Friday, May 30, 2014

Chemophobia and Feeding the Fear

Just a few days after my post on sun exposure, BuzzFeed decided, irresponsibly, to post a video on the "scary facts about sunscreen".  Not only are they contributing to the whole sunscreen conspiracy, but they're exploiting chemophobia to do it.  Here's one of those "scary facts" about sunscreen:

That was it, no explanation of what retinyl palmitate is, just that it's there.  With a skull and crossbones, and the word CHEMICAL.  Retinyl palmitate is a synthetic form of vitamin A, used to treat people with vitamin A deficiency.  It's added to low-fat dairy products to make up for the loss of vitamin A during the fat-removal process, and it is used in eye drops.

File:Retinyl palmitate.png  File:All-trans-Retinol2.svg
       (retinyl palmitate)                                    (vitamin A)

I guess the "scary" part about retinyl palmitate is that, in 2000, it was selected for phototoxicity and carcinogenic testing (which, in and of itself, is not a red-flag, chemicals in all products are frequently randomly selected for testing).  What was reported from the 11 studies on retinyl palmitate:

  • four of those studies showed that a combination of retinyl palmitate and UVA radiation induced reactive oxygen species
  • four other studies showed that the combination of two can be photomutagenic
  • and 3 of those studies showed that the application of retinyl palmitate on mice altered physiological levels of vitamin A.
On first glance, it makes sense that people have become afraid of this compound.  But, some of these studies were done in vitro, which means they were completed on animal cell cultures, rather than animals themselves.  That means that the whole host of antioxidant defenses present in animals, like non-enzymatic (vitamins C and E) and enzymatic (superoxide dismutase, glutathione reductase, etc.) antioxidants, were not present.  These antioxidant systems work together to counteract the reactive oxygen species that are often formed with antioxidants (like retinyl palmitate) are irradiated with UV light.  Therefore, the 8 in vitro studies really don't tell us anything.  The 3 studies on mice did not report increased skin cancer rates with the application of retinyl palmitate and subsequent irradiation.

But this points to a much larger issue in the public perception of science, and specifically chemistry.  We tend to think that chemicals are bad, and that the presence of any chemical spells danger.  This is known as chemophobia.  Between my degrees, I spent two months working retail selling face creams.  I remember vividly an exchange between myself and a particularly challenging customer: I was trying to explain to her how an anti-wrinkle cream worked, and the word chemical slipped out.  She was horrified that there were chemicals in our all-natural, organic face creams!  I backtracked, explaining that what I meant was that everything was a chemical.  She walked out, and I started substituting the word "compound" for "chemical".

A chemical is any substance with a chemical formula.  Everything is a chemical, even water.  Now, I understand that people do the best they can with the knowledge they have, but chemophobia is going way too far!  A woman in the U.S. went after Subway recently over the use of azodicarbonamide in their breads.  This compound (see, still doing it!) is a common food additive used in the bleaching of flour, and it makes dough elastic.  It is also a "chemical foaming agent" (scare quotes NOT mine) that is used in the making of yoga mats, hence its new name "the yoga mat chemical".  Well, the FDA and the WHO have not found it to cause problems in the general public.  In fact, the WHO only found that it causes respiratory problems and skin irritation in workers who handle enormous volumes of it, but your daily exposure, even if you eat a lot of bread, is not a problem.  Eating bread from Subway is not the same as eating a yoga mat!  One is food, the other isn't.  The far greater problem is not what is added to your processed foods, but what is not found in processed foods, like vitamins, nutrients, etc.

In January, Johnson and Johnson was challenged for the presence of formaldehyde in its No More Tears shampoo.  Formaldehyde is classified as a carcinogen, and its uses include disinfectant, tissue embalming, and photograph development.  The level of formaldehyde in the previous recipe for No More Tears shampoo was actually quite low.  So low that you would need to drink 15 or so bottles of shampoo to get the same level of formaldehyde present in one apple!  But consumers started comparing using No More Tears to dipping your baby in a vat of formaldehyde.

Chemophobia wouldn't bother me so much if it was just about people avoiding certain products because of the presence of "chemicals".  But the problem is, chemophobia is one of the reasons people avoid vaccines and Western medicine.  And that really puts everyone at risk.  I mean, come on:

And companies going along with chemophobia are really just contributing to the misinformation of the general public.  People assume that if you can't pronounce the name of a chemical, then it doesn't belong in your body.  But that's just because we've given a lot of "chemicals" more palatable nicknames.  The chemical name for Vitamin C is ascorbic acid (aka 2-oxo-L-threo-hexono-1,4-lactone-2,3-endiol).  What about allyl isothiocyanate?  That's gotta be bad right, it has cyanide in it!  Well, that's actually the oil responsible for the pungent taste of mustard and horseradish.  

I fully understand the desire to know what is in your food and what you are consuming.  But I think this is more of responsibility on the part of the consumer.  It's important for the public to really try to understand what is dangerous and what isn't, rather than jumping on the fear-mongering bandwagon (see picture above).

Thursday, May 22, 2014

Native Species Have a Hard Time Adapting to Climate Change

Did you know that experts estimate that we could lost one-fourth of the world' species by 2050?  In fact, we're discovering species just now, like the 14 new species of dancing frogs discovered in India, that are at risk of extinction.  Polar bears might disappear in the next 100 years because of melting Arctic ice, coral reefs are really feeling rising sea temperatures, and bird migration patterns are changing.  Climate change is occurring at such a fast rate that species just can't keep up, and this may lead to drastic changes in both flora and fauna in the near future.  One concern is that seed banks, which store and conserve seeds of native and heritage species of plants, may be harboring seeds that are no longer suited for their local environment.

When species cannot adapt quickly enough to a changing climate, this is known as adaptational lag.  There is very little evidence of this phenomenon available right now, but it is predicted that this adaptational lag may be mitigated by the migration of species adapted to warmer, southern climates into the ever-warming north.  That is, these southern species may now be better suited for current and future northern climates than the native northern species.  While it may sound cool to think that we might have palm trees up in Ontario, keep in mind that we'd also likely be losing MANY of our awesome native species in the process, and that's not a good thing.

There have been very few experiments that have looked at the role of specific climate factors in shaping the fitness of local populations.  A group out of Brown University looked at adaptational lag in Arabidopsis thaliana, which naturally inhabits a broad climate space in its Eurasian habitat.

File:Arabidopsis thaliana.jpgArabidopsis is like the lab rat of the plant science world.  It has a short life cycle, a small genome, small size, and produces A LOT of teeny tiny seeds (which get EVERYWHERE when you harvest them!).  And it is really easy to make transgenic versions of these plants.  These factors make it an ideal model plant, and its use in the study of plant genetics, physiology, and biochemistry is widespread.

The authors went to seed banks and found a number of Arabidopsis populations (ecotypes) that are native to different European climates: Finland, the UK, Germany, and Spain.  The authors kept plants under different temperature, humidity, and light conditions, and measured fitness as a measure of silique (those are Arabidopsis pods) length and number.  Basically, they found that each ecotype performed best in its own climate (ie, the Spanish ecotype performed best in the Spanish climate, the German ecotype performed best in the German climate), except for the Finnish ecotype.  While the Finnish ecotype performed its best in the Finnish climate, the German ecotype outperformed it, and the English ecotype was almost just as fit.  And the Spanish ecotype was as fit as the English and German ecotypes in their respective climates.

What this essentially means is that the plants that were native to a relatively southern climate were able to thrive in a warmer northern climate, and in the case of Finland, outperform the native species.  The Finnish ecotype displayed adaptational lag to the warming climate, allowing the German ecotype to thrive.  If this occurred naturally (not in a controlled experiment), the German ecotype might have been an invasive species able to choke out the native species.

We've been seeing the effect of climate change on invasive species for a while now: species like purple loosestrife, zebra mussels, and mosquitoes carrying all kinds of viruses, are examples of this.  Invasive species can be harmful to agriculture, the environment, and public health.  Preventing the spread of invasive species at the expense of native species requires an understanding of which species pose threats to the ecosystem, how they propagate, and, once an invasive species becomes established, how to keep it from spreading.

Saturday, May 17, 2014

Gut Microbes Regulate Weight Gain

Humans carry more bacterial cells (10 times more) than human cells - that means that you are more bacteria than you are human.  It sounds gross, but it's a good thing: these little guys do a lot for us.  Human gut microbes (aka gut flora) help keep the immune system healthy, and help us digest our food and absorb the necessary nutrients.  Microbes in the human intestine process undigested carbohydrates into short-chain fatty acids, which are then metabolized.  You can think of the human gut flora as another organ, because they perform metabolic activities like an organ would.  Gut microbes also exert control over the metabolic functions of other organs, like the liver.  Alterations to the gut flora of humans and mice have been associated with obesity, but the exact mechanism for how microbes influence host metabolism and adiposity (accumulation of fatty tissue), was unknown.  Until now.

A new paper out of a group in Ireland, led by Drs. Susan Joyce and Cormac Gahan, has found a link between the bacterial enzyme bile salt hydrolase (BSH) and host lipid metabolism.  Prior to this study, the authors had outlined the role of BSH, an enzyme limited to species of intestinal microbiota, in gut flora survival - this enzyme is an essential reaction in the metabolism of bile acids, allowing the microbes to tolerate bile, which is crucial to the digestion of fats.  BSH activity is often associated with the bacteria used as probiotics for humans and animals, and is considered to be the contributing factor for their survival in the intestines.  The authors took their characterization of BSH one step further by using mice to test the effect of BSH on microbe-mediated host lipid metabolism.

The mice that were used are what is known as "germ-free" mice, which means that they do not have any bacteria living in or on them (learn more here).  The authors found that increasing the expression of BSH was able to regulate the expression of key genes involved in lipid and cholesterol metabolism, and gut equilibrium.  These processes all play a key role in weight gain in the host.  This figure below shows the weight gain, total body fat, and LDL (bad) cholesterol levels in the test mice, with the mice expressing high levels of BSH (ECBSH1) showing significantly lower levels of each:
a 46% reduction in weight gain compared to the mice colonized with normal E. coli not expressing higher levels of BSH (EC), 60% lower LDL cholesterol, and 36.5% lower liver triglycerides.  Food intake was not diminished.

What is also very interesting is that, through a whole-genome microarray analysis of the tissues of these mice, the authors found that expressing high levels of BSH also influenced the expression of genes involved in circadian rhythm.  To put it simply, circadian rhythm is your body clock.  That means that this gut microbe-specific enzyme is able to influence the genes that regulate sleeping and eating patterns, core body temperature, brain wave activity, hormone production, cell regeneration, and other biological activities.  Now, there are a lot of genes involved in the regulation of circadian rhythm, and BSH increases the expression of two of these in the liver.  But this may be an important means of cross-talk between microbes and their hosts, since alterations in circadian rhythm is also linked to alterations in energy metabolism and weight gain.

The authors posit that this may be an effective way to treat obesity globally, by targeting the human gut flora.  They are currently working toward determining how this system works in humans.

Friday, May 16, 2014

What you need to know about sun exposure

It's that time of year again, the weather is getting warmer and everyone is out in the sunshine doing all kinds of fun stuff, and magazines, public health organizations, and everyone else starts cramping your style by talking to you about sunblock and skin cancer.  I am one of those people, by the way.  I have fair skin, and I got a really nasty full-body sunburn when I was 11 years old - blisters and everything - and since then, I've become really intense about protecting my skin.  My friends tease me about being so pale, wearing long sleeves even on the hottest days, and always sitting in the shade, but in my opinion, that is like teasing me for not smoking or for eating well ("haha, you have a healthy habit, what a fool!" is essentially what it sounds like to me when they tease me).  Anyway... I figured now would be as good a time as any to talk about what UV radiation actually does to your body, how it can cause cancer, and how sunblock works.

What do UV Rays do to Your Body?
UV (ultraviolet) light has a smaller wavelength than the visible light spectrum, in the range of 100-400 nanometers (that's 10-9 meters), and contains a lot of energy.  There are three types of UV rays: UVA (long wave), UVB (mid-wave), and UVC (short wave).  UVA and UVB are the ones we are most often exposed to, which can cause DNA damage, sunburn (technical term: erythema), photoaging, immunosuppression, and cancer.  UV initiates a cascade of events in the skin, starting with the absorption of UV rays by chromophores.  This "excites" the chomophores, which can react with other molecules and form free radicals and Reactive Oxygen Species (ROS).  These are highly reactive molecules, which can interfere with DNA and cause DNA damage.  Free radicals and ROS can move freely in the body, which may explain why some melanomas are often found in parts of the body that don't get a lot of sun exposure.  This is known as indirect DNA damage.  When UV radiation directly interferes with DNA, it is called direct DNA damage.  Direct DNA damage creates bonding of thymine nucleotides, which disrupts the strand and prevents DNA replication.  When this happens, it triggers apoptosis.
File:Indirect DNA damage.png
(indirect DNA damage - UVA)

File:Direct DNA damage.png

                             (direct DNA damage - UVB)          

Responding to DNA Damage
When your body senses UV-induced DNA damage, it triggers a signal cascade, which acts like a relay race in the cell that passes along the message and activates all kinds of responses.  These responses include the induction of DNA repair mechanisms and the production of melanin.  This is a simplification of the signalling response to UV radiation:  

Melanin is a skin pigment that absorbs UV light - diverting it away from those pesky chromophores, and protecting your cells from DNA damage.  That's the reason why people say they need to build a base tan before going on vacation.  People with darker skin naturally have more melanin, while us pale folks have less.  Having less melanin means that your skin is less protected from UV rays, which can cause sunburn.  Tanning is your skin's response to sun damage.

When DNA is damaged, a checkpoint protein, known as tumor protein p53, is expressed.  This protein regulates the cell cycle and functions as a tumor suppressor.  Nucleotide excision repair pathways are also induced, as is the alteration of microRNA expression (these are gene silencers).   The diagram at the right shows an example of a DNA repair pathway:

When these DNA repair pathways shut down or become impaired, as they do with age, it leads to immune suppression, inflammation, and cancer.

There are two types of sunblock: physical (they reflect sunlight) and chemical (they absorb sunlight).  Some sunblocks only protect against UVB rays, which is why health care professionals recommend using a broad-spectrum UVA/UVB sunblock.  There has been some controversy over whether or not sunblocks actually prevent melanomas, but authors of these studies often point to the fact that wearing sunblock may encourage people to participate in risky behaviours, such as laying out in the sun longer, and that people often do not use adequate amounts of sunblock.  On the other hand, seeking shade and wearing protective clothing was associated with lower melanoma risk, but that's likely because people who participate in these behaviours are more conscious about their sun exposure risk.

But what about vitamin D?  Vitamin D is the "sunlight vitamin".  It is produced when UVB photons are absorbed in the skin and photolyze 7-dehydrocholesterol, forming previtamin D3, which is isomerized by the body's temperature into vitamin D.  Most people are dependent on sunlight to obtain adequate amounts of vitamin D, and it is estimated that 50% of the global population is vitamin D deficient.  BUT you can get enough sunlight to produce adequate amounts of vitamin D through casual sun exposure, like during your lunch break.  Laying out in the sun for hours is excessive sun exposure, and is the more damaging equivalent of taking multi-vitamins.

Sun protection is important, especially for children.  Skin cancers are on the rise, and this is a huge public health issue.  But just like anything else that has to do with health, there is a lot of pseudoscience and misinformation out there.  There is no consensus on sunblocks causing cancer, and it is largely believed that they are more beneficial than harmful.  The important thing to remember is that there is not a single health organization that does not recommend the use of sunblock.

Saturday, May 10, 2014

Adding letters to the alphabet: scientists create the first living organism with semi-synthetic genetic code

There have been some really interesting reports in genomics and DNA technology this week!  One in particular, has found a way to expand the DNA "alphabet" of Escherichia coli.

File:Base pair GC.svg
File:AT DNA base pair.pngDNA is made up of four nucleotides, each containing a nitrogenous base, a five-carbon sugar backbone (either ribose or deoxyribose), and at least one phosphate group.  The nucleotides form hydrogen bonds with each other, forming base pairs, specific to their hydrogen bonding capacity.  Adenine (A) binds with Thymine (T), and Cytosine (C) binds with Guanine (G).  This is the basis for every single DNA strand we have ever encountered in any living organism on the planet.  All of your genetic information is encoded by these four nucleotides.

A group out of The Scripps Research Institute in California created an additional pair of unnatural (not found in nature) nucleotides, and expressed them stably in E. coli.  That's actually incredibly complicated, because the unnatural nucleotides have to be present in the cell in a concentration that allows them to be integrated into DNA strands, they have to line up alongside the natural nucleotides so as to not interfere with the structure of the DNA strand, and they have to be recognized by the cell's internal DNA replication machinery.  This is what the two unnatural base pairs (UBP) look like, compared to the the G-C bonding at the bottom of that figure:

The authors describe several ideas they had to ensure adequate amounts of UBP in the cells, but ultimately decided to focus on nucleotide triphosphate transporters (NTT), which transport the substrates, or building blocks, of nucleotides across membranes.  Successful transport across the membranes required that the UBP in the growth medium be stable.  Using High Performance Liquid Chromatography (HPLC), were able to quantify the uptake of the UBP into the cells.  The authors also validated that the UBP could be replicated using different DNA polymerases in vitro, finding that DNA polymerase I was suitable for replication.  But since E. coli typically use DNA polymerase III to replicate their genome, the authors engineered a plasmid to focus replication of UBP using polymerase I.  Just to give you an idea of the scope of this project, so far all of this is only the preliminary part of this paper!  I've never seen a paper with such a long methods section, I'm awed by this work.

The researchers then transformed the E. coli cells to express high amounts of NTT, and grew them in media containing the UBP.  After 15 hours, they looked to see how well the plasmids containing the UBP were replicated - meaning that they verified that the cells not only incorporated the UBP in their genome, but copied the DNA that contained them, with a fidelity of 99.4%!  That means that the unnatural nucleotides are not being removed from the genome through the cell's own DNA repair mechanisms - the only way that the UBP were removed from the genome was when they were not being supplied in the growth medium (which means that if these cells were to escape the lab, the UBP would be unlikely to cause any damage to the natural world).  This technology could eventually be applied to create a platform for synthetic biology with a range of applications, from site-specific labeling of nucleic acids in living cells, to the production and evolution of synthetic proteins with unnatural amino acids.

In fact, I am most interested in future studies of this system that look beyond DNA replication to gene expression!  I'm so curious to find out if and how these cells will translate these unnatural nucleotides into amino acids and how the resulting protein will look and function.

Friday, May 9, 2014

Beetles hijack plant defense mechanisms - but for what?

When I was in my second year of my undergrad, I took a really amazing evolutionary genetics class, and one of the topics we talked about is co-evolution, which is the evolution of a species based on a selective pressure from another species.  Often, predators and prey co-evolve, in a process reminiscent of the Red Queen in Through the Looking Glass: "it takes all the running you can do, to keep in the same place."  It's like an arms race, with one species evolving to stay alive, and the second evolving to beat the first.  In my last post about plants (you know, the one where I geeked out), I mentioned the case of tobacco plants changing their flowering time to attract new pollinators and avoid their common herbivore.  Now, the tobacco plants have placed a selective pressure on their herbivorous caterpillar species, which will eventually find a new plant to eat, or evolve to the pollination schedule of their normal plant.
File:Phyllotreta striolata 01.JPGPlants belonging to the order Brassicales (aka cruciferin plants) produce compounds called glucosinolates (left), or mustard oils, which are responsible for the pungent flavour of mustard, cabbage, and horseradish.  These are secondary metabolites that cruciferins use as defense mechanisms against insects and herbivores.  When insects, like the striped flea beetle (Phyllotetra striolata), damage plant tissues, the released glucosinolates are brought into contact with an enzyme called myrosinase.  This enzyme forms isothiocyanates, which are toxic derivatives of glucosinolates, among other compounds.  This adaptation on the part of the plants against herbivory puts a selective pressure on its herbivore, in this case the striped flea beetle, to adapt to this defense.  Some insects are able to sequester plant defense compounds and use them for their own protection.

A group out of the Max Planck Institute in Germany recently reported that not only have flea beetles adapted to the glucosinolates-myrosinase system of cruciferin plants, but they have actually adapted their own system.  They are able to sequester intact glucosinolates from their host plant during feeding, and break it down by expressing their own myrosinase enzymes.  In fact, these beetles have become so efficient in this system, that they can sequester glucosinolates to levels making up 1.75% of their body weight!

This is the first reported case of a crucifer-feeding beetle with this adaptation to dietary exposure to glucosinolates - typically insects that are able to sequester glucosinolates don't chew on plants, they are sucking insects.  The sucking process does not damage the compartments that separate glucosinolates and myrosinase, and so these insects are able to sequester intact glucosinolates, without needing to express myrosinase enzymes to deal with them.  The mechanism in which the beetles handle the glucosinolates without autointoxication is not yet clear, but the authors suggest that there is likely some cellular compartmentalization at play that keeps the glucosinolate-myrosinase system separate.  It's also possible that these flea beetles use these as a defense mechanism; for example, the cabbage aphid stores and releases glycosinolates as a form of "mustard-oil bomb" when they are attacked.

This study leaves us with a whole host of new questions (as good science does), but it contributes significantly to our knowledge of plant-herbivore co-evolution.

Monday, May 5, 2014

Safety in numbers: bees use social cues to avoid predatory threats

There is safety in numbers.  Anyone who has ever had to walk home solo at night knows how vulnerable we can be when alone.  Animals find safety in numbers too: animals often associate into herds to reduce their probability of being attacked and to more easily spot predators.  But even though we know that this happens, very little is known about the behavioural experiences that contribute to the aggregation of individuals in response to predation.

Pollinators, like bees, also have predators, and have also evolved ways to avoid predation.  Bees are threatened by ambush predators, like the crab spider (Family Thomisidae), which does not build webs, but wait on flowers to, you guessed it, ambush their bee prey.  Some species of crab spiders are able to change their coloring, to camouflage themselves on flowers (below, left).  Others sit on leaves, but look like inconspicuous bird droppings, tricking their prey into a false sense of security (below, right).  But most crab spider ambushes are unsuccessful, and so bees are able to learn from their own experiences to avoid risky-looking flowers.  But avoiding sketchy flowers also has the potential to reduce foraging efficiency, so why would this be an adaptive trait?

File:Crab Spider Thomisus Female 5741.jpg   File:Phyrnarachne sp.jpg        

Well, a new study examined this phenomenon.  Bees use social cues to communicate between individuals in a colony.  For example, honeybees use a “waggle dance” to share information about the direction and distance to areas with lots of pollen and nectar, water sources, or new housing locations.  As it turns out, bumblebees also use social cues, like the “lessons learned” of their bee friends, to identify those flowers that are safe and those that are not.

Erika Dawson and Lars Chittka set up three bumblebee colonies in an artificial meadow where pollen sources were held in false flowers whose colors could be interchanged from white to yellow.  Bees were encouraged to forage freely in order to familiarize themselves with the area, prior to being trained to recognize flower color with either a reward or a predation risk.  One half of the bees were exposed to yellow flowers as the “safe” flowers, while the other half had white flowers as their “safe” flowers, to control for color preference.  The “dangerous” flowers were equipped with foam-coated pincers that could rapidly close to trap, but not harm, bees that landed there.  No other cues were provided other than flower color. 

Following training, the authors exposed the bees to only one flower color.  What they found was that the bees were far more likely to choose a “dangerous” flower to gather pollen if that flower was also occupied by other bees.  Those exposed to their identified “safe” flowers were equally likely to choose a free or occupied flower.  The authors conclude that because bees ignore the “safety in numbers” rule in the absence of a threat, and ignore their own personal experiences in the presence of a threat, that they are actively deciding when to use social information to avoid predation.  

I find this study really interesting, because I love to think about the complex lives of other organisms.  The only thing that stands out here, is that the researchers report really small sample sizes (n = 14 for each of the three treatment groups in the “buddy system” part of the experiment).  Now, I’m not a behavioral ecologist, so I’m not sure if this actually is an appropriate sample size or not, but I would be wary of generalizing these results.  But when taken in the context of learning and social interaction among bees, this study definitely strengthens the body of evidence available to us on the secret lives of bees.  It also shows that bee behavior is not a hard-wired set of rules, but can be adapted based on social cues and habitat conditions.

Sunday, May 4, 2014

Bias in genomics research: "charismatic" organisms prioritized in research

Last month, I wrote a post about cute animal conservation, and our aesthetic bias when it comes to which animals we care about and which we do not.  As it turns out, we have the same bias when it comes to the study of microorganisms!  A recent (open access!) opinion piece in Trends in Ecology & Evolution discusses this bias in our understanding of the genomics of microorganisms, with the majority of the information we have being based on the study of eukaryote genomes.

Organisms are divided into two groups based on their main cell type: prokaryotes and eukaryotes. Prokaryotes lack membrane-bound cellular compartments like mitochondria and nuclei:

Since mitochondria and chloroplasts contain DNA and the tools needed for protein synthesis, divide independently of the rest of the cell, and are able to carry out vital metabolic processes, it is believed (though not by all) that eukaryotic cells formed through a process called endosymbiosis, in which a cell engulfed another and retained its function.  

Endosymbiosis in a nutshellProkaryotes are single-celled organisms, and include two major classifications of life: Bacteria and Archaea.  Archaea are involved in the cycling of elements like carbon, nitrogen, and sulfur; they are not human pathogens.  Eukaryotes, on the other hand, can exist as single-celled or multi-celled organisms.  Members of Eukarya include plants, animals, fungi, protists, etc.  Eukaryotes are also the more complex of the three domains of life (Bacteria, Archaea, and Eukarya).  
The identification of eukaryotes in environmental biodiversity studies use 18S ribosomal DNA (rDNA) as a marker.  18S rDNA refers to the genes that encode the RNA of the small ribosomal subunit of eukaryotes.  For prokaryotic studies, 16S rDNA/rRNA is used.  

Genomic studies are key to explaining the evolution of eukaryotic cells, and yet, the authors argue, a significant amount of eukaryotic genomes are being largely ignored.  Research has been biased towards the study of mutli-cellular eukaryotes and their pathogens, ignoring the potentially genetically-rich single-celled eukaryotes.  This bias comes as a result of our very anthropocentric view of life, meaning we are interested in humans and how the natural work harms or benefits us.  The "big three" kingdoms of life (Metazoa, or animals, fungi, and embryophyta, or land plants) contain 96% of the described and studied eukaryotic species, and 85% of the sequenced genomes, to-date.  Yet they only represent 62% of the available 18S rDNA sequences.  The figure on the left describes this bias, as the majority of described species belong to Metazoa, yet significant portions of 18S rDNA belong to protists.

But the study of organisms belonging to the "big three" is also biased.  A lot of invertebrates are not as well-studied as bacteria, and species that are most useful for human survival, medicine, and agriculture are more frequently studied.  The authors note that this leaves gaps in our understanding of the eukaryotic tree of life, running the risk of neglecting the majority of eukaryotic diversity in future ecological and evolutionary studies.  They conclude their article with a call-to-action to the scientific community to fill in the missing parts of the eukaryote phylogenetic tree.

This week in biology/medicine (April 28-May 5, 2014)


Two new species of spiders have been identified this week: the cartwheeling spider and the peacock spider!  Sweet dreams.

Our garbage made it to the ocean floor before we did. (Open Access)

Pig heart transplants for humans could soon become a reality.

Turns out, you can smell sex and sexuality.

Scientists have developed sperm cells from the skin cells of infertile men.

Paper wasps can recognize each other's faces.

Coffee helps prevent eye damage.

Rising ocean acidity is having a drastic effect on shell formation in crustaceans.

Letting your child play with your iPhone may affect their development.

A non-invasive test for lung cancer is being developed that uses breath analysis.

Thursday, May 1, 2014

Improving genome editing specificity

File:Breeding transgenesis cisgenesis.svg
The main goal of biotechnology and genetic engineering is making changes to an organism's genome to make useful products.  Biotechnology is often used in agriculture, food production, and medicine.  Genetic engineering takes on many forms, including breeding to enhance selected traits and the insertion of genes from a related or unrelated organism.

An important tool in biotechnology is the process of genome editing, where a strand of DNA is inserted, replaced, or removed from a genome using specially designed nucleases, which break the phosphodiester bonds between the nucleotides of DNA.  These nucleases are designed to create double-stranded breaks at a desired location within the genome, triggering the cell's natural DNA repair mechanisms, known as homologous recombination (HR) and non-homologous end joining (NHEJ), which can be harnessed to make targeted gene insertions or deletions (indels).

There are specific families of nucleases that are artificially designed for targeted genome editing: zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR associated (Cas) nucleases.  One Cas nuclease from Streptococcus pyogenes, known as Cas9, is a popular tool in genome editing as an RNA-guided DNA endonuclease that cleaves complementary DNA strands.

CRISPRs provide a kind of record of immunity in bacterial genomes.  Infection with viruses caused the evolution of an adaptive method for silencing viral genes.  CRISPRs are short foreign DNA fragments that are incorporated into the host genome.  Processing of these fragments results in CRISPR-RNAs (crRNA) that form endonuclease-RNA complexes to cleave foreign DNA from invaders, acting like an early immune system.  Cas9 is one of these nucleases, creating double-stranded breaks in target DNA.  Recognizing a specific target DNA, called the protospacer adjacent motif (PAM), is the key to Cas9 activity.

The Cas9 nucleases are simple and monomeric, but have high frequencies of off-target indel mutations, making their use in human therapeutic applications suboptimal.  Enhancing their specificity is crucial to future applications of the CRISPR/Cas system.  There has been a significant amount of work done on dimerization of Cas nucleases to improve specificity.  One such study, published recently in Nature Biotechnology, describes RNA-guided FokI nucleases (RFN) for which dimerization is necessary for genome editing:

This dimerization enhances the specificity of nuclease activity because it depends on the binding of the two guide RNA (gRNA) strands to the DNA with a defined orientation and spacing.  This reduces the likelihood of non-specific cutting because it is unlikely that there are multiple regions within the genome that contain both complementary sequences.  

The improved specificity of the Cas9 nuclease was achieved by fusion of the dimerization-dependent FokI nuclease domain to the inactive Cas9 protein with a five amino acid linker, then co-expressing this system with plasmids containing pairs of gRNA.  This improved the RNA targeting range, and also provided the tools for expressing gRNA from RNA polymerase promoters, thus allowing the opportunity for cell-type specific and/or inducible control of genome editing.  The authors also found that non-specific indels coming from partial mismatches or off-site targeting of the gDNA is negligible when using this method.  The longer a gRNA molecule, the lower the probability of finding an identical sequence somewhere else in the genome.  For example, the authors suggest the use of a 45 base-pair long gRNA in human cells, targeting a specific site in the genome.  There is a low probability of having the same 45 base-pair combination somewhere else in the genome.  Using dimerization and two gRNAs decreases this likelihood even more.  Furthermore, the authors found that PAM-orientation was also key to specificity, with the PAM oriented outward.  This reduces the risk of having non-specific indels made in the genome and improves the use of Cas9 for genome editing in human cells.

Now why would we want to use genome editing in human cells?  Relax, it's not about creating superhumans (though that could be fun!).  Rather, genome editing is used in human cells to target HIV infection and for studying disease genotypes in stem cells, to name a few.

The authors also created a program for scanning the genome of interest to find appropriate RFN target sites.  They have made it freely available here.