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ON QUANTUM PARENTING

By | archive, creative

Many adults experience the same suffering, confusion, and joy that quantum physicists have every day. These are the parents of small children — they go through a hard time trying to understand the young, much like a physicist struggling to understand the tiny quantum mechanical systems.

To start, you never know what children are thinking about. In those tiny brains, there could be the idea of pulling a naughty deed or the thought of entertaining a cute dance. Until you go and interact with them, you simply don’t know whether they will do one or the other. Children are therefore simultaneously lovely angels and evil devils. Trying to figure out what’s going on in their head is just like trying to figure out whether the cat in the box is alive or not in Schrödinger’s thought experiment. Until you open that box, the cat is in a superposition of the two states—both dead and alive.

But kids will become devils for sure if you explicitly “tell” them what to do. For example, you might be making a beef stew for dinner. You get a phone call, and in doing so you leave the pot on the stove. You then tell your kids not to touch the pot because it is hot, but they go and touch it anyways. In effect, if you had not bother to tell them of the pot in the first place, these children might not have even notice its existence. Or put another way, the act of ordering them affects their behavior. This is much like measuring a quantum mechanical system — the act of measuring it will affect the behavior of the system and in turn affects the result a physicist will obtain.

It is also impossible to tell children to sit still. Kids can never sit still; they will start moving around randomly after sitting for only a few seconds. Quantum mechanics can explain this: Heisenberg’s Uncertainty Principle tells us that tiny things, which apparently includes children, will never have both a definite position and a definite momentum. This uncertainty drives both parents and physicists crazy.

Children go even crazier when you supply them with toys and candies. Transformer robots and Kit-Kats to them are much like energy to electrons. Both kids and electrons will go into an excited state. And similar to an electron’s quantized energy levels, children are either sad or happy, and are never in a mood that is mediocre. When supplied with toys and candies, kids will most likely become over-excited and start causing troubles, be it playing water gun in the living room or sewing your favorite jeans together. In that case, you as parents might want to “ground” them.

But these excited, troublesome children will eventually get tired. They will fall asleep with a big smile on their face, all the while dreaming about the toys and the candies they just had fun with. Much like electrons emitting various colours when they fall back from the excited state, the sleepy children’s big smiles are probably the most beautiful emission spectrum you will ever see in your life.

And that is why parents still love them despite their confusing thoughts and their diabolic pranks. You realize how pure and beautiful your babies are, just like a physicist who loves the beauty that lies at the heart of quantum mechanics.

About Jimmy Lee

Jimmy Lee is a Grade 12 student at Marc Garneau C.I.. He is currently suffering from Senioritis, a common disease found in high school students that are about to graduate. When he is not studying for a test or browsing GIFs on Tumblr, you can find him conducting Molecular Gastronomy experiments in the kitchen while struggling to sing Mariah Carey songs.

ANIMAL RESEARCH: THE GOOD, THE BAD, AND THE ALTERNATIVES

By | archive, textbook

Animal research is a very controversial topic that has been generating heated arguments and debates all around the world over the past few decades. Recently, there appears to be an overwhelming growth in animal right groups all over the world. Most of these groups don’t just support animal welfare, but they demand a ban on any kind of use of animals in research. At the same time, more and more pro-testing groups that support the humane use of animals in research have been forming – these proponents argue and recognize the essential role that animal research has played in medical advancements and breakthroughs throughout the years.

Imagine for a second, a world where animal research is banned. First of all, medical progress would come to a standstill, with a variety of major setbacks in developing treatments for devastating diseases, including neurodegenerative disorders such as Multiple Sclerosis, Parkinson’s disease, and Huntington`s disease. Yes, we would still be able to use non-invasive techniques on humans (ie Magnetic Resonance Imaging – MRI) and in vitro “test tube” experiments, but will that be adequate? At least in my field of research, Neuroscience, in vitro (Latin for “in glass”) studies are not very representative of the complexity of the human brain. This is a general disadvantage of in vitro studies; in most cases, the natural environment of the cells or the tissue cannot be easily replicated in a petri dish. Of course, in vitro experiments are useful in certain cases and there are various applications for them, but they don’t emulate the environment and complexity of a living “in vivo” tissue (no surrounding tissues, no blood supply, nutrients etc).

The truth of the matter is that the majority, if not all scientists and researchers acknowledge the difficult ethical issues that arise from animal research. To my knowledge there is no scientist that enjoys using animals in research, as it’s been insinuated by various animal extremists. This type of research is demanding, time-consuming, laborious and very expensive. Animals need to be housed, fed, constantly monitored and taken care of by specially trained animal welfare technicians and veterinarians, especially since stressed animals tend not to provide the best experimental results. Regardless, it is necessary, at least for the time being, to rely on such studies to understand how our body functions and to develop new effective drugs for diseases.

The most important question we need to address as researchers and scientists is the following: Why use animal models in research? Animal models can provide a great tool to learn about certain diseases, especially in regards to how they progresses in time and how they can be diagnosed. They also allow us to find new ways to treat diseases without endangering human lives in the process.

What animal activists fail to understand, in my opinion, is that we researchers appreciate the past and present contribution of animals to improving human health, helping cure diseases and saving lives. We take animal welfare very seriously and we are committed to the humane treatment of the animals under our care. Investigators are required to carefully design research projects and protocols, while taking into account the Three Rs (Reduction, Replacement and Refinement) [1]. The Three Rs represent widely accepted ethical principles that are taken into account when designing a research project involving animals. They promote the replacement of animals where possible and the reduction of the overall number of animals used in the other cases through refinement of the techniques and procedures employed. These principles also ensure there is a continuous refinement of the established protocols in order to provide better conditions and care for the animals.

One of the main arguments used by animal activists against the use of animal models in research is the fact that in many cases, the results obtained from animal research and treatments that worked in animals were not successful in clinical trials with humans and vice versa. [2] The fact of the matter is that no animal model can completely reproduce a human disease or a human organ simply because of the biological differences between the different species. However, by choosing the appropriate model you can get a relatively good representation and therefore more accurate and relevant results. This does not necessarily mean that we have never learned anything or provided safe and successful treatments based on animal research. On the contrary, there are countless examples that provide evidence on the merit of such practices. The development of several vaccines we use today is largely based on animal research, like the human papilloma virus (HPV) vaccine [3], the whooping cough and the polio vaccine; as well as the discovery of insulin, the development of organ transplant techniques [4] and anti-transplant rejection medication [5].

Another important argument made by animal activist is that there are several alternative methods to conduct research and gather the results necessary for medical advancements. These alternative methods, however, cannot replace animal research just yet. Some of these methods include imaging techniques, such as MRI and functional MRI (fMRI) scanning, in vitro testing, micro-dosing and computer models.

Imaging techniques like MRI and fMRI scanning allow us to see areas of the brain “light up” under different conditions giving us important information about how the brain works at a large-scale. Nevertheless, imaging techniques have their limitations. The resolution is quite low, which means that you cannot see individual brain cells, but rather whole areas that could contain thousands of different types of brain cells. In order to study brain disorders, it is important to know what cell types are affected and how, which is not possible with the current non-invasive imaging technology. Imaging techniques can provide invaluable information, but once again animal research is needed to understand diseases at the cellular and molecular level.

In vitro testing is based on the use of tissues and cells, a major source of which was and continues to be animals. Immortalized cell lines (that are preserved for many years and used over and over) are not always representative of the physiological functions of cells that are in their normal environment, the body. Even though there are applications for these immortalized cells, using “fresh” cells will yield results that will more closely represent what really happens in the body. We could possibly use human cells to address the need for these “fresh” cells, however, there are tissues and cells that are much more difficult to obtain from humans. One good example is brain cells. Would anyone be willing to go through unnecessary and extremely risky procedures to donate brain cells to science? Probably not. This brings us back to the use of animals to obtain the necessary cells to even conduct in vitro testing.

Micro-dosing (Phase-0 microdosing trials) is a new technique used to study the effect of drugs in humans by administrating very small doses, as the name of the technique suggests. The idea behind micro-dosing is the administration of doses so low that it is unlikely they will cause a large-scale response (throughout the body), but instead cause a small localized response that can be observed and studied. However, this technique has a few limitations. Since it only studies small doses of a drug, it cannot effectively predict the consequences of administrating a higher pharmacological dose. [6] Future studies may be able to exemplify whether the body responds the same to “micro-doses” and pharmacological “therapeutic” doses of a particular compound. Generally, micro-dosing seems to be a very promising tool that might potentially replace the use of some animals in drug testing trials in the upcoming years.

To quote Professor Stephen Hawking – “Computers can do amazing things. But even the most powerful computers can’t replace animal experiments in medical research.” (Quoted by Seriously Ill for Medical Research in 1996). Computers might not be fast or powerful enough yet to simulate and reflect all aspects human physiology, but they are closer than ever. New advancements in the fields of computer science and engineering are making projects like the “Human Brain Project” [7] (human brain simulation) possible. It is important to note that computers can’t possibly replace the study of a live brain, since we don’t understand its complexity to a point where we are able to produce programs that can represent brain function effectively. Computer model simulations could, however, contribute to the optimization of experimental protocols, and thus result in the reduction of animals required for research.

Research is always evolving, improving and progressing. It is possible that in the future we might not need to use animals for research proposes as better, less expensive and time consuming methods may be available. Unfortunately, we are still not at the stage where animal research is obsolete. I do believe that scientists can and should devote the time and effort to adopt such techniques when possible, as well as help develop and refine new techniques and procedures that will help minimize unnecessary use of animals. Finally, it is important to communicate to the general public how animal research is conducted and how carefully animal welfare is being addressed by scientists, as well as the measures that are in place to protect and care for the animals.

References:

1. Russell, W.M.S. and Burch, R.L. The Principles of Humane Experimental Technique. 1959.

2. Perel, P., et al., Comparison of treatment effects between animal experiments and clinical trials: systematic review. BMJ, 2007. 334(7586): p. 197.

3. Peng, S., et al., Development of a DNA vaccine targeting human papillomavirus type 16 oncoprotein E6. J Virol, 2004. 78(16): p. 8468-76.

4. Moore. F.D Give and Take: the Development of Tissue Transplantation. 1964, New York: Saunders.

5. Discoveries in Pharmacology, ed. Parnum, M.J. and Bruinvels, J. Vol. vol 3. 1986, Amsterdam: Elsevier.

6. Garner, R.C. and G. Lappin, The phase 0 microdosing concept. Br J Clin Pharmacol, 2006. 61(4): p. 367-70.

7. Human Brain Project, H.B. 2013; Available from: https://www.humanbrainproject.eu/

About Katerina Othonos

Katerina is a Neuroscience PhD student at UBC. When she isn't in the lab obsessing about making little neurons fat and happy, she enjoys reading sci-fi books and listening to heavy metal.

TYROSINASE GENE ANALYSIS AND PHENOTYPIC COMPARISON IN WOOKIEES WITH OCULOCUTANEOUS ALBINISM TYPE 1

By | archive

(This is the FOURTH paper on a special issue on Wookiee science. You can read the first here, the second here, and the third here).

APCMvol1p39-49front

Annals of Praetachoral Mechanics. (2014). Vol 1. pp39-49 pdf download.

ABSTRACT

Tyrosinase is an enzyme in the melanin biosynthetic pathway. Previous studies have shown that a lack in enzyme activity caused by mutations in the tyrosinase gene (TYR) results in tyrosinase-negative oculocutaneous albinism (OCA1) in humans. The purpose of this study was to investigate if albino Wookiees who lack tyrosinase activity possess similar genetic defects to those seen in humans with mutations in this gene. Fourteen albino Wookiee subjects were assessed for phenotypic clinical attributes related to OCA1 and tyrosinase activity. In addition, TYR of each subject was sequenced and compared to control subjects. It was found that 10 Wookiees possessing OCA1 had a mutation in TYR, while 4 did not. The findings of this study suggest that although Wookiees with OCA1 possess similar phenotypic and genotypic traits to humans with this type of albinism, other factors may be inhibiting the tyrosinase activity in Wookiees.

Keywords: Wookiee wookiee, albinism, tyrosinase, oculocutaneous albinism

Introduction

Mankind has come a long way in understanding the jungle planet Kashyyyk ever since its astounding discovery in 2006 [1]. The sightings of its humanoid-shaped inhabitants, the Wookiees (Wookiee wookiee), in particular have gained considerable attention and interest. Unarguably, the most striking morphological aspect of these arboreal alien species is the even coat of water-shedding hair which is found throughout their entire face and body. The color of this fur generally ranges from black to reddish brown. However, instances of albinism have also been reported. In fact, this condition appears to afflict about 1 in 10,000 Wookiees [2]. Wookiees who sport long, white hair fail to blend with the earth tones of their forest environment and are culturally considered unnatural [2]. As a result, albino Wookiees tend to suffer from social stigma and are often shunned by their family and society at large [2]. This study aims to explore the intricacies of pigment formation in Wookiees. Understanding the genetic underpinnings of albinism in Wookiees may allow us to reduce genetic defects that cause this disorder, and consequently help alleviate the related issues.

In 2008, it was established that convergent evolution had occurred between the human and Wookiee lineages [3]. Whole genomic sequencing in Wookiees and comparative genomics to the human genome demonstrated that Wookiees are highly related to Homo sapiens genetically [3]. In humans, deficient biosynthesis of the melanin pigment in the skin, hair, and eye gives rise to oculocutaneous albinism (OCA) [4]. It was first reported in 1989 that human OCA is triggered by a pathologic mutation of the tyrosinase gene (TYR) [5]. Since then, many studies have continued to analyze TYR and their relation to OCA. Today, it is understood that mutations in 4 genes accounts for the 4 distinct types of oculocutaneous albinism. These include mutations in TYR which leads to oculocutaneous albinism type 1 (OCA1,MIM# 203100), mutations in the OCA2 gene which leads to oculocutaneous albinism type 2 (OCA2, MIM# 203200), and mutations in the tyrosinase-related protein-1 (TYRP1) gene which leads to oculocutaneous albinism type 3 (OCA3,MIM# 203290) [4]. Additionally, it was found that mutations in the solute carrier family 45 member 2 (SLC45A2) or melanin antigen AlM1 contribute to oculocutaneous albinism type 4 (OCA4) [6]. Only the function of tyrosinase and tyrosinase-related protein-1 are identified, but both enzymes play an active role in the melanin biosynthetic pathway [4].

In humans, the TYR gene is cytogenically located in 11q14.3 [7]. More precisely, it is found at base pairs 88,911,039 to 89,028,926, and codes for the production of tyrosinase, the rate-limiting enzyme critical for catalyzing the multiple steps in the synthesis of melanin pigment [8]. Tyrosinase oxidizes the amino-acid tyrosine to dopaquinone, and via a series of additional chemical reactions, dopaquinone is converted to melanin, the substance which provides pigment in skin and hair. [8]. Melanin is also found in the retina where it plays a role in normal vision [9]. Presently, over 100 mutations in the TYR genes have been associated with humans suffering from OCA1 [6]. These mutations disrupt the activity of the tyrosinase enzyme, consequently preventing the production of melanin and causing problems with vision.

OCA1 is the most common type of albinism expressed in humans [8]. Human patients who are diagnosed with OCA Type 1 have reduced skin and hair pigmentation [6]. Consequently, they are extremely photosensitive, are highly prone to sun damage, and at risk of developing skin cancer [6]. Furthermore, their visual acuity is reduced especially in those who have nystagmus, where eye movement is often involuntary [6]. In Wookiees suffering from OCA, similar symptoms are expressed. In general, affected Wookiees have characteristic white fur and bleached irises. Although hypopigmentation makes them more prone to actinic damage than Wookiees without OCA, the dense hair throughout their bodies offers some protection from the sun. As a result, the risk of skin cancer due to sun damage is considerably less in Wookiees with OCA compared to humans with OCA [2].

Little research has been done on albinism in Wookiees. However, based on the genetic similarities previously established between Wookiees and humans, it is hypothesized that albinism in both species is caused by similar mutations. In this paper, we explore mutations of TYR in Wookiees given that OCA1 is the most common in humans [8].

Materials and Methods

Wookiee Subjects and Controls
This study received ethics approval from the Intergalactic Institute of Molecular Biology, Kashyyyr. Informed written consent was obtained from the adult subjects (age > 17) and the parents of minor subjects (age < 18). A total of 14 albino Wookiees with OCA1 phenotype (albino) and 10 wild-type Wookiees with black or brown hair (control group) were recruited from the Woolwarricca and Korrokrrayyo regions of Kashyyyr. All subjects were examined by an Ophthalmologist and a physician to confirm diagnosis of OCA1. Specifically, hypopigmentation of the hair and skin and the presence of eye aberrations, including nystagmus, strabismus, photophobia, and poor vision were evaluated. Hair and peripheral blood samples were collected from all participants.

Tyrosinase Activity
Wookiee hair samples were assayed for the tyrosine hydroxylase activity of tyrosinase by incubating hair preparations (50 mg hair homogenized in 1 ml phosphate buffer) in 0.5 ml of 0.1 mM tyrosine, 0.1 mM L-DOPA, and 5 μCi/ml of [3H]tyrosine in phosphate buffer. Incubations were carried out at 37°C for 1 hr after which the reaction was terminated with 1 ml charcoal (10% w/v, in 0.1 N HCl) and centrifuged at 2000 X g for 10 min at 4°C. Production of 3H2O in the supernatant was measured using the Analytic 6895 scintillation counter equipped with a DPM processor (Technomedia, Montreal, QC, CA).

Genomic DNA Extraction
Genomic DNA from each Wookiee was extracted from 100 μl peripheral blood using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions.

PCR amplification of TYR gene segments
Exon 3 and 5 amplifications were performed as was described previously [3]. All other coding and flanking noncoding TYR exons were amplified in individual PCR reactions using primers designed with Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/) based on the sequence of Wookiee TYR (Gene Bank/EMBL Data bank accession number, W00K133). Sequences of primers used for PCR amplification can be found in Table 1. Amplification was performed in 25 μl volumes with 100 ng genomic DNA, 1 uM of each primer, 2 mM MgCl2, 200 uM of each dNTP (dATP, dGTP, dCTP, dTTP), and 1 U AmpliTaq Gold 360 DNA Polymerase (Applied Biosystems, Grand Island, NY, USA). Thermocycling was performed as follows using BioRad C1000 Thermocycler: initial holding for 10 min at 95°C, 35 cycles of 30 sec at 95°C, 30 sec at 55°C, 30 sec at 72°C, with a final extension at 72°C for 7 min. Absence of non-specific PCR amplicons was confirmed by electrophoresis in a 1% agarose gel. The PCR product was purified using the QIAquick PCR Purification kit following manufacturer’s instructions (Qiagen, Valencia, CA, USA).

APCMvol1p39table01Table 1. (Click to Enlarge) Oligonucleotide primers used for amplification of TYR.

Sequencing of PCR amplicons
We sequenced each of the PCR products in both directions using an ABI 373A automated sequencing system (Perkin-Elmer/Applied Biosystems Division, Foster City, CA, USA). The sequencing reactions were carried out using a Taq FS Dye Terminator Cycle Sequencing Kit (Perkin-Elmer/Applied Biosystems Division, Foster City, CA, USA). The nucleotide sequences of the sequencing primers are summarized in Table 2. All sequences obtained (subjects and controls) were subjected to computer-assisted systematic mutational analysis using AlignX of Vector NTI Suite (Informax, Inc., North Bethesda, MD, USA).

APCMvol1p39table02Table 2. (Click to Enlarge) Oligonucleotide primers used for sequencing of TYR PCR fragments.

Results and Discussion

Clinical Analysis
A total of 14 Wookiees with OCA1 phenotype and 10 wild-type Wookiees with black or brown hair (control group) were recruited from the Woolwarricca and Korrokrrayyo regions of Kashyyyr.

Diagnosis of OCA1 in Wookiees was completed through visual evaluation of ophthalmic and pigmentation phenotypes distinct to this disease. Tyrosinase activity in the hair of each Wookiee subject was completed and results confirmed that all test subjects had no enzyme activity compared to control subjects.

Visual acuity for the Wookiees with OCA ranged from 0.1 to 0.85 (median, 0.28). Ten of the 14 Wookiees with OCA1 (71%) had albinotic fundus with the remaining 4 characterized as normal for this trait. Interestingly, all OCA subjects possessing normal fundus were under the age of five with three of these Wookiees being female. Eleven OCA Wookiees showed foveal hypoplasia, with three having normal foveal reflexes. All Wookiees with OCA1 had nystagmus and photophobia, regardless of their sex and age. The ophthalmic phenotypes observed in Wookiees with OCA1 held close similarity to those characteristics observed in human cases of the disease [11, 12].

In humans, OCA1 is characterized by loss of pigmentation in the skin, hair and eyes [12]. All Wookiees possessing OCA1 lacked pigment in their hair and skin. Although the majority of Wookiees surveyed (71%) had translucent iris colour characteristic of the disease, the same 4 Wookiees that showed normal fundus had grey-purple (n = 3) or grey-green (n = 1) colouring in the iris. This finding is in disagreement with the expression of OCA1 in humans, which all have “bleached” iris expression [13].

Sequence Analysis
We analyzed the sequence of TYR of the 14 OCA1 phenotype and 10 wild-type Wookiees. 4 of the OCA1 Wookiees did not have mutations in TYR (Table 3). Their TYR sequence was comparable to the sequence demonstrated in the dark hair control group. However, 10 of the OCA1 Wookiees had mutations on both alleles. Figure 1 and Table 4 show the compilation of six recurring mutations found on TYR for the 10 OCA1 phenotype Wookiees. Figure 1a is a sequence showing the 1090AC mutation where the A has been replaced with a C nucleotide. The change is a missense mutation where the amino acid isoleucine becomes leucine, both of which are non-polar. However, figure 1b shows another mutation 1138D58 where large fragments of nucleotides are missing between positions 1138 G and 1158 C. This deletion is a frameshift mutation that shifts the grouping of the nucleotides, changing the code for the amino acids downstream from it. This could account for the inactivity of the tyrosinase due to incorrect amino acids being coded. Figure 1c has the 1467T68 mutation where an additional T nucleotide has been inserted into the TYR sequence. This addition also causes a frameshift mutation. Figure 1d shows the 127CT mutation where the change in nucleotides from C to T causes the codon normally for arginine to become a stop codon. This can affect TYR in that protein will be truncated. Figure 1e shows the 1150CG where C has changed to a G nucleotide. This missense mutation results in a change from histidine to asparagine. The histidine is basic and has a ring structure while asparagine is an amide. This drastic change will likely affect the overall structure of the tyrosinase protein. Finally, Figure 1f has 823GT where the expected G is instead a T due to a missense mutation. This causes the amino acid alanine to become a nucleophilic serine.

APCMvol1p39table03Table 3. (Click to Enlarge) TYR allele mutations.

APCMvol1p39table04Table 4. (Click to Enlarge) Mutations in TYR gene and their effects on protein expression.

APCMvol1p39figure1Figure 1. (Click to Enlarge) Direct sequence sections of the 6 mutations found in the tyrosinase gene.

We examined the frequency of these 6 mutations from 10 OCA1 phenotypic Wookiees with our control group of 10 wild-type Wookiees and found that there were no mutations in the 20 alleles surveyed. Therefore, it is possible that the six novel mutations are pathologic and not polymorphic for OCA1. We also found that the missense mutations in TYR gene were the most common from our 10 TYR mutant OCA1 phenotype Wookiees, which is similar in humans [8].

In humans, the structure of tyrosinase contains 529 amino acids, with a signal peptide at the amino end and a transmembrane region at the carboxyl end [5]. The catalytic site of the enzyme contains two copper atoms ligated to six histidine residues. It also has glycosylation sites of asparagine residues at 86, 111, 161, 230, 384, and 499 [5]. The Wookiee TYR 1150CG mutation is located at the 384-residue site, which directly modifies one of these histidine amino acids and may have caused the inactivation of the mutated enzyme. The change in the sites of the asparagine residues would also have been affected by the Wookiee 1138D58 mutation since the nucleotides coding for the area were gone. In addition, the 1467T68 mutation is where there is a T nucleotide insertion changes all the amino acids downstream, thereby possibility affecting the site at 499. The 127CT mutation forms a stop codon relatively early, resulting in an incomplete tyrosinase protein that is unable to function normally. The 1090AC and 823GT mutations result in the change of isoleucine to leucine, and alanine to serene respectively. It is suspected that the change in amino acids caused an inactivation of the enzyme due to an alteration in the tertiary structure at the enzyme’s catalytic sites. This may also be a factor for the 1150CG, 1138D58, and 1467T68 mutations. Further studies will be required to verify why these six mutations cause a lack of tyrosinase activity.

Genotype-Phenotype Comparison
In humans, existing evidence from literature suggests that the genotype-phenotype correlation is strongest in individuals with two null TYR alleles [6]. However, attempts at genotype-phenotype correlations for OCA mutations have also proven difficult and have largely been unsuccessful because same TYR mutations are reported in patients with varying clinical presentations [6]. To date, more than 300 TYR variants have been reported to cause albinism in humans [6]. Furthermore, individuals who are heterozygous for the TYR mutant allele may demonstrate partial albinism wherein melanin is not completely absent and the affected individual is able to still produce some pigmentation. Conversely, our data suggest that partial albinism is potentially not present in Wookiees. As illustrated in Table 3, albinism in Wookiees is demonstrated when both TYR alleles are mutated. We did not observe any cases wherein an albino Wookiee was only heterozygous for the mutant TYR gene.

Previous studies have shown that OCA1 occurs in humans when the TYR activity is disrupted. Results of this study illustrate that this is also true for Wookiees (Table 3). Ten out of 14 albino Wookiee subjects have mutations on both alleles of their TYR genes. Interestingly, our data (Table 3) indicated that 4 of our OCA Wookiees with wild type TYR sequences also demonstrated albinism. Initial screening based on their phenotypic characteristics had suggested that these
albino Wookiees lacked tyrosinase activity, despite the lack of mutations in the TYR alleles. This finding is surprising and suggests that there may be other factors inhibiting the melanin production in Wookiees, by directly inhibiting tyrosinase activity, and that TYR is not the only gene that determines albinism in Wookiees. In humans, various studies have revealed that the mutation of several other genes gives rise to different types of albinism. It is possible that various melanogenic pathways may be present in Wookiees that have yet to be discovered, and that different modes of albinism may also exist.

There was a difference in the visual acuity in OCA Wookiees with the wild type TYR gene and the OCA Wookiees with the mutated TYR genes. Wookiees who expressed OCA1 but do not have a mutation in their TYR genes have a mean visual acuity of 0.75, whereas Wookiees with mutated TYR genes and OCA have a mean visual acuity of 0.27. This difference is statistically significant, having p

It is also interesting to note that the 4 albino Wookiees who had wild type TYR all came from the region of Korrokrrayyo, the tallest and most massive mountainous region in Kashyyyk (Table 5). It is impossible to conclude whether the region actually plays a role in the manifestation of albinism in Wookiees because of our limited knowledge regarding the environment and due to the small sample sized used in this study. However, it is important to acknowledge that environmental factors may certainly be critical in defining phenotypes, especially during early development. More studies would need to be conducted. Genes are not a steadfast blueprint for heredity. Specific environmental factors, which have yet to be determined, may be present in Korrokrrayyo that are affecting and altering gene expression in Wookiees.

APCMvol1p39table05Table 5. (Click to Enlarge) Phenotypic characteristics of Wookies OCA1 subjects.

Acknowledgements
The authors thank the participants and their families for their participation. We also thank Mike Liverspokie for his technical assistance and Dr. Kankertop for critique of the manuscript. This work was supported in part by the Galactic Empire Research Foundation under Grant 226/2012-GERA

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11. Jaworek T, Kausar T, Bell S, Tariq N, Masgood M, Sohail A, Ali M, Igbal F, Rasool S, Shaikh R, Ahmed Z. Molecular genetic studies and delineation of the oculocutaneous albinism phenotype in the Pakistani population. Orphanet J Rare Dis. 2012; 7: 44

12. Tomita Y, Miyamura Y. Oculocutaneous albinism and analysis of tyrosinase gene in Japanese patients. Nagoya J Med Sci. 1998; 61: 97-102.

13. Shibahara S. Mutations of the tyrosinase gene in oculocutaneous albinism. Pigment Cell Res. 1992; 5: 279-83.

About Michelle Chow, Keely Johnston, and Roseanne Lim

Michelle is a Cell Biology and Genetics student. After a long day of fish dissections and running western blots she enjoys curling up with a good book. Keely is a Master's student in Dr. Kevin Allen’s food microbiology laboratory at UBC Vancouver. When she isn’t wearing nitrile gloves and trying to find Listeria monocytogenes in food processing plants she likes to fall asleep during movies, speed walk, and make lists of things she likes to do. Roseanne is a 5th year Animal Biology undergrad student in UBC Vancouver and is also a research assistant at BC Children's Hospital. Her project looks at childhood malnutrition in paediatric surgical patients - more specifically she is interested in determining how the nutritional status of the child and other patient factors would impact surgical outcomes.

DISCOVERING THE WORLD BENEATH OUR FEET: THE SOIL MICROBIAL ECOLOGIST’S TOOLBOX IN THE AGE OF THE METAGENOME

By | archive, textbook

It is seven in the morning and your alarm is chirping away, or maybe it’s the morning sun, or the screech of a parent pressed for time. Regardless of the method from which you awake from your slumber, you crawl out of bed and feel that familiar morning growl emanating from your hungry gut. Some might grab a snack and quickly rush out the door, others might ruminate over an elaborate breakfast, and some are content with a simple bowl of oatmeal. This often first and necessary act of the day is repeated by billions of humans around the globe and should serve to remind us all that we are nothing without food. However, implicit in this realization is another: that there can be no food without that life-giving stuff beneath our feet – the soil.

Soil silently performs a multitude of services critical for life-sustaining ecosystem functions. It controls the cycling of energy (carbon) and nutrients (nitrogen, phosphorus, potassium, sulphur etc.) that sustain the food and fibre we depend on for nourishment and materials. Soils filter our drinking water and with careful management, can fix carbon dioxide and reduce other greenhouse gases to mitigate the effects of climate change. Soil is teeming with life – just one teaspoon of fertile soil can contain 9 billion microbes, larger than the sum of all humans on this planet (Doran et al. 1999). These microbes are largely responsible for the biochemical transformations that provide the above ecosystem functions. In a sense, we can’t live without these microbes, and for this reason, understanding who they are and what they are doing can help us in nearly limitless ways, from increasing crop yields to finding novel enzymes to better understanding climate change.

The challenge, however, remains in how exactly do we study and characterize who these microbes are and what they are doing? Most soil microbes are invisible to the naked eye and less than 1% of microbial life has been cultured in the lab (Torsvik and Øvreås, 2002). For many years soil scientists referred to the soil microbial community as a ‘black box,’ meaning reliable measures of soil processes, such as soil nutrient concentrations, were easily obtained, but how the microbial community adapts to and influences these processes remained a mystery. The soil scientist’s toolbox was not equipped to measure who is out there, much less what they are doing. Today, that black box is beginning to be pried open largely due to advances in human genetics that paved the way forward for microbiologists of all stripes and colours.

The driving force behind the current revolution in understanding soil microbial communities has its roots in the Human Genome Project. A genome is the total complement of DNA from a single organism; it contains all of the genes that produce all of the proteins that make an organism what it is (Clark et al. 2009). The Human Genome Project was an unprecedented investment in genetic research – it cost U.S. taxpayers $2.7 billion but enabled scientists to sequence the human genetic code in its entirety by 2003 (NHGRI 2010). A sequenced genome is an invaluable resource, especially for human health, as mutations within the genetic code that lead to disease can be better understood, potentially leading to alternative medical treatments. The technology used to sequence the human genome is known as Sanger sequencing, a method that became the rate limiting step in sequencing the 3 billion base pairs in the human genome (Mardis 2011). The massive investment in the human genome project, coupled with the drive to increase the speed of DNA sequencing, led to new developments in sequencing technology, known today as ‘next generation sequencing.’

Decoding soil microbial genomes is an important step in understanding the microbial ‘black box.’ From knowledge of the genetic code of a community of soil organisms comes inference of the capabilities of that community. For example, scientists may expect soil samples with a diverse complement of genes that code for enzymes involved in decomposition to more quickly decompose inputs of organic matter such as fallen leaves. The problem though is that DNA extracted from soil does not contain DNA from only one type of microbe; one estimate pegs the diversity of prokaryotes (unicellular microbes without a nucleus) at 52 000 unique species in one gram of soil (Roesch et al. 2007). The average genome size of prokaryotic cells is 2 million base pairs, which would require that 104 billion base pairs be sequenced for a single gram of soil (Gilbert and Dupont 2011). In contrast, the well-funded human genome project took 13 years to complete with only 3 billion base pairs sequenced. It is precisely for this reason that next generation sequencing technology has allowed such advancements in the study of soil microbial communities.

The first high-throughput second generation sequencing platform was introduced in 2005 as the Roche 454 pyrosequencer (MacLean et al 2009). Other platforms are available and in some ways are more widely used today (the Illumina set up for example), but a simplified outline of the method for the 454 sequencing technology is highlighted here:

DNA extracted from a soil sample is first ligated (attached) to universal adapters. The DNA-adapter complex is then immobilized onto reaction beads before sequencing begins. Each bead now contains a unique fragment of extracted DNA, and thousands of beads, each with a different DNA fragment, are loaded onto a plate with thousands of wells where the individual beads reside (MacLean et al. 2009, Mardis 2008). In this way, each well houses a bead that itself is carrying a unique piece of extracted DNA. The soil DNA attached to the beads is single stranded, meaning nucleotide bases (G, A, T and C) remain unpaired and will readily bind with their mate to form a base pair. Sequencing begins by flooding the plate with a single nucleotide. If a fragment in any well has an unpaired adenine (A) that is next in sequence, and thymine (T) is flooded into the plate, an A-T bond will form. The nucleotides that are flooded onto the plate are modified such that when bound to their mate, the reaction triggers the activity of luciferase, the same enzyme responsible for the light emitted from fireflies (Mardis 2008). The activity of luciferase emits light that is detected by a camera. The plate is then washed of nucleotides, another different nucleotide is introduced, light is emitted when the added nucleotide finds its mate and the camera snaps another picture. Every well that has a DNA fragment with an unpaired nucleotide this is the made of the nucleotide being flooded will light up, and the light will be captured by the camera from multiple wells at the same time. In this way, thousands of DNA fragments can be simultaneously sequenced, driving down both the time and cost of sequencing. This is a powerful technique – had this technology been around when the human genome was being sequenced, it would have sequenced the entire human genome in 10 days or less (Nyrén 2007).

With advanced sequencing technologies in their toolkit, soil scientists (who might now call themselves soil microbiologists) can get down to the business of figuring out who is beneath our feet, and what they are doing. Easier said then done. The reality is that genomic DNA extracted from soils can come from hundreds of thousands of uncultured, never characterized, mystery microbes. The DNA extraction procedure produces millions of DNA fragments in a ‘shotgun’ approach such that bits and pieces of the entire soil microbial community are sequenced to produce a profile of all genes distributed throughout the community (Gilbert and Dupont, 2011). This method of study is in contrast to genomic studies where the genome of only one organism is considered. Handelsman et al. (1998) in their study of soil microbes from environmental DNA coined the term ‘metagenomics’ to describe this new branch of genomic research.

Metagenomic studies of soil DNA produce vast quantities of discontinuous sequence data. Post-sequencing data processing involves stitching together the fragments of DNA into something that resembles partial microbial genes. This work is computationally difficult – it is as if you fired a shotgun close-range at a painting and then attempted to re-construct the painting by piecing together all the obliterated bits of canvass without knowing what the painting looked like to begin with. In fact, an entire field – bioinformatics – is devoted to developing algorithms for processing metagenomic sequence data. The first step in data processing is to attempt to stitch together (assemble) the sequence data to form longer DNA fragments (Wooley et al. 2010). Longer fragments are easier to characterize and these fragments are ‘binned’ to assign the DNA fragments to a known microbial species, usually by comparing sample DNA to reference databases using tools such as the Basic Local Alignment Search Tool (BLAST). One downfall is most of the sequences in a metagenomic dataset will remain unassigned because most of the reference database is derived from well-characterized, cultured organisms (Simon and Daniel, 2010). Overall, processing sequence data is the last step when trying to piece together the structure and function of a soil microbial community. Although reference databases are incomplete, they do assign some collected fragments to known microbial species that have well characterized genes with known function. Less than 10 years ago this was a largely impossible or at least a prohibitively expensive task. Advances in sequencing technology, bioinformatics and reference databases will only improve our resolution over time.

With the advances afforded to the study of soil microbial communities through second generation sequencing technology, soil scientists have only just begun pry open the microbial black box. Currently, research is largely focused on microbial decomposition of plant inputs into soil due first to the search for novel enzymes for the biofuels industry, and second, because microbial decomposition has important consequences for global CO2 emissions (Baldrain 2014). The field is also rapidly changing, with emphasis increasingly on characterizing gene products (RNA, proteins) over the metagenome so as to capture the genes and proteins that are active in the soil under various environmental conditions. The rapid advances in sequencing technology over the last 10 years make this an exciting time for soil science research. These methods may one day prove to reveal the secrets held within one of the last frontier in modern science – the soil beneath our feet.

References

Baldrian, P., & López-Mondéjar, R. (2014). Microbial genomics, transcriptomics and proteomics: new discoveries in decomposition research using complementary methods. Applied Microbiology and Biotechnology, 1-7.

Clark, D. P., Dunlap, P. V., Madigan, M. T., & Martinko, J. M. (2009). Brock Biology of Microorganisms.

Doran, J. W., Jones, A. J., Arshad, M. A., & Gilley, J. E. (1998). 2 Determinants of Soil Quality and Health. Soil quality and Soil Erosion, 17.

Gilbert, J. A., & Dupont, C. L. (2011). Microbial metagenomics: beyond the genome. Annual Review of Marine Science, 3, 347-371.

Handelsman, J., Rondon, M. R., Brady, S. F., Clardy, J., & Goodman, R. M. (1998). Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chemistry & Biology, 5(10), R245-R249.

MacLean, D., Jones, J. D., & Studholme, D. J. (2009). Application of’next-generation’sequencing technologies to microbial genetics. Nature Reviews Microbiology, 7(4), 287-296.

Mardis, E. R. (2008). Next-generation DNA sequencing methods. Annu. Rev. Genomics Hum. Genet., 9, 387-402.

Mardis, E. R. (2011). A decade’s perspective on DNA sequencing technology. Nature, 470(7333), 198-203.

National Human genome Research Institute. “The Human Genome Project Frequently Asked Questions.” Genome.gov. http://www.genome.gov/11006943 (accessed Feb 12 2014).

Nyrén, P. (2007). The History of Pyrosequencing®. In Pyrosequencing® Protocols (pp. 1-13). Humana Press

Roesch, L. F. W., Roberta R. F., Alberto R., George, C., Hadwin, A.K.M., Kent, A.D., Daroub, S.H., Camargo, F.A.O., Farmerie, W.G., and Triplett, E.W. “Pyrosequencing enumerates and contrasts soil microbial diversity.” The ISME journal 1, no. 4 (2007): 283-290.

Simon, C., & Daniel, R. (2011). Metagenomic analyses: past and future trends. Applied and Environmental Microbiology, 77(4), 1153-1161.

Torsvik, V., & Øvreås, L. (2002). Microbial diversity and function in soil: from genes to ecosystems. Current Opinion in Microbiology, 5(3), 240-245.

Wooley, J. C., Godzik, A., & Friedberg, I. (2010). A primer on metagenomics.PLoS Computational Biology, 6(2), e1000667.

About Tim Philpott

Tim Philpott is a PhD student in the Faculty of Forestry's Belowground Ecosystem Group at UBC. He is trying to get a handle on the brave new world of metatranscriptomics and apply it to soil ecology, hopefully before a new sequencing technology makes his new skills obsolete.

ISAAC NEWTON, STANDING ON THE SHOULDER OF GIANTS. EXCERPTS FROM HIS DIARY

By | archive, humour

“If I have seen a little further it is by standing on the shoulders of Giants.”
Isaac Newton in a letter to his rival Robert Hooke, 1676

- – -

May 14th, 1665
Went to the post office today, thinking that I’d be picking up a grant proposal from the Royal Society.  Imagine my surprise when I turned up and instead of a grant, there was a giant waiting for me.

May 22nd, 1665
A week later and I’m still a bit confused on what to do with the giant, especially since it follows me relentlessly.  Friends have not been much help in this regard; enemies even less so.  Some have even foolishly suggested my kicking it in the shins or standing on his shoulders.

May 22nd, 1665
Do not kick a giant in the shins.  Ever.

May 27th, 1665
Stood on the giant’s shoulders today.  Surprised to say that it was wonderful.  The world looks so different from this new vantage point, and my head is spinning from new perspectives.

May 28th, 1665
Have decided that I am never coming down.

June 19th, 1665
My cat got stuck in the apple tree today.  Luckily, I can easily reach whilst on the shoulders of the giant.

June 24th, 1665
Cat got stuck in the apple tree again.

July 17th, 1665

Let’s call this a lesson learned in unintended consequences.  In essence, a few weeks ago, I was all pleased with myself since I had just invented the cat door – but you know what?  Turns out, this was not a good idea.  Stupid cat is now letting itself out and getting stuck in the apple tree daily now.  The giant, fed up, has left.

August 27th, 1665
Picked up a new giant today.  This one is Welsh and not averse to cats.

September 16th, 1665
With the fall upon us, we find ourselves very popular amongst all the apple tree owners.  Our height makes us excellent and efficient harvesters. Indeed, I feel a bit like a celebrity, albeit a celebrity paid in bushels of apples.

October 4th, 1665
The giant and I are making apple sauce.  This is actually quite difficult when standing on a giant’s shoulders.

October 11th, 1665
More apple picking today!  More apple bushels in my kitchen!

November 2nd, 1665
I swear if I ever see another apple, I will fucking kill someone.

December 16th, 1665
The giant and I had a grand time at our first Christmas party.  He had fun dressing up as Father Christmas, but it was kind of weird when all my friends wanted to pretend to be little and sit on his lap.

December 19th, 1665
Back from my ninth Christmas party. The giant and I are really popular!

December 21th, 1665
Just had the horrid realization that we have only been invited to these so called “Christmas parties,” on account of our height.  Turns out we are useful for putting star and angel ornaments on the top of really big Christmas trees!  I feel so used.

February 10th, 1666
Now, I am starting to get annoyed by the many many locals who constantly come by and ask for some sort of giant related help. Dusting off ceiling cobwebs, hanging up large paintings, and reaching for books on the high shelf – it all gets a little old after a while.

March 28th, 1666
The giant has accidentally stepped on the cat. This seems to be bittersweet.

March 30th, 1666
The weather is starting to clear a bit, but the giant seems different.  He seems melancholy and distant.  The sadness is especially noticeable when I am standing on his shoulders, as they tend to be hunched these days.

June 6th, 1666
I brought home a new cat today, but the giant seemed not to notice.  I am genuinely worried.  Maybe I should get off his shoulders?  But then again, I don’t want to act too hastily.

September 3rd, 1666
It is fall again.  After a difficult few months, the giant has decided to leave.  In a strange way, being back on solid ground feels right.  Even the apple trees look pretty again.  Maybe, I’ll even try sitting under one tomorrow…

About David Ng

David (@ng_dave) is Faculty at the Michael Smith Labs. His writing has appeared in places such as McSweeney's, The Walrus, and also as an occasional blogger at boingboing.net. If you're looking for a graphic for your next science talk, he encourages you to check out his blog, popperfont.net.