Breaking into the Circadian Clock

Japan Daily Press

Sometime in the future a person could be waking up after a short 16-hour snooze. Preparing a hearty meal to start their “day”, they will probably be thinking about the hours ahead of them. Maybe they have a night shift, a morning to see friends and family, a hobby to indulge in somewhere in-between. Time is cheap when you don’t have to sleep for another 36 hours.

This of course is fantasy, but could it eventually become a possibility  – or even a necessity? After all, society in the 21^st century is “Open 24/7”. In crude terms of thinking, there is a need for us to adapt to our new social patterns.

A growing number of individuals in countries affluent and developing alike are taking on employment that requires work during hours that are deemed “unsociable”. Late-night bar workers, early-morning cleaners and night-shift security  – most people know at least one or two people who work in these sectors.

Working unsociable hours usually comes with better pay and more flexible free time in the day, but research is starting to uncover the dangerous long-term effects of an abnormal day/night cycle.

Following a rough 24-hour period and reacting to simple environmental elements, Circadian rhythms are our biological cycles that adhere to the light/dark cycle of the day. In humans they affect sleep cycles, hormone release, body temperature and many other important physiological factors. Abnormalities in these cycles can cause insomnia, depression and even obesity.

Psychology Blog

People experience the effects of going against their Circadian rhythms quite often; jet lag, lack of sleep and that groggy feeling you get after an afternoon nap are prime examples of what happens when your body is out of sync!

All human necessities are controlled subconsciously by our biological clocks, named so due to their tendency to cause the hand of our Circadian cycles to turn. These clocks drive all life, not just humans, and are genetically rigid in our make-up. Tucked in the hypothalamus part of the brain is a bundle of neurones called the Suprachiasmatic Nucleus (SCN). This construction of neurones is accepted to be the human Master Clock, the conductor of all of the other biological clocks.

Hijacking our own daily rhythms and biological routines would be a very complex ordeal, but we are starting to understand more about Circadian Rhythms.

To avoid the risks of obstructing our natural biological cycles we would need to modify our own physiology - our needs to rest and sleep (accepting that eating and drinking is, pretty much, impossible to avoid). Lots of our Circadian rhythms summate together to produce one large desired effect, which makes the “root” of each Circadian action hard to locate. Scientists at the University of Oxford have recently managed to pinpoint a very significant element involved in Drosophila (fruit fly) Circadian rhythms: a genetic “on-off switch” for sleep. Gero Miesenbock and team used light to turn on genetically modified cells that react to certain wavelengths, due to the presence of the gene. The light caused dopamine release, which inhibited sleep-promoting neurones, thus waking the flies.

Working directly with Drosophila’s neurones, Fang Guo at Brandeis University, Massachusetts, demonstrated an opposite effect: in the same type of fly, after activating neurons in a biological clock, glutamate is release which then turns off neurones in the part of the Master Clock that promotes activity.

Although we already know that dopamine in animals is generally related to active behaviour, and that glutamate inhibits areas of the SCN in humans, we can’t yet assume the same model seen in Drosophila as in more complex organisms. This type of research does, however, demonstrate that we have already started to pick-apart the inner workings of Circadian activity.

Looking at something closer to humans, Circadian rhythms in mice have been found to lengthen when caffeine is involved. We all know that coffee alters our sleep pattern, but researchers Oike, Kobori, Suzuki & Ishida found in 2011 that administering caffeine into the growth media of both human bone-cancer cells and mouse stem cells actually alters the length of the biological cycles. On an intracellular level it was seen that processes associated with high-level activity were extended, causing a lengthening shift in the whole “daily” cycle of the cell. Another experiment with caffeine was attempted which involved the stimulant being added to ex vivo (outside of organism) liver cells, which resulted in signal delays in the SCN – something that could be linked with the lengthening of the cycles.

To try something with a whole organism, the researchers ran a third test with live mice. “Limitless” supplies of caffeine were made available to the subjects for a week under normal light-dark conditions. The findings of this in vivo experiment were that the mice were indeed active later into their dark periods in comparison to the control group.

Although for these mice the administration of a caffeine fountain was a rudimentary way of modifying circadian rhythms, this study raises two interesting points: firstly, as conserved as the genetic foundations of these biorhythms are it is possible to modify them. Secondly, the modification of these cycles can be performed in complex, mammalian organisms and not just isolated tissue.

As our Circadian rhythms are firmly ingrained into our cells’ genetics, the cogitation of altering something so essential and, some could say, ancestral is a daunting task. Surely these axial genes that make up our natural rhythms are hard to get at? Yes, for now, but further specific advances in the field (which if you’re interested is called Chronobiology) have uncovered a clue to unmasking the “cogs” of the biological clock:

Scientists at the Salk Institute (CA) have discovered a tell-tale sign to identify the strength of someone’s Circadian rhythms. They were looking at the REV-ERBa structure, which is a particular receptor found on many genes associated with repressing transcription. The REV-ERBa is considered to be associated with inhibitory effects in biological clock activity, although the mechanism of its workings was previously unknown.

What the team at Salk found surrounding this gene was that 2 proteins work together to change REV-ERBa concentrations. They expected that this is an important mechanism used in biological clocks to lower levels of activity during night time, when an individual is asleep, and then raise levels again in the morning. The 2 proteins perform different tasks to “turn the hand” around on the biological clock; the F box protein FBXW7 selects REV-ERBa for degradation in the morning which causes activity levels to rise, and then Cyclin-dependent Kinase 1 (CDK1) phosphorylates REV-ERBa (a prominent sign of activation in biology) when activity levels become too high. What’s more interesting, is that when CDK1 phosphorylates REV-ERBa it is also signalling to FBXW7. This means that when REV-ERBa levels are very high (i.e. at night, again) FBXW7 is drawn to the component to begin the cycle all over again.

Zhao et al. (2016)

The importance of this study is that it identifies something that has not been seen before: Circadian rhythm amplitude. The strength of gene activation in these biological clocks is being controlled by 2 proteins, both dependent on each-other. If there were drugs that could potentially alter the levels of one, we would be able to not necessarily control our biological rhythms but at least steer them in the right direction. For example, If an early-morning shift worker found that they couldn’t settle-down to sleep early in the evening, they could take a drug that could induce an earlier CDK1 effect. This would lead to REV-ERBa levels increasing earlier on in the day, causing sleepiness earlier than normal and wakefulness earlier the next morning – just in time for a 5am shift.

It is very important that we understand that our biological rhythms have evolved with us over millions of years. They are crucial parts of our physiology, and although the studies explored in this article aim to show that they can be altered it must be stressed that we are a long way off from the future described in the first paragraph. For the time being we are as reliant on our natural Circadian rhythms as much as any other organism, but that doesn’t mean that our increasing knowledge of genetics can one day give us the controls to our own cycles.

Sources:

-         -  Piemental, D., Donlea, J.M., Talbot, C.B., Song, S.M., Thurston, A.J.F., & Miesenbock, G. (2016). Operation of a Homeostatic Sleep Switch. Nature, 536 , 333-337. doi: 10.1038/nature19055.

-        -   Guo, F., Yi, W., Zhou, M., & Guo, A. (2011). Go signalling in mushroom bodies regulates sleep in Drosophila. Sleep, 34 (3), 273-281. Retrieved from http://www.bio.brandeis.edu/rosbashlab/profile.php?imagename=fang.

-        -   Oike, H., Kobori, M., Suzuki, T. & Ishida, N. (2011) Caffeine lengthens circadian rhythms in mice. Biochem Biophys Res Commun, 410 (3), 654-658. doi: 10.1016/j.bbrc.2011.06.049

-        -   Zhao, X., Hirota, T., Han, X., Cho, H., Chong, L., Lamia, K. ... Evans, R.M. (2016). Circadian Amplitude Regulation via FBX27-Targeted REV-ERBa Degredation.Cell, 165 (7), 1644-1657. doi: http://dx.doi.org/10.1016/j.cell.2016.05.012.
a       - Psycology Blog image: http://a2levelpsychology.blogspot.co.uk/2015/06/a2-level-circadian-rhythm.html
         - Japan Daily Press image: http://japandailypress.com/know-your-body-clock-and-lose-weight-2810428/
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-         -  Salk Institute. "Powering up the circadian rhythm." ScienceDaily. ScienceDaily, 26 May 2016. <www.sciencedaily.com/releases/2016/05/160526124908.htm>
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Solid Genetic link to Depression Found Thanks to “Massive Crowd-sourced Depression Study”

For a long time, the study of depression has been from a psychological perspective. Studies investigating the genetic aspects of mental illness have often rendered fruitless outcomes, over-ambitious results and rifts between communities of geneticists. However, recently published in Nature, a study including several datasets of human genomes support the strongest argument yet for a genetic association to Major Depressive Disorder.

Depression is a type of mental illness that can develop in any individual regardless of age, gender or lifestyle. Its exact workings remain a mystery, as each case of depression varies from individual to individual – just like cancer. General belief is that an imbalance of chemicals in the brain, including neurotransmitters, causes depression. As a result, “wrong” genes become expressed (or inhibited) inside cells and different areas of the brain end up functioning less or abnormally.

Research released in collaboration with genome-sequencing company 23andMe earlier this year has posed genetic links to MDD in adult Europeans. The business sells gene-test kits for less than £200, and are easy to obtain through it’s online store.

Until recently most data produced by the organisation was made for customers as a personal, entertaining insight into their ethnic heritages. As well as investigating their genetic miscellanies, users of the service can accept to release their genome for research within a multitude of studies.

The paper, lead by geneticist Dr. Ashley R. Winslow, is the largest study of it’s kind. The research uses a technique called Genome-wide Association Study. This is a commonly-used bioinformatical approach that takes the DNA of people suffering from a disease and compares it, base-by base, to healthy DNA genomes. In this case, the healthy genomes and the genomes of individuals with MDD both came from consenting-users of 23andMe.

Individuals gave important “self-report data” to the organisation, alongside their DNA submissions. Dr. Winslow’s team performed a meta-analysis, combining the 23andMe data with previous MDD association-study datasets. Using the self-reports the researchers confirmed a total of 75,607 individuals declaring some history of clinical depression. This group was transferred to a new dataset and compared to the 231,747 individuals that reported no personal history of depression.

After the comparative assessment, 5 stand-alone “variants” from 4 regions were identified as being unique between the two groups. Upon analysis of loci (their locations in the genome) the team uncovered 17 independent Single Nucleotide Polymorphisms/ mutations from 15 regions that seemed to have a large effect on the genome.

Note: the 17 SNPs were identified after loci with P values more than 1 x 10^-5 were excluded.

In previous studies using whole genome sequences some of these loci had shown associations with psychiatric traits. This means that the regions aforementioned can be scrutinised in individuals to observe whether certain SNPs in these areas can increase the risk of MDD.

The paper aims to set a “diving-board” for more investigations into the biology of depression. As a large undertaking by Winslow et al., this novel study in the field has demonstrated the potential of larger collaborative datasets that are made publicly available. The results could also encourage second-glances at previous studies, such as Cai, Bigdeli, Kretzschmar & Li (2015), which pointed links between MDD and 2 loci in female Chinese populations.

Considering the future implications of public, crowdsourced genetic datasets it would not be unfair to suggest that we could begin to investigate the mechanics of these identified loci. New drugs could be synthesised that target the variants within these regions, and advanced methods of MDD detection could be a possibility. However, the latter may be a long way off. From what is known about its genetic association the risk of depression is down to mutations at hundreds of loci, each having a miniscule effect on the overall outcome.

What can definitely be said is that these findings are a positive re-enforcement, if not an affirmation, that mental illness does has a genetic factor. We now know even-more-so that lifestyle may not be a preventive or causative issue for the development of psychological disorders in some individuals.

References:

·       Winslow, A. R., Hyde, C. L., Nagle, M. W., Tian, C., Chen X., Paciga S. A., Wendland J. R., Tung J. Y., Hinds D. A., Perlis R. H. (2016) Identification of 15 genetic loci associated with risk of major depression in individuals of European descent, Nature. DOI: 10.1038/NG.3613

·                    日本語要約 (2015) Sparse whole-genome sequencing identifies two loci for major depressive disorder, Nature, 588-591. DOI: 10.1038/nature14659

·       “Study points way to finding genes affecting depression risk” – Malcom Ritter (09/2016)http://hosted.ap.org/dynamic/stories/U/US_MED_DEPRESSION_GENETICS?SITE=PASUN&SECTION=HOME&TEMPLATE=DEFAULT

·       “23andMe Pulls Off Massive Crowdsourced Depression Study” – Antonio Regalado (09/2016) https://www.technologyreview.com/s/602052/23andme-pulls-off-massive-crowdsourced-depression-study/

·          Harvard Health Publications http://www.health.harvard.edu/mind-and-mood/what-causes-depression

• Image 1: http://www.wwu.edu/healthyliving/education/Depression/images/causesDNA.png
• Image 2: https://upload.wikimedia.org/wikipedia/commons/thumb/e/ec/23andMe_logo.svg/2000px-23andMe_logo.svg.png

The Rising Tide of Antibiotic Resistance

For the past 70 years state-of-the-art healthcare has been saving countless lives around the world. Since the discovery of antibiotics in the late 1920s, the era of Modern Medicine in which we live in has allowed us to live longer, happier and more fulfilling lives - but all of that could now be in jeopardy.

Theorised and pioneered since the 1880s "antibiotic chemotherapy" is the process in which a drug is used to specifically target a bacterial organism to combat or prevent an infection. Each antibiotic drug works by targeting specific receptor sites on the organism's cell membrane, and bringing about a series of changes that kills the organism. The most well-known antibiotic is Penicillin, discovered accidentally by Alexander Fleming in 1928 and still used today, although in less occurrence.

Before antibiotics many people had a much shorter life expectancy, dying from minor injuries and complications. The problems weren't the ailments themselves but the infections that injured and ill people were more prone to. To give an example, the first patient to be administered Penicillin was Albert Alexander - a British Policeman who was so ridden with bacterial infection that he could not be recognised by his family. His head was covered in abscesses, doctors even had to remove an eye, all because days before he had scratched his face on a rose thorn in his garden.

For the last half-century antibiotics have dissipated the number of deaths from infection. Antibiotic treatment is commonplace in healthcare and even considered the backbone of our medicinal practices. They prevent diseases, work alongside compromise immune systems, allow those who have undergone surgery or other treatments to recover faster and safer.
In the next 50 years, this could all be about to change due to antibiotic resistance.

All life on this planet has evolved to be where it is today, including bacteria. Unlike many multi-cellular organisms they evolve at a much faster, uncomparative rate. With every new generation mutations can occur - some of these bad, some neutral and some even advantageous - and with a generation time of around 20 minutes bacteria can develop (through a build-up of "accidental mutations") advantageous abilities within an extremely short period of time. When a bacterial organism develops an advantageous mutation to survive in the presence of an antibiotic drug it is said to be resistant.

Projections of Antibiotic Resistance

The main factor to address is that, if antibiotic resistance increases enough, many more people will die from infection. A study conducted by RAND Europe in 2014 showed that in the US and Europe 50,000 people die yearly from infections that no drug can help. This is mostly because the vast majority of antibiotics that we use belong to a generation of drugs that were made in the 1980s. The World Health Organisation predicts that by 2050 10 million people will die every year from antibiotic-resistance infection.

Bacterial disease may not just be caused by infections of infamous "hard-hitting" species, in fact a rise of resistance within many species of common bacteria that we live with would become deadly; potentially resistant infections like strep. throat and salmonella could mean death, as it did at the beginning of the 20th century.
Just a few months ago a senior medical official of the NHS reported to GPs that a super resistant strain of Neisseria gonorrhoeae had surfaced in England, meaning many of it's carriers could have a completely non-treatable Gonorrhoea infection.

A Provoked Resistance?

Nature will always find a way to adapt, so the evolution of bacteria to resist harmful drugs that they are being exposed to is inevitable. But that isn't to say that our practises have caused this rate of resistance appearance to sky-rocket.
As briefly mentioned, almost no new antibiotics have been discovered and approved since the 1980s. Back in that time, pharmaceutical companies believed that the various classes of antibiotic drugs were enough to tackle any infection for the foreseeable future. Since then only 1 effective antibiotic has been discovered, and it will not be available for the next 5 years (See Teixobactin). With no other new antibiotics in circulation, 30 years or so has been more than enough time for bacteria to develop a resistance to most of our antibiotic arsenal.

A linked cause (and potential reason) to the non-existing new generations of antibiotics is an economical one - many pharmaceutical companies do not see making new antibiotics a financially beneficial venture. This mainly because bacteria have began to evolve so quickly that it's not in their best interests to fund potentially new antibiotic drugs that could become inadequate in the next 10 years. The figure above backs up their opinions on the matter.

Fourthly, antibiotics are being abused. This is the broadest and most harmful cause of microbial resistance, and the misuse of antibiotics doesn't solely lie with human use.
In a society that relies on industrial farming, many animals are fed a variety of antibiotics on a daily basis. Rather than treating a present infection, most are said to be needed to increase the meat yield and health of the animal. Some antibiotics are even used on plant crops.

In the case of human medicine, is the general practice of some doctors is to prescribe a "shotgun" cocktail of antibiotics for a patient that may have not even been screened for any bacterial infection at all. The UK's National Institute for Care Excellence reported that last year 10 million antibiotics were unnecessarily prescribed - that equates to 1 in every 4 capsules.
The way in which we individually use antibiotic medication is often wrong too. Many people who are given a prescription stop taken it immediately after they start to feel better. In the case of antibiotics this is very dangerous as it gives the trace amounts of the infection left to adapt and grow, bringing back the infection and increasing the chance of this resistant colony to pass on it's genes to other bacteria in the environment.
Taking antibiotics incorrectly or even unjustifiably also harms your body flora - the "friendly" bacteria that live predominantly in your intestines and help you absorb nutrients from food.

What Could We Do to Slow Down the Rate of Resistance?

The easiest ways to tackle this problem are socio-economical. In the here and now, with no scientific development, governments could put pressure on Health services to scrutinise the ways in which antibiotics are prescribed. Institutional audits could question every decision as to whether a patient should be given antibiotic treatment - backed up by regular and commonplace patient screening tests to ensure an specific infection is present and has need for treatment. Changing the public attitude towards typical antibiotic use (targeted equally at non-western countries with less awareness of proper use) could prevent worried parents from demanding that their child has antibiotics for a non-credible, minor illness. These implementations could swiftly slow down the rate of resistance, and reserve many antibiotics for more effective uses in surgery, artificial replacement operations and for those with compromised immune systems.

Another way to look at the problem would be to devise alternatives to antibiotic treatment. Preliminary research into predatory bacteria is currently being conducted by the US Defence Advanced Research Projects Agency, which investigates whether species of bacteria could successfully kill other bacteria which cause infection in the human body.
Similar to antibiotics, genetically produced components in a cell - or peptides - with antibacterial activity have been isolated from several different organisms to see if they can be used pharmaceutically. One drawback to this approach is that just like antibacterial drugs, bacteria would eventually become resistant to many of these peptide drugs.

The most promising alternative to antibiotics is already used in many laboratories on tissue, and some have been trialled for therapeutic use in the past (In the Soviet Union, for example). Phage therapy involves a small virus that attack bacteria (a phage) being introduced into the body of an infected individual. The phage is genetically designed in a way that it should infect and kill the harmful bacteria and that bacteria alone. Progress to clinical trials has been reached in European health institutions although only for one particular treatment (an infection associated with burns).



In all, something has to be done to prevent the impending damages that antimicrobial resistance could cause to our modern civilisation. Since their discovery, these drugs have come with warning predictions from the very pioneers of the field - spelling out a disastrous post-antibiotic era.

"The thoughtless person playing with penicillin treatment is morally responsible for the death of the man who succumbs to infection with the penicillin resistant organism"

- Sir Alexander Fleming, 1946.

- Information researched from many sources, including Mary Mckenna's TED talk "What do we do when antibiotics don't work anymore?"
- Photo of resistance figures taken from M McKenna's TED talk "What do we do when antibiotics don't work anymore?"
- Photo of doctor prescription taken by Anthony Delvin/ PA
- Photo of phage and bacteria taken from http://www.foodsafetynews.com/2016/01/bacteriophages-an-old-antibiotic-alternative-becomes-   new-again/#.Vppc-9SLTIU
- other references http://www.theguardian.com/society/2015/aug/18/soft-touch-doctors-write-10m-needless-prescriptions-a-year-says-nice
- www.nature.com/news/antibiotic-alternatives-rev-up-bacterial-arm-race-117621