. Four control-times were selected, each immediately following the previous 14 day window (days 15–28, 29–42, 43–56 and 57–71). We used conditional logistic regression to determine the RR for acute kidney injury with fluoroquinolone exposure. We sensitized the case-crossover by the distribution of fluoroquinolone use from these time windows in the 10 matched control patients from the main analysis. This analysis, referred to as a “case-time–control,” adjusts for a potential trend toward increased use of all antibiotics before hospital admission.28

Sensitivity analysis

We were concerned that patients taking fluoroquinolones would be more likely to have a genitourinary infection (compared with patients taking one of the control medications), which could make them more likely to have acute kidney injury. We conducted a sensitivity analysis in which we removed patients who had experienced a genitourinary infection during the 6 months before admission, and we repeated the study analysis.

Because the sensitivity of excluding people with chronic kidney disease using ICD-9-CM coding is unknown, we repeated our analyses without excluding patients with previous claims for chronic kidney disease; from this analysis, the changes in the study RRs can be used to assess whether residual confounding from unmeasured chronic kidney disease is a potential concern.


Our nested cohort contained 767 209 patients (162 608 hospital admissions) eligible for matching. We identified 1292 cases with acute kidney injury and 12 651 matched controls. The characteristics of the cases and controls are shown in Table 1. Ciprofloxacin (44.5%) and levofloxacin (43.9%) were the most commonly used fluoroquinolones (Table 2); the most common indications were respiratory (45.6%) or genitourinary infections (27.0%) (Table 3).

Table 1:

Characteristics of cases and controls

Table 2:

Use of oral fluoroquinolones among cases and controls

Table 3:

Indication for the use of antibiotics among cases and controls

We observed an increased risk of acute kidney injury with current use of fluoroquinolones (adjusted RR 2.18, 95% CI 1.74–2.73) and no change in risk with either recent (adjusted RR 0.87, 95% CI 0.66–1.16) or past (adjusted RR 0.86, 95% CI 0.66–1.12) use. There was no association between the use of amoxicillin or azithromycin and acute kidney injury (Table 4).

Table 4:

Nested case–control analysis of the risk of acute kidney injury with the use of fluoroquinolones

When we stratified our analysis by fluoroquinolone product, the largest RR was found for ciprofloxacin (RR 2.76, 95% CI 2.03–3.76), followed by moxifloxacin (RR 2.09, 95% CI 1.04–4.20) and levofloxacin (RR 1.69, 95% CI 1.20–2.39). When levofloxacin was used as a reference, ciprofloxacin had a significantly increased RR (RR 1.73, 95% CI 1.08–2.77), whereas moxifloxacin did not (RR 1.20, 95% CI 0.54–2.65).

The case-time–control analysis confirmed the results from the nested case–control study: we found an increased risk of acute kidney injury with fluoroquinolone use (RR 2.16, 95% CI 1.52–3.18) but not with amoxicillin (RR 0.65, 95% CI 0.38–1.05) or azithromycin (RR 1.06, 95% CI 0.62–1.90) (Table 5). The absolute increase in the incidence of acute kidney injury was 6.5 events per 10 000 person-years with use of fluoroquinolones. We observed 1 additional case of acute kidney injury per 1529 patients who used fluoroquinolone or per 3287 prescriptions dispensed.

Table 5:

Case-time–control analysis of the risk of acute kidney injury with the use of fluoroquinolones or other antibiotics

The addition of a drug–drug interaction to the “current use” models for study drugs found similar main effects. Although renin–angiotensin-system blockers can increase serum creatinine levels, we did not find an increased risk of acute kidney injury with renin–angiotensin-system blocker monotherapy (RR 1.00, 95% CI 0.84–1.18). We did find, however, an interaction between the combined use of fluoroquinolones and renin–angiotensin-system blockers (interaction RR 2.19, 95% CI 1.30–3.69). An interaction can be defined as the additional risk for acute kidney injury from the concomitant use of 2 drugs that is beyond the additive risk of each individual drug. This interaction resulted in a greater than fourfold increase in the RR for acute kidney injury (RR 4.46, 95% CI 2.84–6.99) with active use of both drugs. When we analyzed the data by drug class, a similar increased risk was found with the dual use of fluoroquinolones and either angiotensin-converting-enzyme inhibitors (RR 4.54, 95% CI 2.74–7.52) or angiotensin-receptor blockers (RR 3.80, 95% CI 1.72–8.41).

Adjustment for a genitourinary infection had a negligible effect on all point estimates for fluoroquinolone use and acute kidney injury (< 2% change). When we restricted the nested cohort to only patients with no history of genitourinary infection and repeated the nested case–control analysis, we found similar RRs as in the main analysis between fluoroquinolones and acute kidney injury (current use: RR 2.48, 95% CI 1.92–3.23; recent use: RR 0.95, 95% CI 0.65–1.37; past use: RR 0.98, 95% CI 0.75–1.29). When we included patients with a previous claim for chronic kidney disease, we found similar RRs for all user types (current use: RR 2.08, 95% CI 1.67–2.59; recent use RR 0.95, 95% CI 0.73–1.26; past use RR 0.88, 95% CI 0.67–1.13).


We found a twofold increased risk of acute kidney injury with current use of fluoroquinolones. There were nonsignificant associations between fluoroquinolone use and acute kidney injury among recent and past users (point estimates less than 1.0). The twofold differential in risk between current and both recent and past fluoroquinolone use suggests that acute kidney injury is an acute adverse effect of fluoroquinolones. These results were replicated in the case-time–control analysis, which increases our confidence in these associations because of better control of time-invariant confounding.

Previous evidence of acute kidney injury with fluoroquinolone use comes from case reports. Most case reports result from an allergic or hypersensitivity reaction termed acute interstitial nephritis.29,30 Fluoroquinolones have also been reported to cause granulomatous interstitial nephritis, characterized by infiltration of the renal tissue by histiocytes and T lymphocytes, leading to the formation of granulomas.31,32 Crystalluria has been reported to occur when urine pH is above 6.8,33 and several cases of acute kidney injury from crystal formation secondary to fluoroquinolone use have been documented.34,35 More severe cases of acute tubular necrosis have also been linked to fluoroquinolone use.36,37

Although most published case reports are of ciprofloxacin use,6 this may be an artifact of its high use. Nephrotoxicity may not be entirely dependent on renal elimination,6 and one patient with ciprofloxacin-induced nephrotoxicity did not experience a positive rechallenge after switching to ofloxacin.38 We observed a larger risk of acute kidney injury with ciprofloxacin use, compared with the use of levofloxacin; however, this differential finding was not an a priori hypothesis and should be interpreted with caution until further investigation.

Although fluoroquinolones are thought to induce acute kidney injury through acute hypersensitivity reactions, renin–angiotensin-system blockers affect renal hemodynamics through dilation of the efferent arteriole, reducing intra-glomerular pressure and increasing serum creatinine levels.39 The risk of acute kidney injury with the use of renin–angiotensin-system blockers is thought to increase after a superimposed renal insult, such as that with dehydration or the use of other prescription medications.5,6 Physician monitoring of serum creatinine levels, particularly after starting renin–angiotensin-system blocker therapy, and ascertainment of severe cases of acute kidney injury that require admission to hospital may explain the lack of a signal with renin–angiotensin-system blocker monotherapy.


Because of the transient nature of fluoroquinolone use, we used 3 distinct and nonoverlapping definitions of drug exposure, allowing recent and past users to serve as negative controls. We found similar results after removing patients with genitourinary infections from the nested cohort analysis, thereby reducing concerns about confounding by indication.

We used admission to hospital to ascertain cases of severe acute kidney injury; however, we could not assess milder cases that resulted in mild or asymptomatic kidney injury. This could potentially result in an underestimation of the risk of acute kidney injury. We did not have information about the severity of acute kidney injury, nor did we have sufficient power to assess the risk by dosage or duration of use.

Although we conducted a self-controlled analysis, which has implicit control for unmeasured time-invariant confounders, residual confounding, particularly by time-varying covariates, is always a potential concern in observational research.

There is no reason to think that the proposed mechanism for increased risk of acute kidney injury with fluoroquinolone use is specific only to middle-aged and elderly men; however, this limited population is a key limitation of this study. It is possible that these medications may have different associations in other populations, and verifying this will require further study.


We found a twofold increased risk of acute kidney injury requiring hospital admission with the use of fluoroquinolone antibiotics among adult men, using 2 analytic techniques. We did not find increased risk of acute kidney injury with other antibiotics, supporting the hypothesis that this potential adverse association of fluoroquinolones with acute kidney injury is not a class effect of all antibiotics. We found a strong interaction with concomitant use of fluoroquinolones and renin–angiotensin-system blockers, cautioning against the concomitant use of these 2 drug classes. Although it is clear that the risk of death due to serious infections outweighs the risks associated with the use of fluoroquinolones, the potential for acute kidney injury raises the importance of vigilant prescribing.




Can Fluoroquinolones Cause Acute Kidney Injury?

A case-control study suggests a modest, but significant, association.


Case reports have suggested that fluoroquinolone antibiotics occasionally cause acute kidney injury. In this case-control study, researchers used a claims database of 767,000 men (age range, 40–85) to compare fluoroquinolone exposure in 1300 men hospitalized for acute kidney injury (cases) and 13,000 similarly aged men hospitalized for other reasons (controls).

Cases were twice as likely as controls to have received oral fluoroquinolones during the week before hospital admission (8.4% vs. 3.9%); in analyses adjusted for comorbidities, other medications, and indication for fluoroquinolone use, the association between acute kidney injury and current fluoroquinolone use was significant (rate ratio, 2.18). The RR was similar when patients with urinary infections were excluded. In contrast, less-recent fluoroquinolone use and current use of amoxicillin or azithromycin were not associated with acute kidney injury. One additional case of acute kidney injury occurred per 1529 fluoroquinolone users. Excess risk was noted for all three commonly prescribed fluoroquinolones: ciprofloxacin, levofloxacin, and moxifloxacin (Avelox). Combined use of quinolones and renin–angiotensin-system blockers elevated risk further.


Even if this association between fluoroquinolones and acute kidney injury represents cause and effect, the very small absolute risk should not preclude appropriate prescribing of quinolones. But we should keep the association in mind, particularly when a fluoroquinolone-treated patient unexpectedly is feeling poorly.

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