3D printed model of Cas9 from CRISPR

We need to talk about CRISPR

By Gesa Junge, PhD

You’ve probably heard of CRISPR, the magic new gene editing technique that will either ruin the world or save it, depending on what you read and whom you talk to? Or the Three Parent Baby, which scientists in the UK have created?

CRISPR is a technology based on a bacterial immune defense system which uses Cas9, a nuclease, to cut up foreign genetic material (e.g., viral RNA). Scientists have developed a method by which they can modify the recognition part of the system, the guide RNA, and make it specific to a site in the genome that Cas9 then cuts. This is often described as “gene editing” which allows disease-causing genes to be swapped out for healthy ones.

CRISPR is now so well known that Google finally stopped suggesting I may be looking for “crisps” instead, but the real-world applications are not so well worked out yet, and there are various issues around CRISPR, including off-target effects, and also the fact that deleting genes is much easier than replacing them with something else. But, after researchers at Oregon Health and Science University managed to change the mutated version of the MYBPC3 gene to the unmutated version in a viable human embryo last month, the predictable bioethical debate was reignited, and terms such as “Designer Babies” got thrown around a lot.

A similar thing happened with the “Three Parent Baby,” an unfortunate term coined to describe mitochondrial replacement therapy (MRT). Mitochondria, the cells’ organelles for providing energy, have their own DNA (making up about 0.2% of the total genome) which is separate from the genomic DNA in the nucleus, which is the body’s blueprint. Mitochondrial DNA can mutate just like genomic DNA, potentially leading to mitochondrial disease, which affects 1 in 5000-10000 children. Mitochondrial disease can manifest in various ways, ranging from growth defects to heart or kidney to disease to neuropsychological symptoms. Symptoms can range from very mild to very severe or fatal, and the disease is incurable.

MRT replaces the mutated mitochondrial DNA in a fertilized egg or in an embryo with the healthy version provided by a third donor, which allows the mitochondria to develop normally. The UK was the first country to allow the “cautious adaption” of this technique.

While headlines need to draw attention and engage the reader for obvious reasons, oversimplifications like “gene editing” and dramatic phrases like “three parent babies” can really get in the way of broadening the understanding of science, which is difficult enough as it is. Research is a slow and inefficient process that easily gets lost in a 24-hour news cycle, and often the context is complex and not easily summed up in 140 characters. And even when the audience can be engaged and interested, the relevant papers are probably hiding behind a paywall, making fact checking difficult.

Aside from difficulties communicating the technicalities and results of studies, there is also often a lack of context in presenting scientific studies - think for example of chocolate and red wine which may or may not protect from heart attacks. What is lost in many headlines is that scientific studies usually express their results as a change in risk of developing a disease, not a direct causation, and very few diseases are caused by one chemical or one food additive. On this topic, WNYC’s “On The Media”-team have an issue of their Breaking News Consumer Handbook that is very useful to evaluate health news.

The causation vs. correlation issue is perhaps a little easier to discuss than big ethical questions that involve changing the germline DNA of human beings because ethical questions do not usually have a scientific answer, let alone a right answer. This is a problem, not just for scientists, but for everyone, because innovation often moves out of the realm of established ethics, forcing us to re-evaluate it.

Both CRISPR and MRT are very powerful techniques that can alter a person’s DNA, and potentially the DNA of their children, which makes them both promising and scary. We are not ready to use CRISPR to cure all cancers yet, and “Three Parent Babies” are not designed by anyone, but unfortunately, it can be hard to look past Designer Babies, Killer Mutations and DNA Scissors, and have a constructive discussion about the real issues, which needs to happen! These technologies exist; they will improve and eventually, and inevitably, play a role in medicine. The question is, would we rather have this development happen in reasonably well-regulated environments where authorities are at least somewhat accountable to the public, or are we happy to let countries with more questionable human rights records and even more opaque power structures take the lead?

Scientists have a responsibility to make sure their work is used for the benefit of humanity, and part of that is taking the time to talk about what we do in terms that anyone can understand, and to clarify all potential implications (both positive and negative), so that there can be an informed public discussion, and hopefully a solution everyone can live with.

 

Further Reading:

CRISPR:

National Geographic

Washington Post

 

Mitochondrial Replacement Therapy:

A paper on clinical and ethical implications

New York Times (Op-Ed)

 


A lego Gollum crouches over a metal ring

One ring to rule them all: The cohesin complex

By Johannes Buheitel, PhD

In my blog post about mitosis (http://www.myscizzle.com/blog/phases-of-mitosis/), I explained some of the challenges a human cell faces when it tries to disentangle its previously replicated chromosomes (for an overview of the cell cycle, see also http://www.myscizzle.com/blog/cell-cycle-introduction/) and segregate them in a highly ordered fashion into the newly forming daughter cells. I also mentioned a protein complex, which is integral for this chromosomal ballet, the cohesin complex. To recap, cohesin is a multimeric ring complex, which holds the two chromatids of a chromosome together from the time the second sister chromatid is generated in S phase until their separation in M phase. This decreases complexity, and thereby increases the fidelity of chromosome segregation, and thus, mitosis/cell division. And while this feat should already be enough to warrant devoting a whole blog post to cohesin, you will shortly realize that this complex also performs a myriad of other functions during the cell cycle, which really makes it "one ring to rule them all".

Figure 1: The cohesin complex. The core complex consists of three subunits: Scc1/Rad21, Smc1, and Smc3. They interact to form a ring structure, which embraces ("coheses") sister chromatids.
Figure 1: The cohesin complex. The core complex consists of three subunits: Scc1/Rad21, Smc1, and Smc3. They interact to form a ring structure, which embraces ("coheses") sister chromatids.

But let’s back up a little first. Cohesin's integral ring structure is composed of three proteins: Smc1, Smc3 (Structural maintenance of chromosomes), and Scc1/Rad21 (Sister chromatid cohesin/radiation sensitive). These three proteins attach to each other in a more or less end-to-end manner, thereby forming a circular structure (see Figure 1; ONLY for the nerds: Smc1 and -3 form from long intramolecular coiled-coils by folding back onto themselves, bringing together their N- and C-termini at the same end. This means that these two proteins actually interact with their middle parts, forming the so-called “hinge”, as opposed to really “end-to-end”). Cohesin obviously gets its name from the fact that it causes “cohesion” between sister chromatids, which has been first described 20 years ago in budding yeast. The theory that the protein complex does so by embracing DNA inside the ring’s lumen was properly formulated in 2002 by the Nasmyth group, and much evidence supporting this “ring embrace model” has been brought forth over last decades, making it widely (but not absolutely) accepted in the field. According to our current understanding, cohesin is already loaded onto DNA (along the entire length of the decondensed one-chromatid chromosome) in telophase, i.e. only minutes after chromosome segregation, by opening/closing its Smc1-Smc3 interaction site (or “entry gate”). When the second sister chromatid is synthesized in S phase, cohesin establishes sister chromatid cohesion in a co-replicative manner (only after you have the second sister chromatid, you can actually start talking about “cohesion”). Early in the following mitosis, in prophase to be exact, the bulk of cohesin is removed from chromosome arms in a non-proteolytic manner by opening up the Smc3-Scc1/Rad21 interface (or “exit gate”; this mechanism is also called "prophase pathway"). However, a small but very important fraction of cohesin molecules, which is located at the chromosomes’ centromere regions, remains protected from this removal mechanism in prophase. This not only ensures that sister chromatids remain cohesed until the metaphase-to-anaphase transition, but also provides us with the stereotypical image of an X-shaped chromosome. The last stage in the life of a cohesin ring is its removal from centromeres, a tightly regulated process, which involves proteolytic cleavage of cohesin’s Scc1/Rad21 subunit (see Figure 2).

Figure 2: The cohesin cycle. Cohesin is topologically loaded onto DNA in telophase by opening up the Smc1-Smc3 interphase ("entry gate"). Sister chromatid cohesion is established during S phase, coinciding with the synthesis of the second sister. In prophase of early mitosis, the bulk of cohesin molecules are removed from chromosome arms (also called "prophase pathway") by opening up the interphase between Scc1/Rad21 and Smc3 ("exit gate"). Centromeric cohesin is ultimately proteolytically removed at the metaphase-to-anaphase transition.
Figure 2: The cohesin cycle. Cohesin is topologically loaded onto DNA in telophase by opening up the Smc1-Smc3 interphase ("entry gate"). Sister chromatid cohesion is established during S phase, coinciding with the synthesis of the second sister. In prophase of early mitosis, the bulk of cohesin molecules are removed from chromosome arms (also called "prophase pathway") by opening up the interphase between Scc1/Rad21 and Smc3 ("exit gate"). Centromeric cohesin is ultimately proteolytically removed at the metaphase-to-anaphase transition.

As you can see, during the 24 hours of a typical mammalian cell cycle, cohesin is pretty much always directly associated with the entire genome (the exceptions being chromosomes arms during most of mitosis, i.e. 20-40 minutes and entire chromatids during anaphase, i.e. ~10 minutes). This means that cohesin has at least the potential to influence a whole bunch of other chromosomal events, like DNA replication, gene expression and DNA topology. And you know what? Turns out it does!

Soon after cohesin was described as this guardian of sister chromatid cohesion, it also became clear that there is just more to it. Take DNA replication for example. There is good evidence that initial cohesin loading is already topological (meaning, the ring closes around the single chromatid). That poses an obvious problem during S phase: While DNA replication machineries (“replisomes”) zip along the chromosomes trying to faithfully duplicate the entire genome in a matter of just a couple of hours, they encounter – on average – multiple cohesin rings that are already wrapped around DNA. Simultaneously, cohesin's job is to take those newly generated sister chromatids and hold them tightly to the old one. Currently, we don’t really know how this works, whether the replisome can pass through closed cohesin rings, or whether cohesin gets knocked off and reloaded after synthesis. What we do know, however, is that cohesion establishment and DNA replication are strongly interdependent, with defects in cohesion metabolism causing replication phenotypes and vice versa.

Cohesin has also been shown to have functions in transcriptional regulation. It was observed quite early that cohesin can act as an insulation factor, blocking long-range promoter-enhancer association. Today we have good evidence showing that cohesin binds to chromosomal insulator elements that are usually associated with the CTCF (CCCTC-binding factor) transcriptional regulator. Here, the ring complex is thought to help CTCF's agenda by creating internal loops, i.e. inside the same sister chromatid!

Studying cohesin has, of course, not only academic value. Because of its pleiotropic functions, defects in human cohesin biology can cause a number of clinically relevant issues. Since actual cohesion defects will cause mitotic failure (which most surely results in cell death), most of cohesin-associated diseases are believed to be caused by misregulation of the complex's non-canonical functions in replication/transcription. These so-called cohesinopathies (e.g. Roberts syndrome and Cornelia de Lange syndrome) are congenital birth defects with widely ranging symptoms, which usually include craniofacial/upper limb deformities as well as mental retardation.

It is important to mention that cohesin also has a very unique role in meiosis where it not only coheses sister chromatids but also chromosomal homologs (the two maternal/paternal versions of a chromosome, each consisting of two sisters, which themselves are cohesed). As a reminder, the lifetime supply of all oocytes of a human female is produced before puberty. These oocytes are arrested in prophase I (prophase of the first meiotic division) with fully cohesed homologs and sisters, and resume meiosis one by one each menstrual cycle. This means that some oocytes might need to keep up their cohesion (between sisters AND homologs) over decades, which, considering the half-life of your average protein, can be challenging. This has important medical relevance as cohesion failure is believed to be the main cause behind missegregation of homologs, and thus, age-related aneuploidies, like e.g. trisomy 21.

After twenty years of research, the cohesin complex still manages to surprise us regularly, as new functions in new areas of cell cycle regulation come to light. Currently, extensive research is conducted to better understand the role of certain cohesin mutations in cancers such as glioblastoma, or Ewing's sarcoma. And while we're still far away from completely understanding this complex complex, we already know enough to say that cohesin really is "one ring to rule them all".

 


End Crisis, Bridges and Scattered Genes: Chromatin Bridges and their Role in Genomic Stability

By Gesa Junge, PhD

Each of our cells contains about two meters of DNA which needs to be stored in cells that are often less than 100uM in diameter, and to make this possible, the DNA is tightly packed into chromosomes. As the human cell prepares to divide, the 23 pairs of chromosomes neatly line up and attach to the spindle apparatus via their middle point, the centrosome. The spindle apparatus is part of the cell’s scaffolding and it pulls the chromosomes to opposite ends of the cell as the cell divides, so that every new daughter cell ends up with exactly one copy of each chromosome. This is important; cells with more or less than one copy of a chromosome are called aneuploid cells, and aneuploidy can lead to genetic disorders such as Down Syndrome (three copies of chromosome 21).

In some cancer cells, chromosomes with two centromeres (dicentric chromosomes) can be detected, which can happen when the ends of two chromosomes fuse in a process called telomere crisis. Telomeres are a sort of buffer zone at the ends of the chromosome which consist of repeats of non-coding DNA sequences, meaning there are no genes located here. As one of the DNA strands is not replicated continuously but in fragments, the telomeres get shorter over the lifespan of a cell, and short telomeres can trigger cell cycle arrest before the chromosomes get so short that genetic information is lost. But occasionally, and especially in cancer cells, chromosome ends fuse and a chromosome becomes dicentric. Then it can attach to the spindle apparatus in two points and may end up being pulled apart as the two daughter cells separate, sort of like a rope tied to two cars that drive in opposite directions. This string of chromosome is referred to as a chromatin bridge.

Researchers at Rockefeller University are studying these chromatin bridges and what their relevance is for the health of the cell. A paper by John Maciejowski and colleagues found that the chromatin bridges actually stay intact for quite a long time. Chromosomes are pretty stable, and so the chromatin bridges lasted for an average of about 9 hours (3-20h) before snapping and quickly being pulled back into the original cell (see video). Also, the nucleus of the cell was often heart-shaped as opposed to the usual round shape, which suggests that the chromatin bridge physically pulls on the membrane surrounding the nucleus, the nuclear envelope. Indeed, proteins that make up the nuclear envelope (e.g. LAP2) were seen on the chromatin bridge, suggesting that they take part of the nuclear envelope with them as they divide.  Also, cells with chromatin bridges had temporary disruptions to their nuclear envelope at some point after the bridge was resolved, more so than cells without chromatin bridges.

The chromatin bridges also stained positive for replication protein A (RPA), which binds single stranded DNA. DNA usually exists as two complementary strands bound together, and the two strands really only separate to allow for DNA to be copied or transcribed to make protein. Single-stranded DNA is very quickly bound by RPA, which stabilises it so it does not loop back on itself and get tangled up in secondary structures. The Rockefeller study showed that a nuclease, a DNA-eating enzyme, called TREX1 is responsible for generating the single-stranded DNA on chromatin bridges. And this TREX1 enzyme seems to be really important in resolving the chromatin bridges: cells that do not have TREX1 resolve their chromatin bridges later than cells that do have TREX1.

So how are chromatin bridges important for cells, the tissue and the organism (i.e. us)? The authors of this study suggest that chromatin bridges can lead to a phenomenon called chromothripsis. In chromothripsis, a region of a chromosome is shattered and then put back together in a fairly random order and with some genes facing the wrong direction. Think of a new, neatly color-sorted box of crayons that falls on the floor, and then someone hastily shoves all the crayons back in the box with no consideration for color coordination or orientation. Chromothripsis occurs in several types of cancers, but it is still not really clear how often, in what context and exactly how the genes on a chromosome end up in such a mess.

According to this study, chromothripsis may be a consequence of telomere crisis, and chromatin bridges could be part of the mechanism: A chromosome fuses ends with another chromosome and develops two centromeres. The dicentric chromosome attaches to two opposite spindles and is pulled apart during cell division, generating a chromatin bridge which is attacked by TREX that turns it into single-stranded DNA, the bridge snaps and in the process the DNA scatters, and returns to the parent cell where it is haphazardly reassembled, leaving a chromothripsis region.

The exact mechanisms of this still need to be studied and the paper mentions a few important discussion points. For example, all the experiments were performed in cell culture, and the picture may look very different in a tumor in a human being. And what exactly causes the bridge to break? Also, there are probably more than one potentially mechanism linking telomere crisis to chromothripsis. But it is a very interesting study that shines some light on the somewhat bizarre phenomenon of chromothripsis, and the importance of telomere crisis.

Reference: Maciejowski et al, Cell. 2015 Dec 17; 163(7): 1641–1654.

 

 


The Phase That Makes The Cell Go Round

 

By Johannes Buheitel, PhD

 

There comes a moment in every cell's life, when it's time to reproduce. For a mammalian cell, this moment usually comes at a ripe age of about 24 hours, at which it undergoes the complex process of mitosis. Mitosis is one of the two main chromosomal events of the cell cycle. But in contrast to S phase (and also to the other phases of the cell cycle) it's the only phase that is initiated by a dramatic change in the cell's morphology that, granted, you can't see with your naked eye, but definitely under any half-decent microscope without requiring any sort of tricks (like fluorescent proteins): Mitotic cells become perfectly round. This transformation however, as remarkable as it may seem, is merely a herald for the main event, which is about to unfold inside the cell: An elegant choreography of chromosomes, which crescendoes into the perfect segregation of the cell's genetic content and the birth of two new daughter cells.

 

To better understand the challenges behind this choreography, let's start with some numbers: A human cell has 23 unique chromosomes (22 autosomes and 1 gonosome) but since we're diploid (each chromosome has a homolog) that brings us to a total of 46 chromosomes that are present at any given time, in (nearly) every cell of our bodies. Before S phase, each chromosome consists of one continuous strand of DNA, which is called a chromatid. Then during S phase, a second "sister" chromatid is being synthesized as a prerequisite for later chromosome segregation in M phase. Therefore, a pre-mitotic cell contains 92 chromatids. That's a lot! In fact, if you laid down all the genetic material of a human cell that fits into a 10 micrometer nucleus, end to end on a table, you would wind up having with a nucleic acid string of about 2 meters (around 6 feet)! The challenge for mitosis is to entangle this mess and ultimately divide it into the nascent daughter cells according to the following rules: 1) Each daughter gets exactly half of the chromatids. 2) Not just any chromatids! Each daughter cell requires one chromatid of each chromosome. No more, no less. And maybe the most important one, 3) Don't. Break. Anything. Sounds easy? Far from it! Especially since the stakes are high: Because if you fail, you die (or are at least pretty messed up)...

 

Anatomy of a mitotic chromosome
Anatomy of a mitotic chromosome

To escape this dreadful fate, mitosis has evolved into this highly regulated process, which breaks down the high complexity of the task at hand into more sizable chunks that are then dealt with in a very precise spatiotemporal manner. One important feature of chromosomes is that its two copies – or sister chromatids – are being physically held together from the time of their generation in S phase until their segregation into the daughter cells in M phase. This is achieved by a ring complex called cohesin, which topologically embraces the two sisters in its lumen (we'll look at this interesting complex in a separate blog post). This helps the cell to always know, which two copies of a chromosome belong together, thus essentially cutting the complexity of the whole system in half, and that before the cell even enters mitosis.

Actual mitosis is divided into five phases with distinct responsibilities: prophase, prometaphase, metaphase, anaphase and telophase (cytokinesis, the process of actually dividing the two daughter cells, is technically not a phase of mitosis, but still a part of M phase). In prophase, the nuclear envelope surrounding the cell's genetic content is degraded and the chromosomes begin to condense, which means that each DNA double helix gets neatly wrapped up into a superstructure. Think of it like taking one of those old coiled telephone receiver cables (that's your helix) and wrapping it around your arm. So ultimately, chromosome condensation makes the chromatids more easily manageable by turning them from really long seemingly entangled threads into a shorter (but thicker) package. At this point each chromatid is still connected to its sister by virtue of the cohesin complex (see above) at one specific point, which is called the centromere. It is this process of condensation of cohesed sister chromatids that is actually responsible for the transformation of chromosomes into their iconic mitotic butterfly shape that we all know and love. While our butterflies are forming, the two microtubule-organizing centers of the cell, the centrosomes, begin to split up and wander to the cell poles, beginning to nucleate microtubules. In prometaphase, chromosome condensation is complete and the centrosomes have reached their destination, still throwing out microtubules like it’s nobody’s business. During this whole time, their job is to probe the cytoplasm for chromosomes by dynamically extending and collapsing, trying to find something to hold on to amidst the darkness of the cytoplasm. This something is a protein structure, called the kinetochore, which sits on top of each sister chromatid's centromere region. Once a microtubule has found a kinetochore, it binds to it and stabilizes. Not all microtubules will bind to kinetochores though, some of them will interact with the cell cortex or with each other to gradually form the infamous mitotic spindle, the scaffold tasked with directing the remainder of the chromosomal ballet. Chromsomes, which are attached to the spindle (via their kinetochores) will gradually move (driven by motor proteins like kinesins) towards the middle region of the mother cell and align on an axis, which lies perpendicular between the two spindle poles. This axis is called the metaphase plate and represents a visual mark for the eponymous phase. The transition from metaphase to anaphase is the pivotal moment of mitosis; the moment, when sister chromatids become separated (by proteolytic destruction of cohesin) and subsequently move along kinetochore-associated microtubules with the help of motor proteins towards cell poles. As such a critical moment, the metaphase-to-anaphase transition is tightly safeguarded by a checkpoint, the spindle assembly checkpoint (SAC), which ensures that every single chromatid is stably attached to the correct side of the spindle (we’ll go into some more details in another blog post). In the following telophase, the newly separated chromosomes begin to decondense, the nuclear envelope reforms and the cell membrane begins to restrict in anticipation of cytokinesis, when the two daughter cells become physically separated.

 

Overview over the five phases of mitosis.
Overview over the five phases of mitosis (click to enlarge).

To recap, the process of correctly separating the 92 chromatids of a human cell into two daughter cells is a highly difficult endeavor, which, however, the cell cleverly deals with by (1) keeping sister chromatids bound to each other, (2) wrap them  into smaller packets by condensation, (3) attach each of these packets to a scaffold a.k.a. mitotic spindle, (4) align the chromosomes along the division axis, so that each sister chromatid is facing opposite cell poles, and finally (5) move now separated sister chromatids along this rigid scaffold into the newly forming daughter cells. It's a beautiful but at the same time dangerous choreography. While there are many mechanisms in place that protect the fidelity of mitosis, failure can have dire consequences, of which cell death isn't the worst, as segregation defects can cause chromosomal instabilities, which are typical for tissues transforming into cancer. In future posts we will dive deeper into the intricacies of the chromosomal ballet, that is the centerpiece of the cell cycle, as well as the supporting acts that ensure the integrity of our life's code.

 


How to Live Long and Prosper - a Vulcan's Dream

 

By Jesica Levingston Mac leod, PhD

 

A new Harvard study found that we are living longer and better, too. In fact, the life expectancy for a 65 year old in USA grew a lot in the last 20 years: the life expectancy for females is now 81.2 years and for males it's 76.4 years. The 3 pillars of this improvement are the less smoking, healthier diet and the medical advances. Going straight into the deep science latest developments, two start ups (BioViva and Elysium Health) were in the news recently for their cutting-edge “anti-aging” approaches. The first group to research  telomeres gene therapy is Maria Blasco's group. A study by Bernardes de Jesus et al. demonstrated how telomerese gene therapy in adult and old mice could delay aging and increase longevity, without the collateral effect of increasing the propensity of developing cancer.

In the study, the scientists showed how the treatment of 1- and 2-year old mice with an adeno associated virus expressing mouse telomerase reverse transcriptase (TERT) had beneficial effects on health and fitness, with an increase in median lifespan of 24% and 13%, respectively. Some other benefits included better insulin sensitivity, reduced osteoporosis, improved neuromuscular coordination and improvements in several molecular biomarkers of aging. In cancer cells, the expression of the telomerase is enhanced, giving this protein a bad reputation as having a “tumorigenic activity”. Elizabeth Parrish, the CEO of BioViva, went all the way to Colombia, to receive two gene therapies that her company had developed: one to lengthen the telomeres and the other to increase muscle mass. The results of the treatment were very positive: the telomeres in leukocytes grew from 6.71 kb to 7.33 kb in seven months. As a side note, petite leukocyte telomere length may be associated with several psychiatric disorders (including major depressive disorder) and with poor response to psychiatric medications in bipolar disorder and schizophrenia.

In a nutshell, human telomeres are composed of double-stranded repeat arrays of “TTAGGG” terminating in a single-stranded G-rich overhang. The fidelity of that sequence is maintained by the enzyme telomerase, which uses an intrinsic RNA molecule containing the CAAUCCCAAUC template region and the reverse transcriptase component (TERT), to synthesize telomeric DNA de novo onto the chromosome terminus. The telomeres were named after the greek words télos (end, extremity) and méros (part). Take home message: Telomerase adds DNA to the ends of telomeres and by lengthening telomeres, it extends cellular life-span and/or induces immortalization. The telomerase is not active in normal somatic cells while active only in germ-line, stem and other highly proliferative cells.

 

Last year, Dr. Fagan and collaborators, published in PLoS One that the transcendental meditation and lifestyle variations stimulate two genes that produce telomerase (hTERT and hTR). Even cheerier news were reported in Nature for thanksgiving: the edible dormouse (super cute, small, long tail mouse - Glis glis) telomere length significantly increases from an age of 6 to an age of 9 years. As they state in the paper "the findings clearly reject the notion that there is a universal and inevitable progressive shortening of telomeres that limits the number of remaining cell cycles and predicts longevity".  These species of mouse skip reproduction in years with low food availability, this “sit tight” strategy in the timing of reproduction might pushed "older" dormouse to reproduce, and this could facilitate telomere attrition, this strategy may have led to the evolution of increased somatic maintenance and telomere elongation with increasing age.

The other company, Elysium, co-founded by MIT professor Lenny Guarente, is focus in the mitochondria and the NAD (nicotinamide adenine dinucleotide). Mitochondria are our energy generators and they get crumbly as we age. Dr. Guarente demonstrated in mice how it may be possible to reverse mitochondrial decay with dietary supplements that increase cellular levels of NAD, like nicotinamide riboside (NR, a precursor to NAD that is found in trace amounts in milk), resveratol (a red wine ingredient) or pterostilbene (present in berries and grapes). Elysium has just realized the results of the clinical trial that was placebo-controlled, randomized, and double-blinded, where they evaluated the safety and efficacy of BASIS (the diateary supplement with nicotinamide riboside (NR) and pterostilbene) in 120 healthy participants ages 60-80 over an eight-week period. Participants received either the recommended dose (250 mg NR and 50 mg pterostilbene) or double the dose. In both cases, the intake of Basis resulted in the increase of NAD+ levels in the blood safely and sustainably, 40% and 90% respectively.

 

A former Guarante's postdoc -  Dr. Sinclair - has just published in Science the discovery of a NAD binding area in a protein that regulate NAD's interactions with other proteins related to aging. The Sinclair's lab reported that the binding of NAD+ to DBC1 (Deleted in Breast Cancer 1 protein) prevents it for inhibiting another protein -  PARP1, an important DNA repairing protein. Furthermore, they have shown that as the mice aged, the concentration of NAD+ decreased, and more DBC1 was available to bind to PARP1, culminating in the accumulation of DNA damage. On a brighter note, this process was reversed by restoring higher levels of NAD+. The good news are that NAD+modulation might protect against cancer, radiation and aging.

 

Although all these advances are great, they won’t make you live longer in the next 10 years, so what can you do to live longer/healthier? Science comes again to answer this question! Harvard studies have shown that living “meaningful lives” helping others, having aims/motivations (and been conscious about the fact that we are taking our own decisions), been grateful, enjoying the present and significant relationships with other humans are key aspects to have a happy live. Obviously, exercising and having natural environments around us, as well as healthy eating are crucial points in a healthy life.

It might be an oversimplification, but 70% of your risk of disease is related to diet: soda and processed food are related with shortening the telomeres. Good news: you can slow down aging with a healthier life style: “Switch to a whole-food, plant-based diet, which has been repeatedly shown not just to help prevent the disease, but arrest and even reverse it” claims Dr. Greger’s, author of the Daily Dozen—a checklist of the foods we should try to consume every day. The super food list includes: Cruciferous vegetables (such as broccoli, Brussels sprouts, cabbage, cauliflower, kale, spring greens, radishes, turnip tops, watercress), Greens (including spring greens, kale, young salad greens, sorrel, spinach, swiss chard), other vegetables (Asparagus, beetroot, peppers, carrots, corn, courgettes, garlic, mushrooms, okra, onions, pumpkin, sugar snap peas, squash, sweet potatoes, tomatoes), beans (Black beans, cannellini beans, black-eyed peas, butter beans, soyabeans, baked beans, chickpeas, edamame, peas, kidney beans, lentils, miso, pinto beans, split peas, tofu, hummus),  Berries: (including grapes, raisins, blackberries, cherries, raspberries and strawberries),  other fruit (such as apples, apricots, avocados, bananas, cantaloupe melon, clementines, dates, figs, grapefruit, honeydew melon, kiwi, lemons, limes, lychees, mangos, nectarines, oranges, papaya, passion fruit, peaches, pears, pineapple, plums, pomegranates, prunes, tangerines, watermelon),  Flax seeds, nuts, spices (like turmeric), whole grains (Buckwheat, rice, quinoa, cereal, pasta, bread) and the almighty: water.

As you can expect, a lot of research is needed to get a magic pill that might boost your life expectancy but you can start investing in your future having a positive attitude, healthy diet, exercising and all the other things that you already know you should be doing to feel better, without forgetting that life is too short, so eat dessert first.

 


Can we reprogram adult cells into eggs?

 

By Sophie Balmer, PhD

 

Oogenesis is the female process necessary to create eggs ready for fertilization. Reproducing these keys steps in culture constitutes a major advance in developmental biology. Last week, a scientific group from Japan amazingly succeeded and published their results in the journal Nature. They replicated the entire cycle of oogenesis in vitro starting from adult skin cells. Upon fertilization of these in vitro eggs and transfer in adult females, they even obtained pups that grew normally to adulthood providing new platforms for the study of developmental biology.

 

Gamete precursor cells first appear early during embryonic development and are called primordial germ cells. These precursors then migrate to the gonads where they will remodel their genome via two rounds of meiosis to produce either mature oocytes or sperm depending on the sex of the embryo. For oocyte maturation, these two cycles occur at different times: the first one before or shortly after birth and the second one at puberty. The second round of meiosis is incomplete and the oocytes remain blocked in metaphase until fertilization by male gametes. This final event initiates the process of embryonic development, therefore closing the cycle of life.

 

Up until last week, parts of this life cycle were reproducible in culture. For years, scientists have known how to collect and culture embryos, fertilize them and transfer them to adult females to initiate gestation. This process called in vitro fertilization (IVF) has successfully been applied to humans and has revolutionized the life of millions of individuals suffering specific infertility issues and allowing them to have babies. However only a subset of infertility problems can be solved by IVF.

Additionally, in 2012, the same Japanese group recreated another part of the female gamete development: Dr. Hayashi and colleagues generated mouse primordial germ cells in vitro that once transplanted in female embryos recapitulated oogenesis. Both embryonic stem (ES) cells or induced pluripotent stem (iPS) cells were used for such procedure. ES cells can be derived from embryos before their implantation in the uterus and iPS cells are derived by reprogramming of adult cells. Finally, a couple of months ago, another group also reported being able to transform primordial germ cells collected from mouse embryos into mature oocytes.

 

However, replicating the full cycle of oogenesis from pluripotent cell lines in a single procedure constitutes an unprecedented discovery. To achieve this, they proceeded in different steps: first, they produced primordial germ cells in vitro from either skin cells (following their de-differentiation into iPS cells) or directly from ES cells. Second, they produced primary oocytes in a specific in vitro environment called "reconstituted ovaries". Third, they induced maturation of oocyte up until their arrest in meiosis II. This process took approximately the same time as it would take in the female mouse and it is impressive to see how the in vivo and in vitro oocytes are indistinguishable. Of course, this culture system also produced a lot of non-viable eggs and only few make it through the whole process. For example, during the first step of directed differentiation, over half of the oocytes contain chromosome mispairing during meiosis I, which is about 10 times more than in vivo. Additionally, only 30% complete meiosis I as shown by the exclusion of the 1st polar body. However, analysis of other parameters such as the methylation pattern of several genes showed that maternal imprinting was almost complete and that most of the mature oocytes had normal number of chromosomes. Transcription profiling also showed very high similarities between in vitro and in vivo oocytes.

The in vitro oocytes were then used for IVF and transplanted into mouse. Amazingly, some of them developed into pups that were viable, grew up to be fertile and had normal life expectancy without apparent abnormalities. However, the efficiency of such technique is very low as only 3.5% of embryos transplanted were born (compare to over 60% in the case of routine IVF procedures). Embryos that did not go through the end of the pregnancy showed delayed development at various stages, highlighting that there are probably conditions that could be improved for the oocytes to lead to more viable embryos.

Looking at the entire process, the rate of success to obtain eggs ready for transplant is around 7-14% depending on the starting cell line population. Considering how much time these cells spend in culture, this rate seems reasonably good. However, as mentioned above only few develop to birth. Nonetheless, this work constitutes major advancement in the field of developmental biology and will allow researchers to look in greater detail at the entire process of oogenesis and fertilization without worrying about the number of animals needed. We can also expect that, as with every protocol, it will be fine-tuned in the near future. It is already very impressive that the protocol led to viable pups from 6 different cell line populations.

 

Besides its potential for increasing knowledge in the oogenesis process, the impact of such research might reach beyond the scope of developmental biology. Not surprisingly, these results came with their share of concerns that soon this protocol would be used for humans. How amazing would it be for women who cannot use IVF to use their skin cells and allow them to have babies? Years ago, when IVF was introduced to the world, most people thought that “test-tube” babies were a bad idea. Today, it is used as a routine treatment for infertility problems. However, there is a humongous difference between extracting male and female gametes and engineering them. I do not believe that this protocol will be used on humans any time soon because it requires too many manipulations that we still have no idea how to control. Nonetheless, in theory, this possibility could be attractive. Also, for the most sceptic ones, one of the major reason why this protocol is not adaptable to human right now is that we cannot generate human “reconstituted ovaries”. This step is key for mouse oocytes to grow in vitro and necessitate to collect the gonadal somatic cells in embryos which is impossible in humans. So, until another research group manages to produce somatic gonadal cells from iPS cells, no need to start freaking out ;-)

 

 


In the Life of a Cell

An introduction to the cell cycle

 

By Johannes Buheitel, PhD

Omnis cellula e cellula”. We all heard or read this sentence probably sometime during college or grad school and no, it’s not NYU’s university motto. This short Latin phrase, popularized by the German physician/biologist Rudolf Virchow, states a simple fact, which, however, represents a fundamental truth of biology: “All cells come from cells”. It's so fundamental that we often take it for granted that the basis for all of those really interesting little pathways and mechanisms that we study is life itself; and, moreover, that life is not simply “created” from thin air but can actually only derive from other life. Macroscopically, you (and Elton John) might call this "the circle of life" but microscopically, we're talking about nothing less than the cell cycle. But what is the cell cycle exactly? What has to happen when and how does the cell maintain this order of events?

The cell cycle's main purpose is to generate two identical daughter cells from one mother cell by first, duplicating all its genetic content in order to get two copies of each chromosome (DNA replication), and then carefully distributing those two copies into the newly forming daughter cells (mitosis and cytokinesis). These two major chromosomal events take place during S phase (DNA replication) and M phase (mitosis), which during consecutive cycles alternate, separated by two "gap" phases (G1 between M and S phase and G2 between S and M phase; FYI: everything outside M phase is also sometimes also called interphase). It goes without saying that the temporal order of events, G1 to S to G2 to M phase, must be maintained at all times; just imagine trying to divide without previously having replicated your DNA! And not only the order is important, but each phase must also be given enough time to faithfully fulfill its purpose. How is this achieved?

If you want to boil it down, there are two main principles that drive the cell cycle: timely expression and degradation of key proteins and irreversible switch-like transitions, called checkpoints. So let's try and get an overview over each of these principles.

Cell-cycle
Overview over the different phases of the cell cycle: G1 (“gap”) phase, S (“synthesis”) phase, G2 phase and M (“mitosis”) phase.

In the early eighties, a scientist called Tim Hunt performed a series of experiments, unknowing that these will turn into a body of work, which will ultimately win him a Nobel prize. For these experiments, he radioactively labeled proteins in sea urchin embryos (yes, you read correctly!) and stumbled across one that exhibited an interesting pattern of abundance over time in that it appeared and vanished in a fashion that was not only cyclic but also seemed to be in sync with the embryos' division cycles. Dr. Hunt had just found the first member of a protein family, which later turned out to be one of the main drivers of the cell cycle: the cyclins. What do cyclins do? Cyclins are co-activators of cyclin-dependent kinases or Cdks, whose job it is to phosphorylate certain target proteins in order to regulate their function in a cell cycle-dependent fashion. Since Cdks are pretty much around all the time, they need the cyclins to tell them, when to be active and when not to be. There are a variety of different Cdks, which interact very specifically with various cyclins. For example, cyclin D interacts with Cdk4 and 6 to drive the transition from G1 to S phase, while a complex between cyclin B and Cdk1 is required for mitotic entry. This system allows for enough complexity to explain how the proper length of each phase is assured (slow accumulation of a specific cyclin until the respective Cdk can be fully activated), but also how the correct order of events is maintained; because it turns out that the expression of, say, the cyclin assigned to start replication (cyclin E) is dependent on the activity of the Cdk/cyclin complex of the previous phase (in this example: Cdk4/6-cyclin D) via phosphorylation-dependent regulation of transcription factors.

The second principle I was talking about, are checkpoints. A checkpoint is a way for the cell to take a short breath and check if things are running smoothly so far, and if they are not, to halt the cell cycle in order to give itself some time to either resolve the issue or, if that's not working out, throw in the towel (i.e. apoptosis). Researchers describe more and more checkpoint-like pathways that react to different stimuli all over the cell cycle, but canonically, we distinguish three main ones: the restriction checkpoint at the G1 to S phase transition, the DNA damage checkpoint at the G2 to M phase transition and the spindle assembly checkpoint (SAC) during mitosis at the transition from metaphase to anaphase. What do these checkpoints look for, or in more technical words, what requirements have to be met in order for a checkpoint to become satisfied? The restriction checkpoint integrates a variety of internal and external signals, but is ultimately satisfied by proper activation of S phase Cdk complexes (see above). The DNA damage checkpoint's main function is to give the cell time to correct DNA damage, which naturally occurs during genome replication but can also be introduced chemically or by ionizing radiation. Therefore, it remains unsatisfied as long as the DNA damage kinases ATM and ATR are active. Finally, the SAC governs one of the most intricate processes of the cell cycle: the formation of the mitotic spindle including proper attachment of each and every chromosome to its microtubules. After a checkpoint becomes satisfied, one or more positive feedback loops spring into action and effectively jump re-start the cell cycle.

As one can imagine, all of these processes must be exquisitely controlled to ensure the mission's overall success. In future posts, we will explore those mechanisms in more detail and will furthermore discuss, how a handful of biochemical fallacies can have the potential to turn this wonderful circle of life into a wicked cycle of death.


How a Cancer’s Genome Can Tell Us How to Treat it

By Gesa Junge, PhD

 

Any drug that is approved by the FDA has to have completed a series of clinical trials showing that the drug is safe to use and brings a therapeutic benefit, usually longer responses, better disease control, or fewer toxicities.

Generally, a phase I study of a potential cancer drug will include less than a hundred patients with advanced disease that have no more treatment options, and often includes many (or all) types of cancer. The focus in Phase I studies is on safety, and on finding the best dose of the drug to use in subsequent trials. Phase II studies involve larger patient groups (around 100 to 300) and the aim is to show that the treatment works and is safe in the target patient population, while Phase III trials can involve thousands of patients across several hospitals (or even countries) and aims to show a clinical benefit compared to existing therapies. Choosing the right patient population to test a drug in can make the difference between a successful and a failed drug. Traditionally, phase II and III trial populations are based on tumour site (e.g. lung or skin) and/or histology, i.e. the tissue where the cancer originates (e.g. carcinomas are cancer arising from epithelial tissues, while sarcomas develop in connective tissue).

However, as our understanding of cancer biology improves, it is becoming increasingly clear that the molecular basis of a tumour may be more relevant to therapy choice than where in the body it develops. For example, about half of all cutaneous melanoma cases (the most aggressive form of skin cancer) have a mutation in a signalling protein called B-Raf (BRAF V600). B-Raf is usually responsible for transmitting growth signals to cells, but while the normal, unmutated protein does this in a very controlled manner, the mutated version provides a constant growth signal, causing the cell to grow even when it shouldn’t, which leads to the formation of a tumour. A drug that specifically targets and inhibits the mutated version of B-Raf, Vemurafenib, was approved for the treatment of skin cancer in 2011, after trials showed it lead to longer survival and better response rates compared to the standard therapy at the time. It worked so well that patients in the comparator group were switched to the vemurafenib group halfway through the trial.

While B-Raf V600 mutations are especially common in skin cancer, they also occur in various other cancers, although at much lower percentages (often less than 5%), for example in lung and colorectal cancer. And since inhibition of B-Raf V600 works so well in B-Raf mutant skin cancer, should it not work just as well in lung or colon cancer with the same mutation? As the incidence of B-Raf V600 mutations is so low in most cancers, it would be difficult to find enough people to conduct a traditional trial and answer this question. However, a recently published study at Sloan Kettering Cancer Centre took a different approach: This study included 122 patients with non-melanoma cancers positive for B-Raf V600 and showed that lung cancer patients positive for B-Raf V600 mutations responded well to Vemurafenib, but colorectal cancer patients did not. This suggests that the importance of the mutated B-Raf protein for the survival of the tumour cells is not the same across cancer types, although at this point there is no explanation as to why.

Trials in which the patient population is chosen based on tumour genetics are called basket trials, and they are a great way to study the effect of a certain mutation on various different cancer types, even if only very few cases show this mutation. A major factor here is that DNA sequencing has come a long way and is now relatively cheap and quick to do. While the first genome that was sequenced as part of the Human Genome Project cost about $2.7bn and took over a decade to complete, a tumour genome can now be sequenced for around $1000 in a matter of days. This technological advance may make it possible to routinely determine a patient’s tumour’s DNA code and assign them to a therapy (or a study) based on this information.

The National Cancer Institute is currently running a trial which aims to evaluate this model of therapy. In the Molecular Analysis for Therapy Choice (MATCH) Trial, patients are assigned to a therapy based on their tumour genome. Initially, only ten treatments were included and the study is still ongoing, but an interim analysis after the 500th patient had been recruited in October 2015 showed that 9% of patients could be assigned to therapy based on mutations in their tumour, which is expected to increase as the trial is expanded to include more treatments.

This approach may be especially important for newer types of chemotherapy, which are targeted to a tumour-specific mutation that usually causes a healthy cell to become a cancer cell in the first place, as opposed to older generation chemotherapeutic drugs which target rapidly dividing cells and are a lot less selective. Targeted therapies may only work in a smaller number of patients, but are usually much better tolerated and often more effective, and molecular-based treatment decisions could be a way to allow more patients access to effective therapies faster.


From String to Strand

 

By Jordana Lovett

 

Ask a molecular biologist what image DNA conjures up in the mind. A convoluted ladder of nitrogenous bases, twisting and coiling dynamically. Pose the very same question to a theoretical physicist- chances are that DNA takes on a completely different meaning. As it turns out, DNA is in the eye of the beholder. Science is about perspective. Moreover, it relies on the convergence of distinct, yet interrelated angles to tackle scientific questions wholly.

 

When I met Dr. Vijay Kumar at a Cancer Immunotherapy meeting, I was immediately intrigued by his unique background and path to biology.  Vijay largely credits his family for strongly instilling in him core values of education and assiduousness. He was raised to strive for the best, and was driven to satisfy the goals of his parents, who encouraged him to pursue a degree in electrical engineering. While slightly resentful at the time, he now realizes that this broad degree would afford him multiple career options as well as the opportunity to branch into other fields of physics in the future. As early as his teenage years, Vijay had already begun thinking about the interesting unknowns of the natural universe. With his blinders on, he sought to explore them using physics and math, both theoretically and practically. As he advanced to university in pursuance of a degree in electrical engineering, he strategized and planned what would be his future transition into theoretical physics. He dabbled in various summer research projects and sought mentorship to help guide his career. Vijay ultimately applied and was accepted to a PhD program at MIT, where he studied string theory in a 6-dimensional model universe. He describes string theory as a broad framework rather than a theory that can be related to the world through ‘thought experiments’ and mathematical consistency.  Kumar continued his work in string theory during a post-doc in Santa Barbara, California, where he found himself surrounded by a more diverse group of physicists. Theoretical physicists, astrophysicists, and biophysicists were able to intermingle and share their science.

 

This diversity spurred new perspectives and reconsideration of what he had originally thought would be a clear road to professorship and a career in academia. As one would imagine, the broader impacts of string theory are limited; the ideas are part of a specialized pool of knowledge available to an elite handful. Even among the few, competition was fierce- at the time, there were only two available openings for professors in string theory in the United States. Additionally, seeing the need and presence of ‘quantitative people’ in other fields, such as biology made him increasingly curious about alternatives to the automated choices he had been making until this point. With the support of his (now) wife, and inspiration from his brother (who had just completed a degree in statistics/informatics and started a PhD in biology), he networked with other post-docs and set up meetings with principle investigators (PI’s) to discuss how he, as a theoretical physicist, could play a role in a biological setting. He spent time during his post-doc in Santa Barbara, and throughout his second post-doc at Stony Brook reflecting, taking courses and shifting into a different mindset. Vijay interviewed and gave talks at a number of institutions, and eventually landed in lab at Cold Spring Harbor, where he now is involved in addressing some of the shortcomings in DNA sequencing technology.

 

Starting in a different lab within the confines of a field means readjusting to brand new settings, acquainting with new lab mates and shifting from one narrowly focused project to another. Launching not only into a new lab, but into a foreign field adds an extra unsettling and daunting layer to the scenario.  Vijay, however, viewed this as yet another opportunity to uncover mysteries in nature- through a new perspective.  He recognized an interplay between string theory, wherein the vibration of strings allows you to make predictions about the universe, and biology, where the raw sequence of DNA can inform the makeup of an organism, and its interactions with the world.  It is with this viewpoint that Vijay understands DNA. He sees it as an abstraction, as a sequence of letters that allows you to draw inferences and predict biological outcomes. A change or deletion in just one letter can have enormous, tangible effects. It is this tangibility that speaks to Vijay. He is drawn to the application and broader consequences of the work he is doing, and excited that he can use his expertise to contribute to this knowledge.

 

While approaching a radically different field can impose obstacles, Kumar sees common challenges in both physics and biology and simply avoids getting lost in scientific translation. Just as theory has a language, so too biology has its own jargon. Once past this barrier, addressing gaps in knowledge becomes part of the common scientific core. Biology enables a question to be answered through various assays and allows observable results to guide future experiments- expertise in various subjects is therefore not only encouraged, but necessary. Collaborations between different labs across various disciplines enable painting a complete picture. “I’m a small piece of a larger puzzle, and that’s ok”, says Vijay. His insight into how scientists ought to work is admirable. Sharing and communicating data in a way that is comprehendible across the scientific playing field will more quickly and efficiently allow for scientific progress.

 

If I’ve learned one thing from Vijay’s story, it is to understand that science has room for multiple perspectives. In fact, it demands questions to be addressed in an interdisciplinary fashion. You might question yourself along the way. You might shift gears, change directions. But these unique paths mold the mind to perceive, ask, challenge, and contribute in a manner that no one else can.


The Danger of Absolutes in Science Communication

 

By Rebecca Delker, PhD

Complementarity, born out of quantum theory, is the idea that two different ways of looking at reality can both be true, although not at the same time. In other words, the opposite of a truth is not necessarily a falsehood. The most well known example of this in the physical world is light, which can be both a particle and a wave depending on how we measure it. Fundamentally, this principle allows for, and even encourages, the presence of multiple perspectives to gain knowledge.

 

This is something I found myself thinking about as I witnessed the twitter feud-turned blog post-turned actual news story (and here) centered around the factuality of physician-scientist Siddhartha Mukherjee’s essay, “Same but Different,” published recently in The New Yorker. Weaving personal stories of his mother and her identical twin sister with experimental evidence, Mukherjee presents the influence of the epigenome – the modifications overlaying the genome – in regulating gene expression. From this perspective, the genome encodes the set of all possible phenotypes, while the epigenome shrinks this set down to one. At the cellular level – where much of the evidence for the influence of epigenetic marks resides – this is demonstrated by the phenomenon that a single genome encodes for the vastly different phenotypes of cells in a multicellular organism. A neuron is different from a lymphocyte, which is different from a skin cell not because their genomes differ but because their transcriptomes (the complete set of genes expressed at any given time) differ. Epigenetic marks play a role here.

 

While many have problems with the buzzword status of epigenetics and the use of the phrase to explain away the many unknowns in biology (here, here), the central critique of Mukherjee’s essay was the extent to which he emphasized the role of epigenetic mechanisms in gene regulation over other well-characterized players, namely transcription factors – DNA binding proteins that are undeniably critical for gene expression. However, debating whether the well-studied transcription factors or the less well-established epigenetic marks are more important is no different than the classic chicken or egg scenario: impossible to assign order in a hierarchy, let alone separate from one another.

 

But whether we embrace epigenetics in all of its glory or we couch the term in quotation marks – “epigenetics” – in an attempt to dilute its impact, it is still worth pausing to dissect why a public exchange brimming with such negativity occurred in the first place.
“Humans are a strange lot,” remarked primatologist Frans de Waal. “We have the power to analyze and explore the world around us, yet panic as soon as evidence threatens to violate our expectations” (de Waal, 2016, p.113). This inclination is evident in the above debate, but it also hints at a more ubiquitous theme of the presence of bias stemming from one’s group identity. Though de Waal deals with expectations that cross species lines, even within our own species, group identity plays a powerful role in dictating relationships and guiding one’s perspective on controversial issues. Studies have shown that political identities, for example, can supplant information during decision-making. Pew Surveys reveal that views on the issue of climate change divide sharply along partisan lines. When asked whether humans are at fault for changing climate patterns, a much larger percentage of democrats (66%) than republicans (24%) answered yes; however, when asked what the main contributor of climate change is (CO2), these two groups converged (democrats: 56%, republicans: 58%; taken from Field Notes From a Catastrophe, p. 199-200). This illustrates the potential for a divide between one’s objective understanding of an issue and one’s subjective position on that issue – the latter greatly influenced by the prevailing opinion of their allied group.

 

Along with group identity is the tendency to eschew uncertainty and nuance, choosing solid footing no matter how shaky the turf, effectively demolishing the middle ground. This tendency has grown stronger in recent years, it seems, likely in response to an increase in the sheer amount of information available. This increased complexity, while important in allowing access to numerous perspectives on an issue, also triggers our innate response to minimize cost during decision-making by taking “cognitive shortcuts” and receiving cues from trusted authorities, including news outlets. This is exacerbated by the rise in the use of social media and shrinking attention spans, which quench our taste for nuance in favor of extremes. The constant awareness of one’s (online) identity in relation to that of a larger group encourages consolidation around these extremes. The result is the transformation of ideas into ideologies and the polarization of the people involved.

 

These phenomena are evident in the response to Mukherjee’s New Yorker article, but they can be spotted in many other areas of scientific discourse. This, unfortunately, is due in large part to a culture that rewards results, promotes an I-know-the-answer mentality, and encourages its members to adopt a binary vision of the world where there is a right and a wrong answer. Those who critiqued Mukherjee for placing too great an emphasis on the role of epigenetic mechanisms responded by placing the emphasis on transcription factors, trivializing the role of epigenetics. What got lost in this battle of extremes was a discussion of the complementary nature of both sets of discoveries – a discussion that would bridge, rather than divide, generations and perspectives.

 

While intra-academic squabbles are unproductive, the real danger of arguments fought in absolutes and along group identity lines lays at the interface of science and society. The world we live in is fraught with complex problems, and Science, humanity’s vessel of ingenuity, is called upon to provide clean, definitive solutions. This is an impossible task in many instances as important global challenges are not purely scientific in nature. They each contain a very deep human element. Political, historical, religious, and cultural views act as filters through which information is perceived and function to guide one’s stance on complex issues. When these issues include a scientific angle, confidence in the institution of science as an (trustworthy) authority plays a huge role.

 

One of the most divisive of such issues is that of genetically modified crops (GMOs). GMOs are crops produced by the introduction or modification of DNA sequence to incorporate a new trait or alter an existing trait. While the debate spans concerns about the safety of GMOs for human health and environmental health to economic concerns over the potential disparate benefits to large agribusiness and small farmers, these details are lost in the conversation. Instead, the debate is reduced to a binary: pro-GMO equals pro-science, anti-GMO equals anti-science. Again, the group to which one identifies, scientists included, plays a tremendous role in determining one’s stance on the issue. Polling public opinion reveals a similar pattern to that of climate change. Even though awareness of genetic engineering in crops has remained constantly low over the years, beliefs that GMOs pose a serious health hazard have increased. What’s worse, these debates treat all GMO crops the same simply because they are produced with the same methodology. While the opposition maintains a blanket disapproval of all engineered crops, the proponents don’t fare better, responding with indiscriminate approval.

 

Last month The National Academy of Sciences released a comprehensive, 420-page report addressing concerns about GMOs and presenting an analysis of two-decades of research on the subject. While the conclusions drawn largely support the idea that GMOs pose no significant danger for human and environmental health, the authors make certain to address the caveats associated with these conclusions. Though prompted by many to provide the public with “a simple, general, authoritative answer about GE (GMO) crops,” the committee refused to participate in “popular binary arguments.” As important as the scientific analysis is this element of the report, which serves to push the scientific community away from a culture of absolutes. While the evidence at hand shows no cause-and-effect relationship between GMOs and human health problems, for example, our ability to assess this is limited to short-term effects, as well as by our current ability to know what to look for and to develop assays to do so. The presence of these unknowns is a reality in all scientific research and to ignore them, especially with regard to complex societal issues, only serves to strengthen the growing mistrust of science in our community and broaden the divide between people with differing opinions. As one review of the report states, “trust is not built on sweeping decrees.”

 

GMO crops, though, is only one of many issues of this sort; climate change and vaccine safety, for example, have been similarly fraught. And, unfortunately, our world is promising to get a whole lot more complicated. With the reduced cost of high-throughput DNA sequencing and the relative ease of genome editing, it is becoming possible to modify not just crops, but farmed animals, as well as the wild flora and fauna that we share this planet with. Like the other issues discussed, these are not purely scientific problems. In fact, the rapid rate at which technology is developing creates a scenario in which the science is the easy part; understanding the consequences and the ethics of our actions yields the complications. This is exemplified by the potential use of CRISPR-driven gene drives to eradicate mosquito species that serve as vectors for devastating diseases (malaria, dengue, zika, for example). In 2015, 214 million people were affected by malaria and, of those, approximately half a million died. It is a moral imperative to address this problem, and gene drives (or other genome modification techniques) may be the best solution at this time. But, the situation is much more complex than here-today, gone-tomorrow. For starters, the rise in the prevalence of mosquito-borne diseases has its own complex portfolio, likely involving climate change and human-caused habitat destruction and deforestation. With limited understanding of the interconnectedness of ecosystems, it is challenging to predict the effects of mosquito specicide on the environment or on the rise of new vectors of human disease. And, finally, this issue raises questions of the role of humans on this planet and the ethics of modifying the world around us. The fact is that we are operating within a space replete with unknowns and the path forward is not to ignore these nuances or to approach these problems with an absolutist’s mindset. This only encourages an equal and opposite reaction in others and obliterates all hope of collective insight.

 

It is becoming ever more common for us to run away from uncertainty and nuance in search of simple truths. It is within the shelter of each of our groups and within the language of absolutes that we convince ourselves these truths can be found; but this is a misconception. Just as embracing complementarity in our understanding of the physical world can lead to greater insight, an awareness that no single approach can necessarily answer our world’s most pressing problems can actually push science and progress forward. When thinking about the relationship of science with society, gaining trust is certainly important but not the only consideration. It is also about cultivating an understanding that in the complex world in which we live there can exist multiple, mutually incompatible truths. It is our job as scientists and as citizens of the world to navigate toward, rather than away from, this terrain to gain a richer understanding of problems and thus best be able to provide a solution. Borrowing the words of physicist Frank Wilczek, “Complementarity is both a feature of physical reality and a lesson in wisdom.”

 


CRISPR gene editing in human embryos

Engineering Babies One Crispr at a Time

 

By Sophie Balmer, PhD

Over the past few weeks, the scientific community has been overwhelmed with major advances in human embryonic research. Whether researchers report for the second time the use of Crispr to edit the human germline or extend the conditions of in vitro culture of human embryos (also here), these issues have been all over the news. However, as all topics can not be raised in only one post, therefore, I will focus on genome editing studies.

 

About a year ago, one research group in China reported the first genome editing of human embryos using Crispr technology. Although these embryos were not viable due to one additional copy of each chromosome, this study quickly became highly controversial and raised strong concerns. The public and scientific communities questioned whether editing the human germline for therapeutic benefits was legitimate, leading to numerous ethical discussions. A few of weeks ago, a second study reported genome editing of embryos reinforcing the debate around this issue. Additionally, several research proposal involving genomic modification of healthy human embryos’ DNA have been validated recently in other countries. In this post, I want to address several questions. What are the possible advances or consequences of such work? What is the current legislation on human genome editing worldwide? Are these studies as alarming as what is written in some newspaper articles?

 

The emergence of the Crispr technology a few years ago has revolutionized the way scientists work since this method greatly improves the efficiency of DNA alteration of model organisms. However, this powerful tool has also raised many concerns, notably on the possibility to easily tweak the human genome and generate modified embryos.

In the eyes of the general public, this kind of experiment resonates with science fiction books or movies. Because of the high potential of this technique, it is crucial to inform everyone correctly to avoid clichés. Recently, one of my favorite comedian and television host John Oliver depicted in a very bright and amusing way how small scientific advances are sometimes presented in the media. Although the examples he uses are dramatic, every scientific breakthrough gets its share of overselling to the public. In the case of gene-editing of human embryos, pretending we are about to use eugenics principles to engineer babies and their descendants with beneficial genes is pure fiction. However, to prevent any potential malpractice from happening, clear ethical discussions and regulations need to be established and then explained to the public to prevent misunderstanding of these issues.

Within the scientific community, last year’s results triggered the need for new discussions and regulations on human cloning. Modifying the genome of human embryos involves modifying the germline as well, leading eventually to the transmission of the genetic alteration to future generations. However, the consequences of such transmission are unknown. Potentially, this could resolve a number of congenital genetic diseases for the individual him/herself and be used for gene therapy but would result in generations of genetically modified humans.

 

Because of cultural and ethical differences between countries, the legislation (if there is any) around working with human embryos or cells derived from human embryos (hESC for human embryonic stem cells) is variable. International ethical committees have only been able to establish guidelines as instituting international laws on human cloning is impossible. Ultimately, each country is responsible for enforcing these rules. Most countries and international ethics committees agree on a ban on reproductive and therapeutic human cloning. Moreover, following last year published experiments, a summit held in December 2015 gathered experts from all around the world. The consortium concluded that gene-editing of embryos used to establish pregnancy should not be performed (for now) and to follow up on all-related issues, new sets of guidelines are coming out imminently.

 

Still, it seems difficult to get an idea of the consensus depending on the countries in which scientists perform experiments. There is range of possibilities when working with human samples: some countries completely prohibit any manipulation of human embryos or hESC while others authorize genetic modification of the embryo for research purposes only under specific conditions. In between several nations authorize research exclusively on already derived lines of hESC and others authorize derivation of hESC but no manipulation of the embryos themselves.

Besides these general rules and as of today, three countries have approved proposals for gene-editing of human embryos: China, the UK and Sweden. Research proposals in both European countries have authorized Crispr targeting of specific genes in healthy human embryos to assess the function of these genes during early human development. However, these embryos can not be used for in vitro fertilization (IVF) and have to be destroyed at the end of the study. The purpose of these studies would be to confirm what has been described in hESC and in mammalian model systems and contribute to our knowledge of human development.

 

On the other hand, both published studies from China focused on Crispr targeting towards clinical therapies of an incurable blood disease or HIV. The overall purpose of such projects is to test the use of the Crispr technology for gene therapy. Although rendering embryos immune to several diseases using Crispr is an attractive possibility, it seems more urgent to probe the validity of the technique in humans and assess whether the mechanisms of human embryonic development are similar to what has been hypothesized. Gene therapies have already been successfully attempted in humans using other techniques to modify the genome. Yet, the modifications were targeted towards specific cells in already-born individuals. Again, modifying the genome of embryos implies that the mutation will be inherited in future generations and is in a large part the reason of this debate. Moreover, Crispr targeting still leads to unspecific modification of the genome, although very promising results show that newly engineered cas9 could lead to very specific targeting. The consequences of such off-target modification are unknown and could be disastrous for the following generations.

 

Overall, no research proposal dares to consider genetically modified embryos to establish pregnancy but as research moves faster, increasing demand for ethical discussion and regulations are brought forward. As more studies come out, it will be interesting to follow the evolution of this debate. Additionally, informing clearly the population of the possibilities and outcomes of ongoing projects should be a priority so that they can give an informed consent towards such research. In any case, a clear boundary needs to be established between selecting the fittest embryo by pre-implantation genetic diagnosis, which is routinely performed for IVF and playing the sorcerer’s apprentice with human embryo’s


Genome

How Low Can You Go? Designing a Minimal Genome

By Elizabeth Ohneck, PhD

How many genes are necessary for life? We humans have 19,000 – 20,000 genes, while the water flea Daphnia pulex has over 30,000 and the microbe Mycoplasma genitalium has only 525. But how many of these genes are absolutely required for life? Is there a minimum number of genes needed for a cell to survive independently? What are the functions of these essential genes? Researchers from the J. Craig Venter Institute and Synthetic Genomics, Inc., set out to explore these questions by designing the smallest cellular genome that can maintain an independently replicating cell. Their findings were published in the March 25th version of Science.

The researchers started with a modified version of the Mycoplasma mycoides genome, which contains over 900 genes. Mycoplasmas are simplest cells capable of autonomous growth, and their small genome size provides a good starting point for building minimal cells. To identify genes unnecessary for cell growth, the team used Tn5 transposon mutagenesis, in which a piece of mobile DNA is introduced to the cells and randomly “jumps” into the bacterial chromosome, thereby disrupting gene function. If many cells were found to have the transposon inserted into the same gene at any position in the gene sequence, and these cells were able to grow normally, the gene was considered non-essential, since its function was not required for growth; such genes were candidates for deletion in a minimal genome. In some genes, the transposon was only found to insert at the ends of the genes, and cells with these insertions grew slowly; such genes were considered quasi-essential, since they were needed for robust growth but were not necessary for cell survival. Genes which were never found to contain the transposon in any cells were considered essential, since cells that had transposon insertions in these genes did not survive; these essential genes were required in the minimal genome.

The researchers then constructed genomes with various combinations of non-essential and quasi-essential gene deletions using in vitro DNA synthesis and yeast cells. The synthetic chromosomes were transplanted into Mycoplasma capricolum, replacing its normal chromosome with the minimized genome. If the M. capricolum survived and grew in culture, the genome was considered viable. Some viable genomes, however, caused the cells to grow too slowly to be practical for further experiments. The team therefore had to find a compromise between small genome size and workable growth rate.

The final bacterial strain containing the optimized minimal genome, JCVI-syn3.0, had 473 genes, a genome smaller than any autonomously replicating cell found in nature. Its doubling time was 3 hours, which, while slower than the 1 hour doubling time of the M. mycoides parent strain, was not prohibitive of further experiments.

What genes were indispensable for an independently replicating cell? The 473 genes in the minimal genome could be categorized into 5 functional groups: cytosolic metabolism (17%), cell membrane structure and function (18%), preservation of genomic information (7%), expression of genomic information (41%), and unassigned or unknown function (17%). Because the cells were grown in rich medium, with almost all necessary nutrients provided, many metabolic genes were dispensable, aside from those necessary to effectively use the provided nutrients (cytosolic metabolism) or transport nutrients into the cell (cell membrane function). In contrast, a large proportion of genes involved in reading, expressing, replicating, and repairing DNA were maintained (after all, the presence of genes is of little use if there is no way to accurately read and maintain them). As the cell membrane is critical for a defined, intact cell, it’s unsurprising that the minimal genome also required many genes for cell membrane structure.

Of the 79 genes that could not be assigned to a functional category, 19 were essential and 36 were quasi-essential (necessary for rapid growth). Thirteen of the essential genes had completely unknown functions. Some were similar to genes of unknown function in other bacteria or even eukaryotes, suggesting these genes may encode proteins of novel but universal function. Those essential genes that were not similar to genes in any other organisms might encode novel, unique proteins or unusual sequences of genes with known function. Studying and identifying these genes could provide important insight into the core molecular functions of life.

One of the major advancements resulting from this study was the optimization of a semi-automated method for rapidly generating large, error-free DNA constructs. The technique used to generate the genome of JCVI-syn3.0 allows any small genome to be designed and built in yeast and then tested for viability under standard laboratory conditions in a process that takes about 3 weeks. This technique could be used in research to study the function of single genes or gene sets in a well-defined background. Additionally, genomes could be built to include pathways for the production of drugs or chemicals, or to enable cells to carry out industrially or environmentally important processes. The small, well-defined genome of a minimal cell that can be easily grown in laboratory culture would allow accurate modeling of the consequences of adding genes to the genome and lead to greater efficiency in the development of bacteria useful for research and industry.


Epigenetic Inheritance, Trauma and the Holocaust

 

By Alison Bernstein, PhD

Since my research interests focus on environmental impacts on health and how epigenetic processes mediate those effects, my mother sent me this article, “Study of Holocaust survivors finds trauma passed on to children’s genes“, from The Guardian. This article reports the recent paper, “Holocaust exposure induced intergenerational effects on FKBP5 methylation“, in Biological Psychiatry. I get overly excited by teachable moments so I decided to take the opportunity to teach some more epigenetics (see my pages on Facebook or Google+ for my Intro to Epigenetics series).

Epigenetics literally means “over the genome”. It encompasses all meiotically and mitotically heritable changes in gene expression that are not coded in the DNA sequence itself. If we break that down, there are some key points to note:

  • “Not coded in the DNA”: There is no change in the DNA sequence. Thus, for these to be heritable, there must be mechanisms of inheritance besides DNA replication.
  • “Changes in gene expression”: The underlying assumption of all epigenetic studies should be that these changes alter gene expression (or change how inducible or repressible gene expression is, but that’s harder to measure).
  • “Meiotically and mitotically heritable”: This means heritable through cell division, but not necessarily heritable from parent to offspring.

Epigenetics generally refers to 4 mechanisms: DNA methylation (and other modifications to cytosine), histone modifications, non-coding RNAs, and long-range chromatin interactions (3D structure of chromosomes). In this paper, the authors focused on DNA methylation and identified changes in DNA methylation that occur in people who were in a Nazi concentration camp, witnessed or experienced torture, or hid from the Nazis during World War II. Similar changes were seen in their children. This transmission of a trait from parents to children is called intergenerational inheritance.

The effects of severe stress and other exposures has been shown to be inherited intergenerationally, multigenerationally (to grandchildren) and sometimes even transgenerationally (to great-grandchildren), both in animals and in people. The Dutch famine of 1944 and the polybrominated biphenyl exposure in Michigan in 1978 have provided evidence that exposures that occur prior to conception and in utero can have lasting effects on subsequent generations. However, it is difficult to separate out the different mechanisms that contribute to the inheritance of traits to subsequent generations. Thus, it is an important research question to ask how the effects of trauma, stress and other exposures are passed from generation to generation. This is the question the scientists wanted to address in this paper: is there an epigenetic component to the intergenerational inheritance of the effects of trauma?

Epigenetic Inheritance

This paper provides direct evidence in humans that the epigenetic effects of pre-conception stress can be seen in both parents and offspring. The authors looked at one specific gene only – FKBP5 – because it is known to be involved in the response to high glucocorticoid levels (a biological signal for stress) and is a possible novel target for antidepressant medication. They looked for changes in DNA methylation in glucocorticoid response elements within this gene. Response elements are sequences of DNA that bind to specific transcription factors and regulate transcription of genes. In this case, glucocorticoid response elements are bound by glucocorticoid hormones and their receptors to regulate expression of the gene containing the response element. They found changes in DNA methylation in these specific elements of the specific FKBP5 gene in Jewish Holocaust survivors and their children, but not in other Jewish people of similar age. This observed change in DNA methylation of the FKBP5 gene was in the opposite direction in parents and offspring, yet we do not yet have an explanation as to why this change would be different in parents and offspring. Thus, it is actually impossible to say from the results of this paper if these epigenetic changes are due to direct effects of stress and high glucocorticoid levels (or other shared environmental factors) or to inheritance of epigenetic marks.

Let’s say a woman or girl lived through the Holocaust. She and her eggs were exposed to high glucocorticoid levels, and other effects, due to stress. If a woman was pregnant during this time, she, her eggs and her in utero daughters’ eggs were exposed. So that’s 2, and possibly, 3 generations directly exposed to the stress. Until you get to the 4th generation, there is still a possibility of direct exposure. It might be epigenetic, but it is also possible that it’s still a result of direct exposure. Changes must be observed in the generation the great-grandchildren to definitively say that they are epigenetically inherited and not a result of direct exposure. In general, the great-grandchildren are the first generation that was definitely not directly exposed to the stressor. However, in this case, they looked at preconception stress, so looking at the 3rd generation (grandchildren) would be sufficient to differentiate between epigenetic inheritance and direct exposure.

This paper only looks at parents and their children. So the eggs that produced ALL those children were directly exposed (since females are born with all their eggs) to the trauma. It’s possible that high glucocorticoid levels directly affect the methylation of FKBP5 in the eggs as well in cells of the parent. The discussion of the paper itself goes into this, but the article overlooked this point and it’s a really important point to understand if you are interested in epigenetic inheritance.

From the discussion section of the paper:

“The main finding in this study is that Holocaust survivors and their offspring have methylation changes on the same site in a functional intronic region of the FKBP5 gene, a GR binding sequence in intron 7, but in the opposite direction. To our knowledge, these results provide the first demonstration of transmission of preconception stress effects resulting in epigenetic changes in both exposed parents and their offspring in adult humans. Bin 3/site 6 methylation was not associated with the FKBP5 risk-allele, and could not be attributed to the offspring’s own trauma exposure, their own psychopathology, or other examined characteristics that might independently affect methylation of this gene. Yet, it could be attributed to Holocaust exposure in the F0.

It is not possible to infer mechanisms of transmission from these data. It was not possible to disentangle the influence of parental gender, including in utero effects, since both Holocaust parents were survivors. Epigenetic effects in maternal or paternal gametes are a potential explanation for epigenetic effects in offspring, but blood samples will not permit ascertainment of gamete dependent transmission. What can be detected in blood samples is parental and offspring experience-dependent epigenetic modifications. Future prospective, longitudinal studies of high risk trauma survivors prior to conception, during pregnancy and postpartum may uncover sources of epigenetic influences.”

The paper reports evidence that the epigenetic effects of stress and trauma can be seen in both parents and offspring. However, there are a lot of variables that may cause similar epigenetic changes in parents and offspring. Further studies are needed to really know what the mechanism of these shared epigenetic marks are, before we can confidently assert that the epigenetic changes observed in parents and offspring are due to epigenetic inheritance. As with all good science, this paper answers a question while, at the same time, raising additional questions for future research.

This article was originally published on The Sound of Science blog in August 2015.


Leaving Your Mark on the World

By Danielle Gerhard

 

The idea that transgenerational inheritance of salient life experiences exists has only recently entered the world of experimental research. French scientist Jean-Baptiste Lamarck proposed the idea that acquired traits throughout an organism’s life could be passed along to offspring. This theory of inheritance was originally disregarded in favor of Mendelian genetics, or the inheritance of phenotypic traits isn’t a blending of the traits but instead a specific combination of alleles to form a unique gene encoding the phenotypic trait. However, inheritance is much more complicated than either theory allows for. While Lamarckian inheritance has largely been negated by modern genetics, recent findings in the field of genetics have caused some to revisit l’influence des circonstances, or, the influence of circumstances.

 

Over the past decade, efforts have shifted towards understanding the mechanisms underlying the non-Mendelian inheritance of experience-dependent information. While still conserving most of the rules of Mendelian inheritance, new discoveries like epigenetics and prions challenge the central dogma of molecular biology. Epigenetics is the study of heritable changes in gene activity as a result of environmental factors. These changes do not affect DNA sequences directly but instead impact processes that regulate gene activity such as DNA methylation and histone acetylation.

 

Epigenetics has transformed how psychologists approach understanding the development of psychological disorders. The first study to report epigenetic effects on behavior came from the lab of Michael Meany and Moshe Szyf at McGill University in the early 2000s. In a 2004 Nature Neuroscience paper they report differential DNA methylation in pups raised by high licking and grooming mothers compared to pups raised by low licking and grooming mother. Following these initial findings, neuroscientists have begun using epigenetic techniques to better understand how parental life experiences, such as stress and depression, can shape the epigenome of their offspring.

 

Recent research coming out from the lab of Tracy Bale of the University of Pennsylvania has investigated the heritability of behavioral phenotypes. A 2013 Journal of Neuroscience paper found that stressed males went on to produce offspring with blunted hypothalamic pituitary (HPA) axis responsivity. In simpler terms, when the offspring were presented with a brief, stressful event they had a reduction in the production of the stress hormone corticosterone (cortisol in humans), symptomatic of a predisposition to psychopathology. In contrast, an adaptive response to acute stressors is a transient increase in corticosterone that signals a negative feedback loops to subsequently silence the stress response.

 

The other key finding from this prior study is the identification of nine small non-coding RNA sperm microRNAs (miRs) increased in stressed sires. These findings begin to delve into how paternal experience can influence germ cell transmission but does not explain how selective increases in these sperm miRs might effect oocyte development in order to cause the observed phenotypic and hormonal deficits seen in adult offspring.

 

A recent study from the lab published in PNAS builds off of these initial findings to further investigate the mechanisms underlying transgenerational effects of paternal stress. Using the previously identified nine sperm miRs, the researchers performed a multi-miR injection into single-cell mouse zygotes that were introduced into healthy surrogate females. To confirm that all nine of the sperm miRs were required to recapitulate the stress phenotype, another set of single-cell mouse zygotes were microinjected with a single sperm miR. Furthermore, a final set of zygotes received none of the sperm miRs. Following a normal rearing schedule, the adult offspring were briefly exposed to an acute stressor and blood was collected to analyze changes in stress hormones. As hypothesized, male and female adult offspring from the multi-miR group had a blunted stress response relative to both controls.

 

To further investigate potential effects on neural development, the researchers dissected out the paraventricular nucleus (PVN) of the hypothalamus, a region of the brain that has been previously identified by the group to be involved in regulation of the stress response. Using RNA sequencing and gene set enrichment analysis (GSEA) techniques they found a decrease in genes involved in collagen formation and extracellular matrix organization which the authors go on to hypothesize could be modifying cerebral circulation and blood brain barrier integrity.

 

The final experiment in the study examined the postfertilization effects of multi-miR injected zygotes. Specifically, the investigators were interested in the direct, combined effect of the nine identified sperm miRs on stored maternal mRNA. Using a similar design as the initial experiment, the zygote mRNA was collected and amplified 24 hours after miR injection in order to examine differential gene expression. The researchers found that microinjection of the nine sperm miRs reduced stored maternal mRNA of candidate genes.

 

This study is significant as it has never been shown that paternally derived miRs play a regulatory role in zygote miR degradation. In simpler terms, these findings contradict the conventional belief that zygote development is solely maternally driven. Paternal models of transgenerational inheritance of salient life experiences are useful as they avoid confounding maternal influences in development. Studies investigating the effects of paternal drug use, malnutrition, and psychopathology are ongoing.

 

Not only do early life experiences influence the epigenome passed down to offspring but recent work out of the University of Copenhagen suggests that our diet may also have long-lasting, transgenerational effects. A study that will be published in Cell Metabolism next year examined the effects of obesity on the epigenome. They report differential small non-coding RNA expression and DNA methylation of genes involved in central nervous system development in the spermatozoa of obese men compared to lean controls. Before you start feeling guilty about the 15 jelly donuts you ate this morning, there is hope that epigenetics can also work in our favor. The authors present data on obese men who have undergone bariatric surgery-induced weight loss and they show a remodeling of DNA methylation in spermatozoa.

 

Although still a nascent field, epigenetics has promise for better understanding intergenerational transmission of risk to developing a psychopathology or disease. The ultimate goal of treatment is to identify patterns of epigenetic alternations across susceptible or diagnosed individuals and develop agents that aim to modify epigenetic processes responsible for regulating genes of interest. I would argue that it will one day be necessary for epigenetics and pharmacogenetics, another burgeoning field, to come into cahoots with one another to not only identify the epigenetic markers of a disease but to identify the markers on an person by person basis. However, because the fields of epigenetics and pharmacogenetics are still in the early stages, the tools and techniques currently available limit them. As a result, researchers are able to extract correlations in many of their studies but unable to determine potential causality. Therefore, longitudinal, transgenerational studies like those from the labs of Tracy Bale and others are necessary to provide insight into the lability of our epigenome in response to lifelong experiences.


Dr. Thomas Gregor

Development On the Fly: An Interview with Dr. Thomas Gregor

By John McLaughlin

 

Thomas Gregor is a biophysicist and Professor at Princeton University. His Laboratory for the Physics of Life uses both Drosophila melanogaster and Dictyostelium discoideum as model systems to understand developmental processes from a physical perspective.

 

Could you briefly describe your educational path from undergraduate to faculty member at Princeton?

TG: As an undergraduate, I studied physics in Geneva, and then moved into theoretical physics and math. I came to Princeton, initially for a theoretical physics PhD; I switched during my time here to theoretical biophysics and then realized that it makes sense to combine this with experiments. I ended up doing a PhD between three complementary disciplines. My main advisor was Bill Bialek, a theoretical physicist. My other two were David Tank, an experimental neuroscientist, and Eric Wieschaus, a fly geneticist. So I had both experiment and theory, from a biological and a physical side. I then went to Tokyo for a brief post-doc, during which I continued in that interface. But I changed model organisms: I switched from a multicellular, embryonic system to looking at populations of single cells [the social amoeba Dictyostelium discoideum]. As a physicist you’re not married to model organisms. When I came back to start my lab at Princeton in 2009, I kept both the fly and the amoeba systems.

 

What is the overall goal of your lab’s research program?

TG: Basically, to find physical principles behind biological phenomena. How can we come up with a larger, principled understanding that goes beyond the molecular details of any one particular system? I mostly look at genetic networks and try to understand their global properties.

 

Do you think the approaches of biologists and physicists are very different, and if so are they complementary?

TG: I’m driven by the physical aspects of things, but I’m also realistic enough to see what can now be done in biological systems, in terms of data collection and what we can test. To find the overlap between them is kind of an art, and I think that’s where I’m trying to come in.

 

Do you have any scientific role models who have shaped how you approach science?

TG: The three that I mentioned: Bialek influenced me in the types of questions that speak to me; Tank had a very thorough experimental approach that taught me how to make real, physics-style measurements; and Wieschaus brought a lot of enthusiasm and knowledge of the system.

 

Your lab has been studying developmental reproducibility and precision, in the patterning of the fly Drosophila melanogaster. In a 2014 paper1, you showed that levels of the anterior determinant bicoid mRNA vary by only ~9% between different embryos. This is a very similar value to the ~10% variation in Bicoid protein levels between embryos, which you demonstrated several years earlier2. So it seems that this reproducibility occurs even at the mRNA level.

TG: Before going into this, the general thought in the field is that things were very noisy initially, and as the developmental path goes along it becomes more refined and things become more precise. This paper basically asked whether the precision is inherited from the mother, or the embryo needs to acquire it. Because the fluctuations in mRNA, from the mother, completely mimic the fluctuations in protein that the zygote expresses, that told us that the mother lays the groundwork, and passes on a very reproducible pattern. So there’s no necessity for a mechanism that reduces fluctuations from the mRNA to the protein level.

 

Continuing on the theme of precision: in a separate paper from the same year3, your lab showed that the wing structure among different adult flies is identical to within less than a single cell width. Did you have any prior expectations going into this study, and did the results surprise you?

TG: Before looking at the wing, I had kind of made up my mind. I had first seen single cell precision in patterning of gene expression boundaries in the embryo. But I also knew that it’s always better to make a measurement first, and it seems that things are much more precise and reproducible in biology than we think, given the idea of “sloppiness” that we have.

 

Do you think that a high level of reproducibility is a general feature of development, or varies widely among different types of species?

TG: It’s a philosophical question in a way, because I haven’t looked. I think what we found in the embryo is not special to the fly; specific mechanisms for getting there might be unique to the fly. For instance, we have also shown in a recent paper from 2013 that transcription is just as noisy in flies as it is in bacteria, hugely noisy. So, physical mechanisms like temporal and spatial averaging seem enough to reduce the high ubiquitous noise that transcription has to the very fine, reproducible patterns that you see in the fly. The specific mechanisms that reduce noise will be very different from species to species, but I think overall the fact that development is precise and reproducible is something we may one day be able to call a principle.

 

If you could make any changes to scientific institutions, such as the current funding system, journal peer review, etc. what would they be?

TG: One thing that might be nice is if we didn’t have to fund graduate students for the first five years of their career; it would be nice to have more streamlined training grants, not only for U.S. but also international graduate students. And so, graduate students wouldn’t have to worry. They should be free to choose a school based on their scientific interests.

For peer review in journals, the problem is the sheer volume of output is becoming so high. One way to keep a peer review system, is either to pay the reviewers money, or to put everything on the bioRxiv [bio archive is a pre-print server for the life sciences] and let some other means determine how to evaluate a paper. I don’t read papers from looking at the top journals’ table of contents every week, I read them because I see people talk about it on Twitter, or my colleagues tell me I should look at that paper, or because I hear about the work in a talk and decide to see what else the guy is doing.

A lot of people are advocating the new metrics – citations, citation rates, H-index – which are so dependent on the particular field and not necessarily a good measure of impact. In 100 years, are we going to look more at those papers than the ones that currently get very few citations? We don’t know. I don’t think the solution is out there yet.

 

Do you have any advice for young scientists – current PhD students or post-doctoral fellows – for being successful in science?

TG: My advice would be to focus on one very impactful finding. If it’s very thorough and good science, it will be seen. Also, nothing comes from nothing. You need to put in the hours if you want to get a job in academia. And I think that’s one of the ways to measure a good scientist, because knowledge in experimental science comes from new, good data.


What are some future goals of your lab’s research?

TG: We’ve been looking at the genetic network in the fly embryo, trying to understand properties of that network. Medium term, we want to incorporate a slightly different angle, which is looking at the link between transcriptional regulation and the 3D architecture of the genome. In the living embryo, we want to look at how individual pieces of DNA interact, and how that influences transcription and eventually patterning. In the longer term, I don’t know yet; I just got tenure, so I need to sit back. Everything is open. That is what’s nice about being a physicist; you’re not married to your biological past so much.

 

In your opinion, what are the most exciting developments happening in biology right now, whether in your own field or elsewhere?

TG: It’s definitely the fact that so many different disciplines have stormed into biology, making it a very multidisciplinary science. I think it makes the life sciences a very vibrant, communal enterprise. Hopefully the next decades will show the fruits of those interactions.

 

This question is asked very often: How do you balance your lab and family life?

TG: When you start thinking about having a family in science, things become much more complicated. Since I’ve had children, my workload went down a lot. My wife is also a scientist, and for her it’s much harder because she’s not yet tenured. As much as people look at the CV and see how many high-profile papers you have, they should also look at it and see your family and life situation. And for women in science, despite all the efforts that have been made, I don’t think we’re there yet.

 

References

[ordered_list style="decimal"]

  1. Petkova, MD et al. Maternal origins of developmental reproducibility. Current Biology. 2014. 24(11).
  2. Gregor, T et al. Probing the limits to positional information. Cell. 2007. 130(1).
  3. Abouchar, L et al. Fly wing vein patterns have spatial reproducibility of a single cell. J R Soc Interface. 2014. 11(97).

[/ordered_list]