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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)

 

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Paperfuges and Foldscopes: The Case for Low-Tech Science

 

By Gesa Junge, PhD

 

If you have ever been inside a lab you will know that centrifuges and microscopes come in various shapes and sizes and degrees of sophistication, but in some form they are used every day in most research labs around the world. Microscopes and centrifuges are pretty basic lab equipment, although some versions can be very high-end, for example high-speed centrifuges that can cool down to fridge temperatures, or electron microscopes that can magnify structures up to 2 million times. But even basic centrifuges and microscopes cost a few thousand dollars, and they require electricity and maintenance. These are not big issues for most universities and established research institutes, but for scientists working in the field, or in developing countries, money and electricity can be hard to come by.

With this in mind, Manu Prakash from Stanford University developed a centrifuge and a microscope made of paper. Yes, you read that right. The centrifuge is basically a paper disk on two strings that you pull to make the disk spin (kind of like a whirligig Saw Mill, remember those?) – check out this video from Wired Magazine. The whole thing costs 20 cents and fits into a jacket pocket, but it can spin samples up to 12500rpm, which is fast. Fast enough, for example, to separate blood into blood cells and plasma, which is a key step in many diagnostic procedures.

And the foldscope is basically origami. It is printed on paper, you cut out the parts and fold them up and insert a lens. The microscope does need electricity, but it can run on a battery for up to 50 hours, and the sample can be mounted on a piece of tape, as opposed to a glass slide. The lens determines the magnification, and they can go up to 2000x. For reference, we can distinguish individual human cells easily at 10x, nuclei become clearly visible at 20x and bacteria at 40x. Using different color LEDs, this can even be converted into a fluorescent microscope, meaning it can be used to analyse different stains of tissues.

The paperfuge and the foldscope are the implementation of an emerging concept called “frugal science”, and aim to bring scientific advances to inaccessible and under-developed regions. And while Manu Prakash’s ideas are very low-tech approaches, the idea of making science useful to everyone also benefits from innovation and advanced technology. For example, Dr Samuel Sia at Columbia University has developed a smart phone dongle technology called mChip which can diagnose HIV from a finger prick’s worth of blood. This device contains all the necessary reagents which mix at the push of a button, and it plugs into the headphone jack of a phone as a power supply. Testing takes about 15 minutes and costs about $1 (the dongle is $100), which is a huge improvement over current methods. In a similar concept, a company called QuantumMDx in Newcastle in the UK is developing a handheld DNA testing tool, which could be used to identify strains of pathogens. And electronics company Phillips has come up with the MiniCare I-20, a handheld device that can measure troponin I levels from a single drop of blood taken from a pinprick. Troponin I is a marker of a damaged heart muscle, and is often measured in emergency departments.

All of these innovations address a really important, and sometimes overlooked, point: science and technology, in all their greatness and cool fascination, will only benefit humanity if applied in the community in a way that leads to real-life changes. As with so many resources, scientific expertise and technology, and therefore the benefit of science, are distributed incredibly unevenly among the world’s society. For example, malaria and AIDS drugs are still not reaching many of the people who need them, be it for financial, infrastructural, political, or organisational reasons. Diagnostic tests often require well-equipped labs and trained technicians. And while they are limited in their applications for research, the paperfuge and the foldscope have the potential to revolutionize diagnostics as well as education around the world. Cutting-edge research may require more sophisticated centrifuges that spin faster, microscopes that have better resolution, computers to store the images, and teams of scientists analyzing the data. But the frugal science approach is well-suited for the diagnosis of diseases, or to help a high school science class understand what cells are.

If you would like to find out more about the foldscope, check out Manu Prakash’s very cool TED talk. More information on Dr Sia’s mChip can be found here.

 

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Immunotherapy: Using Your Own Cells to Fight Cancer – Part 2

 

By Gesa Junge, PhD

 

Part 1 of this post described passive immunotherapies like antibodies and cytokines, but there are also active immunotherapies, which re-target our immune system towards cancer cells, for example cancer vaccines. These can be preventative vaccines, offering protection against cancer-associated viruses such as Hepatitis B (liver cancer) or Human Papilloma Virus (HPV, cervical cancer). The link between HPV and cervical cancer was first described in 1983, and a vaccine was approved in 2006. By 2015, the incidence of HPV infections in women under 20 had decreased as much as 60% in countries that had 50% vaccination coverage, although it may still be too early to tell what the impact on HPV-associated cancer incidence is. There are also other factors to consider, for example screening programmes are also likely to have a positive impact on HPV-associated cancers.

Vaccines can also be therapeutic vaccines, which stimulate the immune system to attack cancer cells. To date, the only cancer vaccine approved in the US is Provenge, used for the treatment of metastatic prostate cancer. For this therapy, a patient’s white blood cells are extracted from the blood, incubated with prostatic acid phosphatase (PAP, a prostate-specific enzyme) and granulocyte macrophage colony stimulating factor (GM-CSF) in order to produce mature antigen presenting cells which are then returned to the patient and search and destroy tumour cells.

Many other therapeutic cancer vaccines are in development, for example OncoVax, which is an autologous vaccine made from a patient’s resected tumour cells. OncoVax has been in development since the 1990s and is currently in phase III trials. Another example is GVAX, an allogenic whole-cell tumour vaccine currently being studied in phase I and II trials or pancreatic and colorectal cancer. As an allogenic vaccine, it is not made from the patient’s own blood cells (like an autologous vaccine), and it does not target specific antigens but rather increases the production of cytokines and GM-CSF.

Another therapy which is based on re-programming the patient’s immune system is adoptive T-cell transfer. As with some cancer vaccines, a patient’s T-cells are isolated from the blood, and the cells with the greatest affinity for tumour cells are expanded in the lab and the re-infused in the patient. A recent modification of this technique is the use of chimeric antigen receptor (CAR) T-cells, where the T-cell receptors are genetically engineered to be more tumour-specific before re-infusion. This approached was especially promising in chronic lymphocytic leukaemia, where some patients experienced remissions of a year and longer. Later, CAR T-cells were also tested in acute lymphocytic leukaemia, where response rates were as high as 89%.

Finally, a new class of cancer drugs called immune checkpoint inhibitors has been making headlines recently, some of which are now approved for the treatment of cancer. Immune checkpoints are part of the mechanism by which human cells, including cancer cells, can evade the immune system. For example, the programmed cell death (PD) 1 receptor on immune cells interacts with PD1 ligand (PDL1) on cancer cells, which inhibits the killing of the cancer cell by the immune cell. Similarly, CTLA-4 is a receptor on activated T-cells which downregulates the immune response.

The first checkpoint inhibitor was an antibody to CTLA-4, ipilimumab, which was approved for the treatment of melanoma in 2011. PD1 antibodies such as pembrolizumab and nivolumab were only approved in 2014, and the only PDL1 antibody (atezolizumab) in 2016, so it is difficult to tell what the long-term effects of checkpoint inhibitor treatment will be. Numerous checkpoint inhibitors are still undergoing trials, most of the advanced (phase III) ones being targeted to PD1 or PDL1. However, there are other compounds in early trials (phase I or II) that target KIR (killer-cell immunoglobulin-like receptor) which are primarily being studied in myeloma, or LAG3 (lymphocyte activation gene 3), in trials for various solid tumours and leukaemias.

Immunotherapies all come under the umbrella of biological therapies. Biologics are produced by organisms, usually cells in a dish, unlike synthetic drugs, which are manufactured using a chemical process in the lab. This makes biologicals more expensive to manufacture. Ipilimumab therapy, for example, can cost about $100 000 per patient, with pembrolizumab and nivolumab being only slightly less expensive at $48 000 – $67 000. This puts considerable financial strain on patients and insurance companies. From a safety perspective, biologicals can cause the immune system to overreact. This sounds odd, as the whole point of immunotherapy is to activate the immune system in order to fight tumour cells, but if this response gets out of control, it can lead to potentially serious side effects as the immune system attacks the body’s organs and tissues.

All of these therapeutic approaches (antibodies, interleukins, vaccines, and checkpoint inhibitors) are usually not used alone but in combination with each other or other chemotherapy, which makes it difficult to definitively say which drug works best. But it is safe to say that collectively they have improved the lives of a lot of cancer patients. If you are interested in finding out more about the fascinating history of immunotherapy, from the discovery of the immune system to checkpoint inhibitors, check out the CRI’s timeline of progress on immunology and immunotherapy here.

 

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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 😉

 

 

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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

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Dr Frankenstein’s Modern Guide to Body Building

By Sally Burn, PhD

Square head, green skin, bolt through the neck, and plagued by misconceptions: “Frankenstein” is a popular choice at the Halloween costume store. This erroneously named costume is based on the monster from Mary Shelley’s Frankenstein, the titular character of which is Dr Frankenstein, the scientist who creates the in fact unnamed creature. Modern depictions of the monster tend to hold little resemblance to that in the book where we are told that “His yellow skin scarcely covered the work of muscles and arteries beneath; his hair was of a lustrous black, and flowing; his teeth of a pearly whiteness…”

A final misconception – born from the movies – is that the monster was created from stolen body parts, animated to life with electricity. Shelley did not go into details of how exactly the monster was made, instead leaving us with just an indication that Frankenstein had the lifestyle of a postdoc (“I had worked hard for nearly two years, for the sole purpose of infusing life into an inanimate body. For this I had deprived myself of rest and health”) and a Nature Letter-size methods section: “With an anxiety that almost amounted to agony, I collected the instruments of life around me, that I might infuse a spark of being into the lifeless thing that lay at my feet.”

Clearly this is an insufficient Materials & Methods section to permit study replication. So, in the spirit of spooky science, Scizzle presents Dr Frankenstein’s Modern Guide to Body Building – how to build a brain, kidney, gut, and more in the comfort of your own lab:

 

* Building Brains:

The first thing your creation will need is a brain. While we don’t yet have the technology to grow a whole brain in the lab, we can make cerebral organoids. Scizzle first reported on these brain-like spheres two years ago, when they were published in Nature. Cerebral organoids are generated from human pluripotent stem cells (hPSC) or induced pluripotent stem (iPS) cells – so no need to go raiding the graveyard for spare body parts anymore, wannabe Frankensteins! The cells are aggregated into embryoid bodies, which are then differentiated into neuroectoderm and cultured in a spinning bioreactor, resulting in 3D cerebral organoids. After around a month in culture the organoids contain distinct brain regions and a cerebral cortex – the seat of consciousness, memory, and language.

 

* Growing Guts:

Upon waking, your monster will need a good meal so you’re going to need to build a digestive system. The gut is composed of a number of functionally, physiologically, and histologically distinct organs, including the stomach and small and large intestines. The liver and pancreas also play vital roles in digestion. Progress has been made on growing all of these tissues in the lab. Stomach-like “gastric organoids” can be generated by exposing hPSCs to a specific cocktail of proteins and growth factors. These mini stomachs even act in an organotypic manner, responding to H. pylori infection as a human stomach would. Moving down the digestive tract, our next requirement is an intestine. While we can’t yet grow an entire intestine in the lab (that would be one big culture dish), our old friend the organoid is here to help again – this time in the form of intestinal organoids, grown from crypt-derived stem cells (crypt as in small intestinal, not as in the spooky Halloween place). And yes, you will be pleased to know it is even possible to engineer your monster an anal sphincter. Even monsters need to poop.

 

* Crafting Kidneys:

To keep your creation in peak operational form, it will need to be able to process and remove toxins from its body. For drug processing look no further than iPS-derived liver buds (iPS-LBs), which can successfully metabolize drugs. Excretion of waste from the monster’s body will require kidneys. The generation of kidney organoids is a hot topic – earlier this month Melissa Little’s lab in Australia reported their iPS-derived kidney organoid system. While other groups have made similar organoids, this newest report is exciting as their kidney organoids contain numerous cell types and tissue structures found in human kidneys, arranged in an organotypic fashion. Furthermore, the engineered organs exhibit kidney-like function and nephrotoxin sensitivity.

 

So could a modern Dr Frankenstein make a monster using these techniques? No, obviously not – and thank goodness for that – but by exchanging grave-digging tools for iPS cells they could certainly have a good attempt at making replacement body parts for real humans. Such an endeavor may be possible in the near future, but already right now these lab-grown organoids offer a number of other benefits.

One use is pharmacological screening – does a new drug adversely affect the human kidney, for example? The human origin of organoids also allows researchers to gain insights into the development and disease of their related organs, in a way not possible with animal models. Lastly, by using patient-specific iPS or stem cells to generate organoids, scientists can better understand the etiology and treatment prospects of an individual’s disease. Earlier this year, Hans Clever’s group reported the generation of gut organoids from colorectal cancer patients, which recapitulate characteristics of their tumor of origin and are amenable to high throughput drug screening.

For a more in-depth look at the growing field of organoid science, see Cassandra Willyard’s article in Nature and stay tuned to Scizzle for future tales of “Frankenstein” science!

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Mice are Not Men

On the use of rodent models in neuroscience research

By Franchesca Ramirez

We depend on animal models for biomedical research. In so doing we work diligently under the assumptions that these animal models will provide us insight into the development of novel treatment approaches. Specifically, the rodent is become flagship of animal research. We gleened a great deal from the study of rodent models that seem to recapitulate the symptoms of psychiatric diseases and yet not consistently been led to clear therapeutic insight as a result. Essentially, clinical trials are necessary because animal studies do not predict the efficacy of treatment in human populations with sufficient certitude.

Let us consider what we ask of our rodent models. We expect for such animals to model the etiology of psychiatric disorders, confer some congruence with molecular markers confirmed in humans and ideally to respond to available treatment. Indeed, we are witness to robust mouse models of disorders like, Rett syndrome, fragile X and Down’s syndrome to name a few. However, these human brain disorders have discrete and recognizable genetic  causes. Why then do behaviorally induced animal models of other neurocognitive disorders, like depression or schizophrenia fall short, one may ask. The thing that makes an animal model bad is that perhaps we should not be modelling a psychiatric disorder with cerebral and cognitive diagnostic criteria in a bottom-up fashion. That is to say by focusing on the details of complex biological systems in animals we can not know the landscape of cognition in humans.

Perhaps neuroscientists must be reticent to draw analogies between animals showing symptoms of psychiatric illness without knowledge of the cognitive process underlying the behavior, agrees neuroscientist, Erik Klann at New York University in NYC. Individual humans do not at all display identical symptomatology so how then do the animal symptoms add up to a recognizable human psychiatric disorder? How would we model hallucinations, sadness and guilt in animals, says Klann. These are legitimate concerns for the future of biomedical neuroscience research. We cannot know what it is like to experience the conscious state of another organism, we may however, ask what brain systems are conserved across taxa and how do they work to elicit behaviors that mimic symptoms of psychiatric disorders.

Perhaps the effort should be devoted to research on humans to map the progression and symptomatology of psychiatric disease to then probe the neural circuitry in our beloved rodent models, says Yadin Dudai, a neuroscientist at Weizmann Institute of Science in Rehovot, Israel. It is logical to conclude that it is not the fault of the model in question but rather the approach we take to probe the system for answers to our research questions. One approach Dudai endorses is to start with the human components of a disorder but not the entire spectrum. In this case the top-down approach is key- let us study humans as models for our animal systems. Just so, this is largely how we attained such robust disease animal models in the first place.

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The Most Scizzling Papers of 2013

 

The Scizzle Team

Bacteriophage/animal symbiosis at mucosal surfaces

The mucosal surfaces of animals, which are the major entry points for pathogenic bacteria, are also known to contain bacteriophages. In this study, Barr et al. characterized the role of these mucus associated phages. Phages were more commonly found on mucosal surfaces than other environments and adhere to mucin glycoproteins via hypervariable immunoglobulin like domains. Bacteriophage pre-treatment of mucus producing cells provided protection from bacterial induced death, but this was not the case for cells that did not produce mucus. These studies show that there may be a symbiotic relationship between bacteriophages and multicellular organisms which provides bacterial prey for the phages and antimicrobial protection for the animals.

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Interlocking gear system discovered in jumping insects

Champion jumping insects need to move their powerful hind legs in synchrony to prevent spinning. Burrows and Sutton studied the mechanism of high speed jumping in Issus coleoptratus juveniles and found the first ever example in nature of an interlocking gear system. The gears are located on the trochantera (leg segments close to the body’s midline) and ensure both hind legs move together when Issus is preparing and jumping. As the insect matures, the gear system is lost, leaving the adults to rely on friction between trochantera for leg synchronization.

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HIV-1 capsid hides virus from immune system

Of the two strains of HIV, HIV-1 is the more virulent and can avoid the human immune response, whereas HIV-2 is susceptible. This may be due to the fact that HIV-2 infects dendritic cells, which detect the virus and induce an innate immune response. HIV-1 cannot infect dendritic cells unless it is complexed with the HIV-2 protein Vpx, and even then the immune response isn’t induced until late in the viral life cycle, after integration into the host genome. Lahaye et al. found that only viral cDNA synthesis is required for viral detection by dendritic cells, not genome integration. Mutating the capsid proteins of HIV-1 showed that the capsid prevents sensing of HIV-1 cDNA until after the integration step. This new insight into how HIV-1 escapes immune detection may help HIV vaccine development.

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Transcription factor binding in exons affects protein evolution

Many amino acids are specified by multiple codons that are not present in equal frequencies in nature. Organisms display biases towards particular codons, and in this study Stamatoyannopoulos et al. reveal one explanation. They find that transcription factors bind within exonic coding sequences, providing a selective pressure determining which codon is used for that particular amino acid. These codons are called duons for their function as both an amino acid code and a transcription factor binding site.

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Chromosome silencing

Down syndrome is caused by the most common chromosomal abnormality in live-born humans: Trisomy 21. The association of the syndrome with an extra (or partial extra) copy of chromosome 21 was established in 1959. In the subsequent fifty years a number of advances have been made using mouse models, but there are still many unanswered questions about exactly why the presence of this extra chromosome should lead to the observed defects. An ideal experimental strategy would be to turn off the extra chromosome in human trisomy 21 cells and compare the “corrected” version of these cells with the original trisomic cells. This is exactly what a team led by Jeanne Lawrence at the University of Massachusetts Medical School has done. Down syndrome is not the only human trisomy disorder: trisomy 13 (Patau syndrome) and trisomy 18 (Edward’s syndrome), for example, produce even more severe effects, with life expectancy usually under one to two years. Inducible chromosome silencing of cells from affected individuals could therefore also provide insights into the molecular and cellular etiology of these diseases.

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Grow your own brain

By growing organs in a dish researchers can easily monitor and manipulate the organs’ development, gaining valuable insights into how they normally develop and which genes are involved. Now, however, a team of scientists from Vienna and Edinburgh have found a way to grow embryonic “brains” in culture, opening up a whole world of research possibilities. Their technique, published in Nature, has also already provided a new insight into the etiology of microcephaly, a severe brain defect.

[box style=”rounded”]Scizzling extra: In general, 2013 was a great year for growing your own kidneys, potentially a limb and liver. What organ will be next? [/box]

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Sparking metastatic cell growth

A somewhat controversial paper published in Nature Cell Biology this year showed that the perivescular niche regulates breast tumor cells dormancy. The paper showed how disseminated breast tumor cells (DTC) are kept dormant and how they can be activated and aggressively metastasize. Based on the paper, this is due to the interaction of interaction with the microvascularate, where thrombospondin-1 (TSP-1) induces quiescence in DTC and TGF-beta1 and periosstin induces DTC growth. This work opens the door for potential therapeutic against tumor relapse.

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Fear memories inherited epigenetically

Scientists showed that behavioral experiences can shape mice epigenetically in a way that is transmittable to offspring.  Male mice conditioned to fear an odor showed hypomethylation for the respective odor receptor in their sperm; offspring of these mice showed both increased expression of this receptor, and increased sensitivity to the odor that their fathers had been conditioned on.  Does this suggest that memories can be inherited?

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Grid cells found in humans

Scientists have long studied rats in a maze, but what about humans?  An exciting paper last August demonstrated that we, like out rodent counterparts, navigate in part using hippocampal grid cells.  Initially identified in the entorhinal cortex of rats back in 2005, grid cells have the interesting activity pattern of firing in a hexagonal grid in the spatial environment and as such are thought to underlie the activity of place cells. Since then grid cells have been found in mice, rats, and monkeys, and fMRI data has suggested grid cells in humans.  This paper used electrophysiological recordings to document grid cell activity in humans.

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Sleep facilitates metabolic clearance

Sleep is vital to our health, but researchers have never been entirely sure why.  It turns out part of the function of sleep may be washing waste products from the brain, leaving it clean and refreshed for a new day of use.  Exchange of cerebral spinal fluid (CSF), which is the primary means of washing waste products from the brain, was shown to be significantly higher when animals were asleep compared to waking.  This improved flow was traced back to increased interstitial space during sleep, and resulted in much more efficient clearance of waste products.  Thus, sleep may be crucial to flushing metabolites from the brain, leaving it fresh and ready for another day’s work.

[box style = “rounded”] Robert adds: As a college student my friends and I always had discussions about sleep and it was also this mysterious black box of why we actually need to sleep. Studies could show the effects of lack of sleep such as poor cognition and worse memory but this paper linked it to an actual mechanism by which this happens. First of all I found it very impressive that the researchers trained mice to sleep under the microscope. On top of that showing the shrinkage of the neurons and the flow of cerebrospinal fluid which cleans out metabolites finally linked the cognitive aspects of sleep deprivation to the physical brain. [/box]

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Poverty impedes cognitive function

People who are struggling financially often find it difficult to escape poverty, in part due to apparently poor decision making.  Investigators demonstrated that part of this vicious cycle may arise from cognitive impairment as a direct result of financial pressures.  The researchers found that thinking about finances reduced performance on cognitive tasks in participants who were struggling, but not in those who were financially comfortable.  Furthermore, farmers demonstrated poorer cognitive performance before harvest, at a time of relative poverty, compared to after harvest when money was more abundant.

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Gut Behavior

2013 has definitely been the year of the gut microbiome! Studies have shown that diet affects the composition of trillions of microorganisms in the human gut, and there is also a great deal of evidence pointing towards the gut microbiome affecting an individual’s susceptibility to a number of diseases. Recently published in Cell, Hsiao and colleagues report that gut microbiota also affect behavior, specifically in autism spectrum disorder (ASD). Using a mouse model displaying ASD behavioral features, the researchers saw that probiotic treatment not only altered microbial composition, but also corrected gastrointestinal epithelial barrier defects and reduced leakage of metabolites, as demonstrated by an altered serum metabolomic profile. Additionally, a number of ASD behaviors were improved, including communication, anxiety, and sensorimotor behaviors. The researchers further showed that a particular metabolite abundant in ASD mice but lowered with probiotic treatment is the cause of certain behavioral abnormalities, indicating that gut bacteria-specific effects on the mammalian metabolome influence host behavior.
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Your skin – their home

A paper published in Nature examined the diversity of the fungal and bacterial communities that call our skin home. The analysis done in this study revealed that the physiologic attributes and topography of skin differentially shape these two microbial communities. The study opens the door for studying how the pathogenic and commensal fungal and bacterial communities interact with each other and how it affects the maintenance of human health.

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Discovery of new male-female interaction can help control malaria

A study published in PLOS Biology provided the first demonstration of an interaction between a male allohormone and a female protein in insects.  The identification of a previously uncharacterized reproductive pathway in A. Gambiae has promise for the development of tools to control malaria-transmitting mosquito populations and interfere with the mating-induced pathway of oogenesis, which may have an effect on the development of Plasmodium malaria parasites.

[box style = “rounded”]Chris adds: “My friend chose this paper to present at journal club one week, because he thought it was well written, interesting etc etc. Unbeknownst to him, one of the paper’s authors was visiting us at the time. We sit down for journal club and one of the PIs comes in, sees the guy and exclaims (with mock exasperation) “you can’t be here!” Me and the presenter look at one another, confused. He presents journal club, and luckily enough, the paper is so well written, that he can’t really criticize it!” [/box]

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Using grapefruit to deliver chemotherapy

Published in Nature Communications, this paper describes how nanoparticles can be made from edible grapefruit lipids and used to deliver different types of therapeutic agents, including medicinal compounds, short interfering RNA, DNA expression vectors, and proteins to different types of cells. Grapefruit-derived nanovectors demonstrated the ability to inhibit tumor growth in two tumor animal models. Moreover, the grapefruit nanoparticles used in this study had no detectable toxic effects, could be manipulated or modified to target specific cells/ tissues, and were economical to create. Grapefruits may have a bad reputation for interfering with drugs, but perhaps in the future we will be using grapefruit products to deliver drugs more effectively!

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Getting CLARITY

In May, a new technique called CLARITY to effectively make tissue transparent through a new fixation technique was published in Nature. This new process has allowed them to clearly see neuron connection networks not possible before because they can now view the networks in thicker tissue sections. This new advancement will help researchers be able to better map the brain, but this new technology can also be to create 3-D images of other tissues such as cancer. This new ability allows us to gain better insight into the macroscopic networks within a specific tissue type.

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Crispier genome-editing

This year, the CRISPR technique was developed as an efficient gene-targeting method. The benefit of this method over the use of TALENS or a zinc-finger knockout is it allows for the rapid generation of mice that have multiple genetic mutations in just one step. The following review gives even more information on this new technique and compares its usefulness to that of TALENS and zinc-finger knockouts. Further, just couple of weeks ago, two back-to-back studies in Cell Stem Cell using the CRISPR-Cas9 system to cure diseases in mice and human stem cells.  In the first study the system was used in mice to correct the Crygc gene that causes cataracts; in the second study the CRSPR-Cas9 system was used to correct the CFTR locus in cultured intestinal stem cells of CF patients. These findings serve as a proof-of-concept that diseases caused by a single mutation can be “fixed” with genome editing using the CRISPR-Cas9 system.

What was your favorite paper this year? Let us know! And of course – use Scizzle to stay on top of your favorite topics and authors.

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5 Killer Ways to Use Your Smartphone for Science

 

Celine Cammarata

 

Take your smartphone from toy to tool with these simple tricks!

 

512px-App_Store_icon.svg1. Increase you APPtitude for Science

Want to calculate dilutions, figure out what that blob under your microscope is, or know which buffer to use?  There’s an app for that!  There are hundreds of great apps to help you with your research, but here are a few of our favorites:

[unordered_list style=”green-dot”]

  • Protocolpedia gives you offline access to hundred of protocols in the palm of your hand, as well as a set of useful calculators, a new lab timer feature, and even forums to discuss your work with other researchers.
  • Dilution is a super simple app that does exact what it sounds like – calculates dilutions.  This no frills app is perfect if you want find your dilution quickly without having to do math in your head or fill your lab notebook with scribbled numbers.
  • Beakr aims to give the one things that scientists often struggle to find: instant gratification. Like any great game, you can level up by completing tasks – in this case experiments – track your success, and even share (or compete) with friends.  Demotivation be gone!
  • Bacteria identification is unimaginatively named but incredibly handy, not to mention cool.  This little app combines Advanced Bacterial Identification Software based on morphology, growth conditions, etc. with an encyclopedia of bacterias to figure out just what it is you’re seeing on those plate.

[/unordered_list]

 [box style=”rounded”]Bonus tip: For even more great apps, check out the collections at BioTechniques and Life Technologies [/box]

 

Credit: katerha via flickr
Credit: katerha via flickr

  2. Read Papers Without Deforesting a Small Country

Forget teetering stacks of printed out papers – your smartphone is you new best friend for scientific reading.  Many journals, including Science, Nature, and Cell, now have their own apps allowing you to browse and access papers, and there are numerous apps for using PubMed on your mobile device.  The screen may seem small at first, but the trade off is being able to zoom in on figures and keep them looking great – no more monotone, impossible to interpret images courtesy of your printer.  Finally, apps such as Notability let you mark up PDFs of papers freely, so you can highlight, annotate, and doodle (and even add voice recordings) to your hearts content.

[box style=”rounded”]Bonus tip: Discover new and relevant papers on Scizzle and stay tuned for the Scizzle app! BTW, Did you see our new home page?[/box]

 

 

Credit: Jmak via WikiMedia Commons
Credit: Jmak via WikiMedia Commons

3. Collaboration Elaboration

Don’t let distance keep you apart – a little smartphone handiwork can make collaborators feel like they’re right in the lab with you, no more attempting to describe exactly what that weird result looked like.  To keep your hands free to work while still allowing your smartphone a good view of your experiments, try mounting it on a tripod – you can even make one yourself!  Use FaceTime or Skype for real-time collaboration, or snap pictures and video to share later – most phones let you edit videos easily so you can send only the relevant parts.  Time for lab meeting? Google+ Hangouts lets you discuss your work with multiple people at once – it’s simple, free, and gets the job done.  If you want to share a presentation with others, Fuze Meeting and Join.Me let you share screens and slides.

4. Present in the Present (Not in the Past)

Credit: WikiMedia Commons
Credit: WikiMedia Commons

Tired of dragging your computer around when it’s your turn to give lab meeting or journal club?  Present from your phone!  Making presentations from scratch is fairly simple on an iPhone using Keynote, though it’s still tricky on Android devices, and either platform features numerous ways to display presentations once created.  You can buy cables allowing connection to a projector, follow instructions to hook your iPhone or Android  to a larger screen, or even make a projector yourself.  Even if you do still lug in the laptop, you can liberate yourself from the podium by turning your iPhone or Android into a remote control, letting you advance the presentation from anywhere in the room, get a preview of upcoming slides, and even see presenter notes.

 

5. Keep it Clean

Credit: Karen/ karpatchi (Flickr)
Credit: Karen/ karpatchi (Flickr)

Ahh lab notebooks – they start out so pristine and organized, but somehow they always end up as a jumbled mess of scribbles and scotch-taped gel pictures.  Not anymore!  Now your lab notebook can stay crisp and clean, and can fit in your pocket.  Labguru is designed specifically for scientists, and lets you not only easily take notes – including pictures – but also pull up protocols, keep a running shopping list for supplies, and more.  The more general Evernote is also widely popular, and several blogs give in-depth tips for using Evernote in the lab.

[box style=”rounded”]Bonus tip: electronic lab notebooks are becoming so popular that a group at NYU did what any good scientists would and wrote a paper on it![/box]

Do you have a favorite app worth sharing? Did we miss any cool app?

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A Peek Inside Mouse Development

Sally Burn

The humble laboratory mouse is one of the greatest tools researchers have to model human development and disease. A common approach is to create a transgenic model of a human disorder, often by “knocking out” a gene in mice and then examining the effects. Transgenic mouse models are of particular use for characterizing disorders that disrupt embryonic development. When a disease progresses through childhood or adult life we can gather information about its pathogenesis, even taking samples from the patient for research along the way. However, genetic diseases that disrupt embryonic development often result in death during gestation or at birth, limiting opportunities to observe how the disease manifested. By examining embryonic development in mouse models we can get an idea of the timeline of events involved.

Unfortunately mouse embryonic development can usually only be examined in a fairly spasmodic manner. The roughly twenty days of mouse gestation cannot be observed in a single fluid motion; instead researchers must euthanize the mothers and remove the embryos for examination at set points throughout gestation. The embryos cannot survive outside the mother and so all that can be achieved is a snapshot of that moment in development. Imagine that instead of watching a movie all you get is a series of film stills, which you must piece together to try to get the full story, potentially missing out on key plot twists. Now, in an effort to address this problem, researchers are turning to a non-invasive imaging technique used routinely in humans: high frequency ultrasound.

In utero ultrasounds were first reported in mice nearly twenty years ago but are still not that widely use