Getting to the Heart of the Matter: “Fish are friends, not food”

https://www.flickr.com/photos/shany_410/1125438164

Repeat after me: “Fish are friends, not food”

When most people think about “Finding Nemo,” they likely think about Nemo, the adventurous young clownfish who got caught up in a fishy situation (no pun intended) and ended up in a dentist’s fish tank. Or, they might remember Marlin, the overprotective father. However, what about Dory? Though some might characterize Dory as loopy, she was the real hero who saved Marlin and Nemo’s lives and reunited father and son. Who would have thought that the loopy blue tang fish would be the unsung hero?

https://commons.wikimedia.org/wiki/File:Zebrafish_(26436913602).jpg

Zebrafish (Danio rerio), possibly the key to treat/cure heart disease

Just like Nemo, humans have their own fish hero too: the zebrafish. The zebrafish is a tropical freshwater fish that is currently used in many research labs as a powerful model organism for studying various diseases such as heart disease, cancer, diabetes, and gastrointestinal disease. You might be thinking that there is no way a 3-5 cm fish, with fins and no lungs, can model human disease; humans are extremely different from zebrafish. However, zebrafish are much more similar to people than one might initially think. In fact, according to a paper published in Nature, 84% of genes known to be associated with human disease have a zebrafish counterpart. You might also be wondering why not use mice? After all, they’re at least mammals. Good question!

Zebrafish models actually have many advantages over mouse or rat models:

  1. Zebrafish reproduce more frequently (every week) than mice and rats.
  2. Zebrafish can easily be genetically manipulated to study a disease; it takes less time to generate a zebrafish mutant line than a mouse mutant line.

    https://www.flickr.com/photos/nihgov/29229249924

    Picture above shows zebrafish blood vessels labelled in red and lymphatic vessels labelled in green

  3. In zebrafish, important pathways can be easily blocked and examined by simply adding the drug to the water. This allows for more cost effective and faster identification of new drugs and new uses for current drugs.
  4. The zebrafish is see-through; fluorescence markers can be used to “highlight” various cells in tissues and organs. Imagine being able to see individual cells migrate from one location to the next and form an entire heart that begins to beat 24 hours later- in real time. THAT’S AMAZING!

Other advantages of the zebrafish are disease specific. For example, zebrafish are an advantageous model for studying congenital heart disease because they can survive severe cardiac defects that are typically lethal in mice. Therefore, the zebrafish model allows scientists to follow a disease longer than they would be able to in a mouse model. Zebrafish models of heart failure have been found to exhibit similar defects to those found in patients with heart disease. In addition, zebrafish heart models have provided genetic evidence that certain signaling pathways protect humans against heart problems.

https://www.flickr.com/photos/uwiscseagrant/7250836704

Zebrafish organs or tissues can be easily visualized because they are see-through

One of the major reasons why people succumb to heart attacks is because heart cells get damaged and die; heart cells have little to no capability to regenerate (make more of themselves). The zebrafish heart, on the other hand, has the capability to regenerate and replace injured heart tissue after damage. Therefore, scientists are using the zebrafish to figure out what factors or pathways are involved in that process, so they may be able to help the human heart heal itself after being damaged during a heart attack. Heart disease is the leading cause of death in the U.S. and worldwide; so, many lives can be saved with the help of a little striped fish, like Dory.

With that in mind, it’s probably safe to say that fish are friends (and not just food).

 

Peer edited by Breanna Turman.

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CRISPR-edited Plants and Regulation

https://www.pexels.com/photo/young-tomato-5808/

Pictured above are young tomato plants.  Some vegetable plants (such as corn and  sugar beets) are being genetically engineered to generate tastier and larger quantities of food

If you wanted to get a genetically modified organism (GMO) through the regulatory process, you can expect to dish out about $35.1 million and wait at least five and a half years. This doesn’t even include the money and time it takes to discover and develop a new crop. It’s no wonder then that the agricultural biotechnology industry was historically dominated by big agribusinesses that have had the resources and power to get over this regulatory hurdle. This is changing with the introduction of new techniques such as CRISPR that are cheaper to use and are starting to bypass expensive regulation.

 

Many criticize the existing regulatory process, which was last updated in 1992, as being unreasonable considering the nature of the new technologies that have emerged. The USDA recognizes this and released a statement just last week saying they will not regulate plants developed through genome editing. The responsibility of regulating crop biotechnology is shared between the EPA, FDA and USDA, although the EPA and FDA have not disclosed their stance on new methods of crop modification. The USDA ensures safety to grow a crop, the EPA ensures the environmental safety of crops, usually with pest-resistance genes, and the FDA ensures safety for crop consumption. The USDA explains in their press release that “methods, such as genome editing, expand traditional plant breeding tools because they can introduce new plant traits more quickly and precisely, potentially saving years or even decades in bringing needed new varieties to farmers”. Importantly, these new tools are “indistinguishable from those developed through traditional breeding methods” and therefore do not warrant regulation.

Image Credit: Amala John

The genome can be thought of like a Word document, where the sequence of letters can be edited to modify the meaning of the document to the reader

To understand the differences in these new technologies, imagine entering the genetic code of a plant into a word document like in the figure above. First generation genetic modification of crops in the 90s primarily involved making plants resistant to certain pests or herbicides. This would be analogous to copying DNA sequences from an organism like bacteria and pasting it randomly into your plants’ genome, which allows it to be immune to the effects of certain plant pests. These are the typical GMO corn and soy crops you might be familiar with. They are contentious and highly regulated since the random “pasting” process may disturb genes and they contain foreign DNA. Genome editing, on the other hand, allows us to search through our word document for a specific sequence, make an edit, and let spell-check take over from there. In a plant, we can cut a specific DNA sequence and allow the cells’ repair mechanism to fix the mistake, which is imperfect and normally disables that gene. This process is cleaner and more reliable compared to traditional breeding methods, which is why the USDA sees no need to regulate it. Both of these techniques are a huge step up from traditional breeding, which can involve random mutagenesis and introduction of undesirable genes, which have been shown to happen through the domestication process. Furthermore, newer techniques enable this whole process to be DNA-free, so there is no foreign DNA ever inserted into these edited plants. Critics point to the unintended biological consequences of both of these processes, which is possible but has not been shown to occur in the past 30 years.

Genome editing tools include Zinc-finger nucleases like TALEN, which has been used since the 90s, and CRISPR, which is only a few years old. CRISPR especially has made dramatic advancements in the past few years due to its thrift, ease of use and reliability. Academic labs and biotech start-ups have been developing crops using genome editing and therefore are bypassing costly regulation that they normally would not have been able to afford. Browning-resistant mushrooms, for example, were developed at Penn State and were the first CRISPR-edited crops given the go-ahead for commercialization. High-fiber wheat and high oleic soybeans are currently being developed by a startup called Calyxt using TALEN. These are just some of the crops on the horizon and there are many more that are expected to emerge in the next few years. If the U.S. government doesn’t develop guidelines for proper regulation of these biotech products, they won’t be able to handle the increased rate at which they’re currently being produced. Without unnecessary regulation slowing things down, the future of genome editing in crops looks promising; there is a greater emphasis on developing nutritional foods that consumers desire while also helping farmers grow more efficiently. However, just because these new technologies don’t fit under the current definition of a “GMO” for the government, they should still require some level of oversight appropriate for the technology. Genome editing is changing the landscape of agricultural biotechnology; hopefully, the regulation of these crops follows suit.

Peer edited by Julia DiFiore.

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The Terminator of the Genome

https://www.flickr.com/photos/bagogames/19869372178

The real Terminator

“Listen. Understand. The Terminator is out there. It can’t be reasoned with, it can’t be bargained with… it doesn’t feel pity or remorse or fear…and it will absolutely not stop. Ever. Until you are dead.” In the movie “The Terminator”, the Terminator’s one job was to kill a woman whose son will later hurt his cyborg people. You may wonder what the Terminator has to do with molecular biology but it’s actually quite relevant. We have a ‘terminator’ in our body called p53; its job is to kill rogue cells and prevent cancer from spreading through our bodies at all costs.

p53 works as a gatekeeper in the cells of our body. Just like how gates prevent criminals from getting out when they’re not supposed to, our cells have gates and security to prevent rogue cells, like cancerous cells, from squeezing past checkpoints that make sure cells are normal and healthy. Cells grow, mature and reach a point where they need to divide to make new cells. Each of these steps have checkpoints that make sure the cells are normal and healthy so that the new cells aren’t abnormal or cancerous. Without this security, cancer cells would be running rampant in our bodies. Many cancers are caused by a loss of security in the cell, allowing cancer cells to grow and divide without being stopped at these checkpoints. p53 is one of these gatekeepers. Its function is crucial for cell regulation, cell death and checking cells to make sure they’re not abnormal and cancerous.

When a cell grows and divides, it stops at certain checkpoints in that process to make sure nothing is abnormal and that it is a healthy cell. p53 acts at these checkpoints in the cell to inspect and ensure that the cell isn’t mutated or messed up in any way. If the cell passes inspection, then it moves along and keeps growing and then exits the cycle of growing and dividing. If p53 catches a rogue cell trying to sneak past a checkpoint with mutated DNA or too many chromosomes, it calls in a whole arsenal of molecules called caspases that kill the cell through a process called apoptosis or prevent the cell from making new cells.

Gatekeeper molecules are very important to the cell because they prevent mutated cells from growing and making more mutated cells, eventually leading to cancer or neurodegenetive disease. p53 is activated by cell stress, like external heat or toxins. Stress signals that there is something wrong with the cell division process and that there needs to be more stringent inspection of cells to determine the cause of cell stress and to get rid of it. The problem becomes when gatekeeper molecules themselves are mutated like how border guards can be corrupted or bribed. When p53 in a cell is corrupted, nothing else is checking the cell’s genome to see if it is mutated. These cells get a free pass to divide, thrive and build up in places they shouldn’t be, which is what causes cancer.

 https://commons.wikimedia.org/wiki/File:P53.png

p53 (blue) interacting with DNA (orange)

p53 is mutated in over 50% of cancers like ovarian cancer, breast cancer and colorectal cancer. When working properly, p53 is a tumor suppressor but when it is mutated, is becomes an oncoprotein, a protein that promotes cancer. Two copies of p53 or two gatekeepers are needed in each cell in order for p53 to inspect and shut down rogue cells properly. However, in cancer, one of these copies of p53 is mutated and the other copy is unable to keep up with all the inspections that need to be done. In aggressive cancers, the remaining copy of p53 can become mutated into an oncoprotein and help rogue cells grow, divide and spread to other parts of the body.  

There are various ways that the body tries to prevent mutant p53 from enabling cancer to grow. One of these is through the protein Mdm2 which targets p53 for degradation. When the cell is happy and under low stress, p53 does not need to cause cell death. However, if p53 is mutated it will try to kill the cell. At this point, Mdm2 destroys p53 because it can see that p53 isn’t working properly. Mdm2 is responsive to p53. If p53 levels go up, Mdm2 levels go up as well so that the cell is working properly and is regulated. However, in cancer, Mdm2 can be shut down by other mechanisms so that it can’t shut down p53.

At UNC, Yanping Zhang’s lab studies the Mdm2-p53 pathway. In particular, they study what happens when Mdm2 is mutated and can’t degrade abnormal p53. Thus far, they have found that under conditions of low stress, Mdm2 can be mutated and not have any adverse effects on the cell. However, mice that do not have any Mdm2 were not viable because, left unregulated, p53 constantly killed the cells no matter if they were abnormal or not.

p53 is an amazing protein that works hard to terminate all the abnormal cells in our body. Hopefully, through more study, we can find ways to prevent the mis-regulation of p53 and thus help treat cancers due to p53 mutations.

Peer edited by Samual Honeycutt and Mimi Huang.

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Epigenetics: The Software of the DNA Hardware

https://pixabay.com/en/dna-string-biology-3d-1811955/

Scientists identified the genes of the human genome to understand how the genes influences the function and physical characteristics of human beings.

The Human Genome Project (HGP) was an amazing endeavor to map the full human genome, and so intense an effort that it required an international collaborative research team. One of the ultimate goals of this project was to shed light on human diseases and find the underlying genes causing these health issues. However, the HGP ended up creating more questions than answering them. One thing we found out is that most diseases are complex diseases, meaning that more than one gene causes the disease. Obesity is one such example of a complex disease. This is in contrast to cystic fibrosis which is a disease caused by a mutation in a single gene. To further complicate diseases, there are gene and environment interactions to consider. A gene-environment interaction is a situation in which environmental factors affect individuals differently, depending on their genotype or genetic information. The possible number of gene-environment interactions involved in complex diseases is daunting, but the HGP has given us the information necessary to start better understanding these interaction.

Although the HGP did not end up giving us the answers we were looking for, it pointed us in the direction we needed to take. We needed to consider the role of environmental factors on human health and disease. For not only are complex diseases not fully explained by genetics alone, but another aspect of these diseases remains unexplained by genetics: the health disparities seen within diseases like obesity, diabetes, cancer, etc. The HGP showed us that not all diseases are caused by single mutations and that genetic diversity does not explain the differences in health outcomes. The environment plays a big part.

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Epigenome  is the software that controls gene expression to make the different cells that make up the human body.

Gene-environment interactions begin to answer why genetics alone cannot explain  varying health outcomes by considering that the environment may have varying effects on our genetic data. However, there is another dimension to our genetic background that could better answer why genes often can’t be mapped directly to a disease. Imagine that our genome is our computer hardware, with all the information necessary to create the cells we are composed of. However, something needs to configure the genome to differentially express genes so as to make skin and heart cells from the same DNA. Skin and heart cells have the same information (DNA) in their nucleus but only express what’s necessary to function as a skin or heart cell. In other words, software is needed for the hardware. For us, that software is the epigenome. The epigenome consists of a collection of chemical compounds, or marks, that tell the genome what to do; how to make skin and heart cells from the same information. The epigenome, unlike the genome, is flexible. It can change at key points in development and even during the course of one’s lifetime. This flexibility makes the epigenome susceptible to environmental factors and could explain: (1) Why our genome alone cannot explain the incidences of diseases such as obesity, (2) the health disparities within these complex diseases, and (3) the transgenerational inheritance of complex diseases like metabolic syndrome, defined as a cluster of conditions such as high blood pressure and high blood sugar that increase your risk for heart disease and diabetes.

Now of course, the more we find out the more questions are left unanswered. As stated before, the epigenome can change due to lifestyle and environmental factors which can prompt chemical responses. However, the mechanisms by which things like diet and smoking induce these chemical responses is unclear. But researchers have started to fill in the gap. For example, certain types of fats, like polyunsaturated fatty acids (corn oil is high in these), can generate highly reactive molecules and oxidative stress, which can cause epigenetic alterations. Tobacco smoke contains a mixture of chemicals that have been independently investigated with mixed results on the epigenetic effects. Psychological stress, more specifically child abuse, has been seen to cause increased methylation (a sort of mark on the genome) of a receptor for hormones responsible for metabolism (glucocorticoid receptor) in suicide victims. This has also been seen in mouse models where higher maternal care of pups decreased methylation of the glucocorticoid receptor. Increased methylation usually decreases the expression of the glucocorticoid receptor, and decreased methylation would increase the glucocorticoid receptor’s expression.

The HGP was an amazing endeavor of science and has given us amazing insight into the structure, organization, and function of the complete set of human genes. It has also helped point us in a new direction to better understand chronic diseases and seek to find the solutions to address the burden of disease.

Peer edited by Mejs Hasan and Emma Hinkle.

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Cloned Monkeys: Another Human Creation

http://english.cas.cn/head/201801/t20180123_189488.shtml Image credited to Qiang Sun and Mu-ming Poo, Institute of Neuroscience of the Chinese Academy of Sciences

First cloned none-human primates: Zhong Zhong and Hua Hua (Image credited to Qiang Sun and Mu-ming Poo, Institute of Neuroscience of the Chinese Academy of Sciences)

Cloned primates are here! Over three decades have passed since the birth of Dolly, the sheep, scientists have now tackled cloning mammals that are even closer to us on the evolutionary tree: macaque monkeys. What does this mean for a society that witnesses dramatic changes day by day: computers are outperforming doctors in calling out heart abnormalities in patients; 3D-printed organs are bringing us one step closer to tissue restoration; genome sequencing has become an online product easily available for anyone curious about their ancestry, bodybuilding, or just simply wine tastes. Breakthroughs in science and technologies are so prevalent in our life that by now, we probably shouldn’t be surprised by any new discovery. Yet when the two cute, little, cloned monkeys were born, the whole world was, once again, shaken.

Published in Cell on January 24th, 2018, a study from a group of scientists in China reported their methods in generating two non-human primates that are genetically identical. To clone the two identical macaque monkeys, the scientists applied Somatic Cell Nuclear Transfer, the same method that generated Dolly in 1996. The key idea behind cloning is that a new organism, be it sheep or monkey, is generated without sexual reproduction. Asexual reproduction is not as uncommon as one would think, plenty of plants do so. For example, Bryophyllum shed plantlets from the edge of the leaves to produce new plants. Some insects, such as ants and bees, also exploit asexual reproduction to clone a huge working class army. Since asexual reproduction is essentially an organism duplicating itself, the offsprings are all genetically identical. Evolution, however, doesn’t favor asexual reproduction as identical offsprings don’t prevail in a fast changing environment. On the other hand, sexual reproduction combines different sperms and eggs to create diverse offsprings, of which some may survive. To combat challenges from the mother nature, higher organisms, such as mammals, almost exclusively reproduce sexually. This is why a cloned monkey, an anti-evolution human creation, is mind blowing.

https://commons.wikimedia.org/wiki/File:Kalanchoe_plantlets.JPG

The succulent, genus Kalanchoe, uses asexual reproduction to produce plantlets.

To clone mammals, scientists came up with the idea of transferring the nucleus of a somatic cell to an enucleated egg (an egg that lacks nucleus). Unlike  germ cells (sperm and eggs), somatic cells refer to any cells that don’t get passed onto the next generation. These cells have the full genome of an organism that is split equally in germ cells during sexual reproduction. Carrying half of the genome, sperm and egg need to fuse their genetic materials to make one viable embryo. Technically, the nucleus of a somatic cell holds all the genetic information an organism needs. Thus, by inserting the somatic cell nucleus into an egg, scientists could generate a functional embryo. But why not into a sperm? Evolution has trimmed mammalian sperm tremendously so that it can accomplish its only job better: swim faster to fertilize the egg. As a result, not much other than the sperm’s genetic information is incorporated into the fertilized egg and the embryo relies on the cellular machinery from the egg to finish development. Using this technology, the scientists generated over 300 “fertilized” embryos. Of these embryos, 260 were transferred to 63 surrogate mothers to finish developing. 28 surrogate mothers became pregnant, and from those pregnancies, only 2 healthy monkey babies were born. Although they were carried by different surrogate mothers, every single piece of their genetic code is the same as the the somatic nucleus provider, a real-life demonstration of primate-cloning. Followed by millions of people since their debut to the world, these two macaque superstars are the living samples of a revolutionary breakthrough in our science and technologies.

 

Despite the extremely low success rate, this technology erects another monument in the history of mankind’s creations. Carrying identical genetic information, cloned monkeys like these two can be a very powerful tool in biomedical research and diseases studies. Co-author Mu-ming Poo, director of the Chinese Academy of Sciences’ Institute of Neuroscience in Shanghai, said that these monkeys could be used to study complicated genetic diseases where environmental factors also play a significant role, such as Alzheimer’s and Parkinson’s diseases. While there are ethical concerns on this technology and its easy application to human cloning, it is worth noting that almost all human creations (explosives, GMO food, the internet, etc.) are double-sided swords. It is up to the hand that wields this sword to decide whether to do good or bad. It is wise to be cautious with the development of new technologies, but it’s also important not to constrain our creativity. After all, it is our creative minds that drive us toward creating a better life for everyone.

Peer edited by Cherise Glodowski.

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Cambridge Researchers use Mouse Embryonic Stem Cells to Grow Artificial Mouse “Embryos”

Let’s start at the very beginning. When a mammalian egg is successfully fertilized by a single sperm, the result is a single cell called a zygote. A zygote has the potential to grow into a full-bodied organism. It is mind-boggling that this single cell, containing the genetic material from both parents, can divide itself to make two cells, then four cells, then eight cells, and so on, until it becomes a tiny ball of 300-400 stem cells.

https://upload.wikimedia.org/wikipedia/commons/3/3c/Stem_cells_diagram.png

Early Development and Stem Cell Diagram, modified by author to include ESC and TSC labels.

At these early stages, these stem cells are totipotent, meaning that they have the potential to become either embryonic stem cells (ESCs), which will eventually become the fetus itself, or extraembryonic trophoblast cells (TSCs), which go on to help form the placenta. That ball of ESCs and TSCs develops into a blastocyst with a tight ball of ESCs on the inside, and a layer of TSCs on the outside (See Figure 1).

You might imagine the blastocyst as a pomegranate, with the seeds representing the ESCs and the outer skin representing the TSCs. The ESCs have the potential to transform, or differentiate, into any type of cell in the entire body, including heart cells, brain cells, skin cells, etc., which will ultimately become a complete organism. The outer layer TSCs have the ability to differentiate into another type of cell that will ultimately attach itself to the wall of the uterus of the mother to become the placenta, which will provide the embryo with proper nutrients for growth. 

Scientists in the field of developmental biology are absolutely bonkers over this early stage of embryogenesis, or the process of embryo formation and development. How do the cells know to become ESCs or TSCs? What tells the ESCs to then differentiate into heart cells, or brain cells, or skin cells? What signals provide a blueprint for the embryos to continue growing into fully-fledged organisms? The questions are endless.

The challenge with studying embryogenesis is that it is incredibly difficult to find ways to visualize and research the development of mammalian embryos, as they generally do all of their growing, dividing, and differentiating inside the uterus of the mother. In recent years, there have been multiple attempts to grow artificial embryos in a dish from a single cell in order to study the early stages of development. However, previous attempts at growing artificial embryos from stem cells face the challenge that embryonic cells are exquisitely sensitive and require the right environment to properly coordinate with each other to form a functional embryo.

Enter stage right, several teams of researchers at the University of Cambridge are successfully conducting groundbreaking research on how to grow artificial mouse embryos, often called embryoids, in a dish.

In a paper published in Development last month, David Turner and colleagues in the Martinez-Arias laboratory report a unique step-by-step protocol developed in their lab that uses 300 mouse ESCs to form tiny balls that mimic early development.

https://www.biorxiv.org/content/early/2016/05/13/051722

Mouse embryonic stem cell aggregates with polarized gene expression in a dish (4 days in culture). Image courtesy of authors.   doi.org/10.11.01/051722.

These tiny balls of mouse ESCs are collectively termed “Gastruloids” and are able to self-organize and establish a coordinate system that allows the cells to go from a ball-shape to an early-embryo shape with a head-to-tail axis. The formation of an axis is a crucial step in the earliest stages of embryo development, and it is exciting that this new model system may allow scientists to better study the genes that are turned on and off in these early stages.

In a paper published in Science this past April, Sarah Harrison and her team in the Zernicka-Goetz laboratory (also at Cambridge) report another technique in which mouse ESCs and TSCs are grown together in a 3D scaffold instead of simply in a liquid media. The 3D scaffold appears to give the cells a support system that mimics that environment in the uterus and allows the cells to assemble properly and form a blastocyst-like structure. Using this artificial mouse embryo, the researchers are attempting to simulate the growth of a blastocyst and use genetic markers to confirm that the artificial embryo is expressing the same genes as a real embryo at any given stage.

The researchers found that when the two types of stem cells, ESCs and TSCs, were put together in the scaffold, the cells appear to communicate with each other and go through choreographed movement and growth that mimics the developmental stages of a normal developing embryo. This is enormously exciting, as models like this artificial embryo and the Gastruloid have the potential to be used as simplified models to study the earliest stages of embryo development, including how ESCs self-organize, how the ESCs and TSCs communicate with each other to pattern embryonic tissues, and when different genetic markers of development are expressed.

It is important to note that this artificial embryo is missing a third tissue type, called the endoderm, that would eventually form the yolk sac, which is important for providing blood supply to the fetus. Therefore, the artificial embryo does not have the potential to develop into a fetus if it is allowed to continue growing in the dish. The fact that these artificial embryos cannot develop into fully-fledged organisms relieves some of the controversial ethical issues of growing organisms in a dish, and will allow researchers to study critical stages of development in an artificial system.   

These techniques and discoveries developed by these teams of researchers have the potential to be applied to studies of early human development. These models may prove especially useful in studying how the maternal environment around the embryo may contribute to fetal health, birth defects, or loss of pregnancy. In the future, artificial embryos, coupled with the not-so-futuristic gene editing techniques that are currently in development to fix disease genes, may prove key in the quest to ensure healthy offspring. 

Peer Edited by Nicole Smiddy and Megan Justice.

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Get Alternative with Epigenetics

Our bodies are marvels of precise control, synchronization and design. Every one of our cells has the same genetic sequence, but we have many different types of cells – heart, muscle, lung, skin. Amazingly, our body has a mechanism to determine which cell is which even though they all share the same code. The field of epigenetics dives into this phenomenon. Epigenetics is a study of changes to DNA that does not change the actual sequence but modify it by repressing or activating certain parts of DNA. In short, epigenetics can reversibly turn genes on and off without changing the DNA sequence.

The genes in our body are like words that have to be spelled a certain way in order for them to work properly. All genes are made up of “base” molecules, which are assigned a specific letter (A, C, G, or T). These bases combine to form 3-letter “words,” or amino acids.  Amino acids serve as the “words” that form the “sentences” or proteins in our body that govern all the biological processes necessary for life. However, none of these biological phenomena could be produced if there are misspellings in the genetic code. Mutations are a misspelling of the original genetic code through deleting, duplicating, substituting or inverting parts of a gene. Mutations are permanent changes to the DNA code which can be passed on from generation to generation. This is the cause of many heritable diseases.

For a long time, genetic changes were thought to be permanent, but reversible epigenetic changes were uncovered around 1950 and have led to an explosion of knowledge in understanding the human body. Conrad Waddington was the first scientist to propose the concept of epigenetics. He studied embryonic development and saw how an embryo gave rise to all the different types of cells, even though every cell had the same genetic sequence. He visualized this model with “Waddington’s landscape,” which used the analogy of a marble rolling down a hill into different troughs to represent the developing cell becoming a muscle cell, heart cell or any other cell.

https://upload.wikimedia.org/wikipedia/commons/5/54/Paisagem_epigenetica.jpg

The marble example that Waddington used to describe an embryonic stem cell becoming other cells.

Alternative splicing is one epigenetic mechanism that allows for cells to be able to choose multiple fates. This can happen all over the body, such as in the brain, heart, and muscle. Our body has many genes, but we only use 2% of those genes to code for proteins, the other 98% are genes that help regulate the protein-coding genes. Alternative splicing is one way that we fully utilize the 2% of our genes that code for protein and accounts for our complexity. Splicing allows for the “word” of one gene to be broken up into many different ways to make many other genes. The word “lifetime” can be broken up into ‘life’ and ‘time,’ but can also be rearranged to make the words ‘fit,’ ‘lie,’ and ‘tile.’ The parts of protein-coding genes can be also be broken down and mixed and matched to produce different proteins. The sites for splicing are determined by the tightness of DNA, the accessibility to DNA, and other epigenetic factors that are still being actively researched.

Emma Hinkle

An example of how alternative splicing can produce different protein products.

Dr. Jimena Guidice at the University of North Carolina at Chapel Hill is actively investigating the epigenetics of alternative splicing in the heart to try to determine why certain heart diseases cause the heart to revert back to fetal alternative splicing as opposed to adult alternative splicing. A few weeks postnatal, the muscle cells needed to contract the heart are not yet mature and have a different alternative splicing pattern to facilitate growth into adult muscle cells. Eventually, the muscle cells are spliced with a different alternative splicing pattern which is a mark of adult muscle cells since these cells are large and can pump blood to the heart more efficiently.

If you’re interested in reading more about epigenetics and its history, I highly recommend Nessa Carey’s Epigenetics Revolution and Siddhartha Mukherjee’s The Gene.  

Peer edited by Deirdre Sackett.       

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One in a Million: The Importance of Cellular Heterogeneity and the Power of Single Cell Sequencing

One of the most overwhelming aspects of modern-day biomedical research is the overarching heterogeneity that consumes all realms of biology. Ranging from cell to cell to human to human, we have become increasingly aware of the important differences that drive divergent responses to therapeutics and biological stimuli.  The complexity of cancer is one such example.   

A landmark paper published by Gerlinger et al. in The New England Journal of Medicine demonstrated that analyzing multiple biopsies from a single patient’s tumor gives a much different picture of what the biology driving that tumor is, compared to examining a single biopsy alone. In addition, many studies characterizing the cellular heterogeneity of cancer have revealed that a tumor is much more than a mass of identical cells all growing out of control. Rather, a tumor is comprised of cancer cells at different stages of the cell cycle, engaged in different cell signaling pathways, as well as other cell types including immune cells and endothelial cells (the cells lining blood and lymphatic vessels).  

https://vimeo.com/157377539

Figure 1: A schematic showing the multiple biopsy sites in a single patient tumor. The study found that the number of private mutations (i.e., a mutation found only at a single biopsy site and not at any of the other sites) are immense, suggesting that looking at a single biopsy alone greatly undermines the mutational diversity of a given patient’s tumor.

New technologies are emerging to enable us to better dissect the heterogeneity of disease and biology.  One such technique is single cell sequencing. Sequencing is a technique with which we are able to get a readout of the entire genetic composition of a cell. In its most basic form, sequencing can be performed to get a readout of DNA or RNA, which are two types of molecules  involved in different levels of genetic regulation.  Sequencing has traditionally been performed on bulk samples comprised of hundreds to millions of cells in aggregate. While we have made remarkable advances in our understanding of biology using bulk sequencing, the emergence of single cell sequencing has now allowed us to begin to  probe the genetic profile of a single cell at varying levels of complexity. The technology often takes advantage of molecular indexing, which is a technique whereby individual mRNA or DNA molecules are labeled with a molecular tag that is associated with a single cell, and then the cells are all pooled together into one tube and sequenced. During the post-sequencing analysis, the molecular tags are re-associated with each single cell, and then the profiles of each of the single cells are compared to one another.

This novel technique has allowed us to begin investigating and understanding biology with a higher degree of resolution than we ever could have imagined before, and will undoubtedly lead to the discovery of many new exciting realms of biological regulation. For example, the biomedical company Becton and Dickson have performed a research study analyzing single cells from tumors of mouse models of cancer. They found that within each tumor sample, there were distinct populations of cells with unique gene expression profiles, and these profiles were associated with vastly different biological functions. Understanding how these different populations work together as a community to promote tumor growth may help us better understand how to develop new treatments for cancer.

However, like all cutting-edge technologies, there are still limitations that need to be overcome. One concern is inefficient collection of sample, because the amount of genetic material in a single cell is much smaller than the amount of material from a large group of cells. An additional confounding variable is uncertainty of whether a low yield of genetic information from a single cell is the result of technical error, inefficiency of small sample collection, or simply the lack of expression of a gene in that particular cell. Discerning the difference between a true, biological negative result and simply a technical deficiency are often difficult to parse out.

Especially in fields such as cancer biology, we have increasingly begun to realize that heterogeneity has largely been an obstacle in our ability to develop effective therapies and to truly understand the mechanisms of regulation of biological processes. Advances in single cell sequencing research have allowed us to further realize that there is not one function or process driving disease progression, but rather a network of cells with distinct roles. Uncoupling the forces dictating the progression of this heterogeneity is what will help us to make the next great advances in therapeutic development in cancer and many other diseases.

Peer edited by Chiungwei Huang and Richard Hodge.

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H What N What? A Designer Protein Hits the Science Runway

Image ID: 10073 https://phil.cdc.gov/phil/details.asp

TEM Image of Influenza Virion. Content Providers: CDC/ Erskine. L. Palmer, Ph.D.; M. L. Martin, 1981.  Photo Credit: Frederick Murphy

Influenza is a virus that straddles two worlds: that of the past and that of the future. Responsible for more deaths than HIV/AIDS in the past century, the flu is one of the world’s’ most dangerous infectious diseases though it may not seem so, especially in the United States. However, the flu is responsible for millions of cases of severe illness and approximately 250,000 to 500,000 deaths worldwide each year.

Influenza Pandemics
Influenza A and B circulate each flu season, but it is the emergence of new influenza A strains that have been responsible for worldwide epidemics, or pandemics, in the past such as the 1918 ‘Spanish Flu’ pandemic and the 2009 H1N1 pandemic. There are 2 ways a new influenza virus can emerge. Every time the virus replicates, small genetic changes occur that result in non-identical but similar flu viruses: this is called “antigenic drift”. If you get infected with a certain flu virus, or get a vaccine targeting a certain flu virus, your body develops antibodies to that virus. With accumulating changes, these antibodies won’t work against the new changed virus, and the person can be infected again. The other source of change is “antigenic shift”, which results in a virus with a different type of hemagglutinin and/or neuraminidase, such as H3N2 to H1N1. The 2009 H1N1 virus is cited by some as a result of antigenic shift, because the virus was so different than previous H1N1 subtypes; however, as there was no change in the actual hemagglutinin or neuraminidase proteins, it was technically a case of antigenic drift.

Image ID: 13469 https://phil.cdc.gov/Phil/details.asp

This diagram depicts how the human cases of swine-origin H3N2 Influenza virus resulted from the reassortment of two different Influenza viruses. The diagram shows three Influenza viruses placed side by side, with eight color-coded RNA segments inside of each virus. The virus from the 2009 pandemic (right) has HA/NA proteins and RNA from Eurasian and North American swine instead of from humans like in previous years (left two viruses).
Content Provider: CDC/Douglas Jordan, M.A. 2011.

 

Challenges in Studying the Flu
Scientists and policymakers face many challenges when studying the influenza virus. For instance, the virus can be transmitted among people not showing symptoms and cough, sneeze, or handshake can spread infectious droplets from someone who doesn’t know they’re sick. Scientific study is further complicated by the virus itself: there are 3 antigenic types of influenza virus that infect humans, (A, B, C), with various subtypes and strains. Each year, government agencies work with scientists to decide which strains to target in that year’s vaccine manufacturing. The lag time between production in the spring and the flu season in the winter provides time for unexpected types to emerge.

Image ID: 17345 https://phil.cdc.gov/Phil/details.asp

This is a 3D illustration of a generic Influenza virion’s fine structure. The panel on the right identifies the virion’s surface protein constituents. Content Provider: CDC/ Douglas Jordan, Dr. Ruben Donis, Dr. James Stevens, Dr. Jerry Tokars, Influenza Division. 2014. Illustrator: Dan Higgins.

The H#N# nomenclature for influenza A subtypes refers to the hemagglutinin (H) and neuraminidase (N) proteins that sit on the surface of the virus. There are 18 types of hemagglutinin and 11 types of neuraminidase. Hemagglutinin aids the virus in fusing with host cells and emptying the virus’ contents inside. Neuraminidase is an enzyme embedded in the virus membrane that facilitates newly synthesized viruses to be released from the host cells to spread the infection from one cell to another.

Targeted Therapy
In studying ways to prevent and battle influenza, research scientists have focused their efforts on blocking the actions of neuraminidase and hemagglutinin. Antiviral drugs, such as oseltamivir (Tamiflu®) and zanamivir (Relenza®), bind neuraminidase, both interact with neuraminidase at sites crucial for its activity. The drugs act to render the virus incapable of self-propagating. A computational biologist at the University of Washington in Seattle, David Baker and his team know the hemagglutinin protein well. In 2011, they utilized nature’s design by studying antibodies that bind hemagglutinin in order to design a protein that targets the glycoprotein’s stem in H1 subtype flu viruses and prevent the virion from infecting the host cell. However, antiviral resistance contributed to by antigenic drift, is a serious issue. Researchers much constantly develop new drugs to keep up with changes in the virus.

David Baker and his team now focus their research on the hemagglutinin protein. Utilizing a computational biology approach, they designed a protein that fits snugly into hemagglutinin’s binding sites. They tested their designer protein on 10 mice and found that in mice exposed to the H3N2 influenza virus, their protein worked both as a preventative measure and as a treatment.  Though there is a long road to human testing, this binding protein shows promise for bedside influenza diagnosis as well as a model for possible treatments.

Want to know more? 

Image ID: 8675 https://phil.cdc.gov/phil/details.asp

This photograph depicts a microbiologist in what had been the Influenza Branch at the Centers for Disease Control and Prevention (CDC) while she was conducting an experiment.  Content Provider: CDC/Taronna Maines. 2006. Photo Credit: Greg Knobloch.

Learn how scientists monitor circulating influenza types and create new vaccines each year.

See flu activity and surveillance efforts with the CDC’s FluView and vaccination trends for the United States using the FluVaxView.

 

Peer edited by Richard Hodge and Tyler Farnsworth.

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Science and Ethics

So let’s say, hypothetically, that your lab receives blood samples from a group of individuals to study genetic links with diabetes.  However, these samples would also provide important insights into other diseases.  But the researchers did not get consent from the blood samples donors for the extra research.  For researchers at Arizona State University (ASU) and the University of Arizona (U of A), this was not a hypothetical situation.  

https://www.flickr.com/photos/neeta_lind/3572379084

DNA from blood samples provide the information needed to potentially cure many diseases that plague us today.  But if the proper procedure is not followed, these scientific breakthroughs may never leave the courtroom.

They collected 400 blood samples from the Havasupai Tribe around 1990 to understand if there was any connection between genes and diabetes, at the tribe’s request. This particular tribe is from an isolated area of the Grand Canyon, with a restricted gene pool contributing to genetic diseases.  This Native American tribe has a high-incidence with diabetes.  The researchers did investigate this problem with diabetes, but they also wrote a grant proposal for researching schizophrenia in the Havasupai Tribe, which the tribe was not aware of nor gave consent for.

The main issues raised in this case are:

  • What is informed consent?  In this case, the consent form stated that the samples were to be used for studies on behavioral and medical diseases. But, meetings between the researchers and tribe members indicated that only diabetes was to be studied.  Using broad or vague language in consent forms can lead to miscommunication between scientists and subjects.
  • What information in the medical records can be accessed and by who?  Some researchers gained access to medical records without permission. Files should be kept in a secured place where only the authorized users have access.
  • Who has control of the samples?  This is a question that needs to be discussed with the subjects before samples are collected.  Researchers might want to contact their university’s research center for more information on sample ownership.

 

As scientists, we have a set of standards, or ethics, that help members coordinate their actions and establish trust with the public. Below are four ethical norms (or goals) that affect graduate students:

https://upload.wikimedia.org/wikipedia/commons/d/d9/March_for_Science%2C_PDX%2C_2017_-_29.jpg

Scientists build and maintain credibility with the public by conducting research responsibly and with integrity.

  1. Promote the goals of scientific discovery, such as furthering knowledge and truth.
  2. Advocate collaboration between scientists; diversity and collaboration create new and novel discoveries that we can all benefit from.
  3. Promote accountability to the Public; it’s essential that the Public can trust the scientists to do their best work and avoid misconduct, conflicts of interest, and ensure that human/animal subjects are properly handled.
  4. Build Public support, without federal funding many of us graduate students would not be able to do our research.

For the misuse of their DNA samples, the  Havasupai Tribe filed a lawsuit against Arizona Board of Regents and ASU researchers in 2004, which eventually led to a settlement in 2010.  The tribe received $700,000 and their blood samples were returned.  The situation with ASU and U of A researchers has left an air of mistrust in Native American communities.  As scientists, it’s our responsibility to build trust with the public and maintain open and honest communication.  

 

Peer Edited by Bailey DeBarmore

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