Spinal muscular atrophy and Zolgensma

Screenshot 2021-06-10 at 10.28.50Reports have appeared in the UK media of the NHS administering the most expensive drug in the world to a 5-month-old boy. Onasemnogene abeparvovec (sold as Zolgensma), costing around two and a half million US-dollars (£1.79 million),* is a gene therapy agent to treat spinal muscular atrophy. The headlines quoted the price but gave scant details on what Zolgensma is and how it works. And when you look a little deeper, it turns out there are some parallels with Covid-19 vaccines.

Spinal muscular atrophy is a serious genetic disorder that affects around 1 in 10,000 people worldwide. Being relatively rare, it’s classified in the pharmaceutical jargon as an orphan disease and until recently received comparatively minor attention. Zolgensma was originally developed by a biotechnology company called AveXis, funded largely from charities and the National Institute of Health in the USA and before this, basic research was carried out by the French Muscular Dystrophy Association. In 2018, Novartis bought AveXis for $8.7 billion, and now manufactures and markets Zolgensma.

Different species possess different numbers of chromosomes. The male Australian ant (Myrmecia pilosul) has just one chromosome, the Adder’s tongue fern (Ophioglossum reticulatum) has 1260 and humans have 46. Our 46 chromosomes are arranged as 26 pairs, one in the pair inherited from the father and the other from the mother. (Pears incidentally, have 34 chromosomes arranged in 17 pairs). Within the chromosomes is DNA, and DNA is arranged into genes. The genes comprise a sequence of DNA bases that code for proteins, and proteins make up the stuff of life for every living organism from bacteria to you and me. The human DNA code is around 3-billion bases long and given this complexity, now and again something goes wrong with one of the genes. This is not usually a problem because if there’s one faulty gene then there’s a duplicate in the other paired chromosome that can still do the job. Occasionally however, the genes on both chromosomes go wrong, in what’s called “autosomal recessive inheritance” and then there can be a problem.

On human chromosome number-5 there are a series of genes collectively known as survival motor neuron genes (SMN). There are two types – SMN1 and SMN2 which make SMN protein, one protein in a group called the SMN complex, which is important in maintaining motor neuron cells. These are the nerve cells which transmit signals from the brain and spinal cord to control muscle contractions. The role of the SMN complex is… well,… complex. It’s involved in processing messenger RNA (mRNA), which is the intermediary molecule between the DNA code and its associated protein. mRNA starts out as a “rough draft” known as pre-mRNA and has to go through a number of processing steps before it’s transformed into the working copy. With a faulty SMN complex that final working copy never emerges and so motor neuron-associated proteins are not made. Over time motor neurons therefore degenerate which leads to progressive muscle wasting and within a year or two, it’s usually fatal.

The body has a back-up to the SMN1 gene – SMN2, but SMN2 typically only makes 10% of the SMN1 protein. Some people with spinal muscular atrophy might have multiple copies of the SMN2 gene, in which case the disease is not as severe. On the whole however, most patients have one inoperative SMN1 gene and two SMN2 genes which only make a fraction of the SMN protein required.

Not that long ago there was not only no treatment for spinal muscular atrophy, but most physicians would say there could never be an effective treatment because the root cause goes back to genetics. They were wrong. Zolgensma is not a drug in the normal sense. It comprises two parts, a length of laboratory-made DNA to replace the faulty SMN1 gene and the husk of a virus called vector adeno-associated virus 9 (AAV9). The viral vector is manufactured in isolated Human Embryonic Kidney cells (HEK293) originally grown by Dutch biologist Alex Van der Eb in the early 1970s. The viral genetic material is removed and the SMN1 replacement gene is placed into the empty shell which acts as a vector, delivering DNA to motor neurones. (Without the vector the body would rapidly break the DNA down). This is similar to the Covid-19 vaccine from the Jenner Institute at Oxford, where they developed technology using adenoviruses and modified Vaccinia Ankara (MVA) virus as vectors for genetic material to code for spike proteins on SARS-Cov-2. As the body translates the code into spike protein, so it primes the immune system against future SARS-Cov-2 infection. If you’ve had the Astra-Zeneca vaccine (developed at the Jenner) then you would have received this type of vector vaccine.

The SMN1 gene delivered by Zolgensma is not incorporated into human DNA, but sits within the cell’s nucleus where the normal molecular machinery translates it to SMN protein. The intention is to administer Zolgensma just once to replace the faulty gene but as experience grows over time, the necessity for further administrations cannot be ruled out.

Currently Zolgensma is approved for children under 2-years of age but timing appears important and it should be given as soon after diagnosis as possible. It is administered by intravenous infusion but research continues to develop delivery systems directly into the cerebrospinal fluid, which could make Zolgensma available to a wider patient base.

Zolgensma is not the first gene therapy agent to be approved, there are around a dozen others in use. To give just one as an example, about one in a million people have a deficiency in their lipoprotein lipase gene leading to elevated levels of triglycerides and ultimately liver and pancreatic disease. A gene therapy agent called alipogene tiparvovec uses the adeno-associated virus serotype 1 as a vector to deliver a copy of the human lipoprotein lipase gene to muscles, thereby bypassing the faulty one.

All gene therapy agents are expensive but they are also state-of-the-art pharmacotherapy based on science which goes back to at least 1953, when the structure of DNA was first elucidated. Science is like that. It’s a series of dots that join together from many sources over many years and at the start it’s impossible to predict what the dot-to-dot picture will look like. Let’s hope we are entering a new age of genetic therapy giving hope where before there was none. And then from there, who knows?

* a confidential deal was struck between the NHS and Novartis Gene Therapies which reportedly reduced the price tag. 

A Crisis of Infection

AntibioticsIt’s amazing what humankind can achieve with the right amount of political will and resources. After the attack on Pearl Harbor in December 1941, Roosevelt oversaw the rebuilding of the Pacific fleet in only 6-months. Driven by rivalry with the Soviet Union, Kennedy announced in May 1961, “…we choose to go to the moon …” and just 8-years later Neil Armstrong left his indelible footprint in the lunar dust. December 2019 saw the genesis of Covid-19 and within a year the UK approved the first vaccine. Other vaccines quickly followed, an astonishing achievement facilitated by an estimated global expenditure of 8.5 billion US dollars. Yet behind this story lies a long history of under-investment in vaccine development, regardless of the inevitability of recurring pandemics. And there’s another inevitable pathogenic crisis heading our way under the political radar. We are running out of effective antibiotics.

Despite claims that Covid-19 vaccines materialised through “greed and capitalism” in reality, global public investment fuelled that one year achievement. A pandemic it seems, catalyses political will like little else. Meanwhile, funding applications took up much of the development time towards vaccines such as a BCG* replacement and the recently approved malaria vaccine. Some might say, Covid-19 aside, vaccine development is more to do with accounting than science.

In 1941, a 43-year-old policeman named Albert Alexander scratched his face on a rose bush whilst gardening. The scratch became septic and he became the first person to be treated with one of the earliest antibiotics – penicillin. Unfortunately, supplies at that time were too scarce to save his life. The story marks the start of an antibiotic revolution, but also illustrates how easily death came from even the most minor of wounds. Thirty years after Albert Alexander’s death, we entered what became known as the golden era of antibiotics. It is however, frighteningly poignant to remember that the last truly novel antibiotics, the quinolones, arrived in the 1970s and discovery since this time has been dire.

The trouble with new antibiotics is not science but, once again, political will and resources. Big pharmaceutical companies have all but abandoned development of antibiotics because the “greed and capitalism” model doesn’t work in this instance – anymore than it did for vaccines. This is not some Marxist view of the world – far from it, but a recognition that free markets are not a one-size-fits-all panacea. Over the years many small biotechs have entered the antibiotic market only to go bust. The dilemma is despite an estimated 700,000 deaths worldwide from antibiotic-resistant strains, high development costs make the price per dose prohibitively expensive. The situation is perhaps exemplified by the case of Melinta Therapeutics, which had four antibiotics on the market, one of them for the treatment of much feared hospital acquired resistant infection. Melinta Therapeutics nevertheless declare bankruptcy in late 2019.

Unlike Covid-19, the number and impact of resistant strains of bacteria build up over time. They arise by stealth until we realise we’re in a crisis that politicians can no longer avoid. Once we’ve reached crisis, reactive solutions are sought as people die, but with a little foresight and vision, science has the know-how to find solutions before we reach a critical point. If Covid-19 has taught us anything, it’s what we can bring about when we put our minds to it. And I end as I started – it’s amazing what humankind can achieve with the right amount of political will and resources.

* BCG stands for Bacillus Calmette-Guérin, a vaccine used to treat tuberculosis, made from attenuated Mycobacterium bovid.

Magic mushrooms and depression

Screenshot 2021-04-16 at 11.28.56Depending upon where you get your news, you may have seen reports that the active ingredient of magic mushrooms, psilocybin, is being studied as a possible treatment for depression.

Being of a certain age, I remember the 1960s and 70s when magic mushrooms were all the rage. The law at the time was ambiguous in that picking the mushrooms wasn’t illegal but extracting psilocybin was. There were reports of people exploiting the loophole by crawling on all fours to graze directly on the mushrooms. A somewhat risky business, as those people were perhaps not the best at distinguishing psilocybin species from those far more toxic. But back to the topic of this blog post – what’s the link between psilocybin and antidepressants?

Sometimes dismissed with a “pull yourself together,” in reality depression is as real a condition as any biochemical disorder from diabetes to Gaucher disease. By far the most commonly prescribed antidepressants today are the Selective Serotonin Receptor Uptake Inhibitors or SSRIs. An early SSRI was fluoxetine, better known as Prozac, which is still widely used*. Although an effective drug, it’s not without some controversy as its development was based on what’s known as the serotonin hypothesis of depression. Serotonin is a hormone which interacts with receptors in the brain. There are 15 known receptors, with 5-HT1A and 5-HT1B being the most extensively studied (5-HT stands for 5-hydroxytryptamine – the chemical name for serotonin). Serotonin receptors are proteins which when bound to serotonin, mediate the release of a range of neurotransmitters such as dopamine (amongst others). Serotonin is transported across the brain and its concentration partly depends on re-uptake by nerve cells. Inhibiting re-uptake by a SSRI results in higher concentrations of serotonin outside the cells and hence, so the theory goes, there’s more available to bind onto the 5-HT receptors.

The serotonin hypothesis of depression is controversial because the biochemistry of serotonin interactions is complex and not fully understood. There may also be genetic aspects, particularly in sub-types of proteins involved in serotonin transport (known in genetic-pharmacology jargon as polymorphisms). Moreover, evidence of an association between higher levels of serotonin in the brain and depression is not compelling. Even though the pharmacological mechanism isn’t well understood, SSRIs are effective in clinical trials and so they clearly have some influence on all this intricate biochemistry.

Interestedly, psilocybin also acts on 5-HT receptors, 5-HT2A in particular. To be more precise, psilocybin contains a phosphate group on its molecular structure, making it pharmacologically inactive. The phosphate group is removed either by stomach acid or in the bloodstream to form psilocin which is the active substance. It crosses into the brain and reacts with a variety of receptors, including the 5-HTs. It leads to complex reactions in cellular signalling, resulting in cascade effects which ultimately cause alterations to sensory perception. Apart from 5-HT receptors, how psilocybin might affect depression is not understood but, like SSRIs, there appears, perhaps, to be an effect in clinical trials. Now is the time to be very cautious because the clinical trials have so far been very preliminary. The first trial appears to be in 2016, conducted at the Centre for Psychedelic Research at Imperial College, London, with a more recent study this year. The number of participants was small, 59 in the 2016 study and 80 in this years. Other institutions have likewise been studying the effects of psilocybin on depression and Imperial have also been looking at its use to treat Anorexia nervosa. In addition, some imaging studies have been performed using functional-MRI (fMRI) but so far, no extensive trials have taken place.

If psilocybin and its related compounds are eventually developed to treat depression, then I suspect the chemical structure will be modified in an attempt to optimise it’s effectiveness and reduce side effects. I also suspect that the choice of dosages will be challenging. Nevertheless, an interesting area of pharmacology might be on the rise – we will wait and see.

* – Stanley Feldman’s From Poison Arrows to Prozac (how deadly toxins changed our lives forever) tells the fascinating story.

What has opioid addiction got to do with cats?

Screenshot 2021-02-04 at 15.06.19Cat lovers will know the effect of catnip on their beloved pets only too well. Catnip (Nepeta cataria) is a member of the mint family and along with a similar plant called silver vine (Actinidia polygamy) it elicits a feline euphoria followed by a period of placid tranquillity, lasting perhaps 15-30 minutes. The effects were first reported by the Japanese botanist E Kaihara in 1709 but we haven’t really learned that much more about the reason for catnip intoxication since that time – that is until recently.

What we thought we knew was that the euphoria-causing constituent of catnip was nepetalactone a member of the iridoid family of plant chemicals. The scientific consensus was that the odour of nepetalactone was like certain cat pheromones, but this was largely supposition. More recently however, scientists identified a related but more potent chemical in catnip called nepetalactol. Plants biosynthesise nepetalactone, nepetalactol, and indeed all iridoids, from a plant chemical called geraniol that gives geraniums their smell. I’ve blogged on this class of compounds before describing how the substances that give aromatic plants their fragrance travel down a biochemical pathway ending up as rubber and even cannabinoids. Such is the versatility of plant biochemistry.

Plants don’t spend their precious metabolic resources merely to pleasure cats and the iridoids act as repellents to certain insects such as aphids. Catnip doesn’t just affect domestic cats, but experimenters have demonstrated its euphoric properties on lions, jaguars, leopards and the lynx. Scientists believe these animals exploit the insect repelling properties of the iridoids, which coincidently then also make the animals high! Until recently the pharmacology of iridoids was largely unknown but a recent paper has shed some surprising light on the subject.

It turns out opioids taken by humans and iridoids in cats both bind onto receptors in the brain which are triggered by endorphins. The body produces endorphins in response to stress and pain and when bound to their brain receptors they elicit the release of dopamine – sometimes (rather inaccurately) called the pleasure chemical*. Opioids and endorphins bind to the same brain receptors but opioid compounds cause a much greater dopamine release. It’s this trigger of dopamine by opioids that is the prime reason for addiction. While opioids trigger dopamine in humans, so iridoids do the same in cats. The pharmacology has in fact now been demonstrated by blocking feline opioid receptors which results in the cat losing interest in catnip.

There is still much we don’t know about how catnip works. It seems it has no effect on other species such as dogs and mice. Presumably therefore, there’s something special about feline opioid receptors?  Not all cats are susceptible to the narcotic effects of catnip and so there may well be a genetic component involved. If humans can become addictive to opioids, can cats become addicted to catnip? The jury is out on that one partly because addiction in animals is more difficult to study than it is in humans. The best advice is to use catnip sparingly. But if you do give catnip to your cat, your pet might end up seeing you as the local drug dealer – be warned!

* – opioid pharmacology is a little more involved than this – if you want to know more here’s a great short video.

Maradona and a Biochemist

I start this blog post with, “I don’t want to sound bitter but…” and then I’ll go on to probably do just that. The media is full of tributes to Diego Maradona; some call him a genius, some a deeply flawed legend. Screenshot 2020-11-30 at 11.23.29For me personally, he was a cocaine addict connected to the Mafia who had a talent for kicking a football – when he wasn’t cheating. Does that sound bitter? Well, actually it’s not Maradona specifically I have a problem with but more the celebrity-culture which worships its heroes no matter how damaged their persona becomes.

Some years ago I had the great privilege of meeting Fred Sanger. Who, you might ask? Fred Sanger was a British biochemist and one of only three people to receive two Nobel Prizes in Science; one in 1958 for his work on proteins and another in 1980 for the way DNA stores its code in a sequence of bases. Both proteins and the genetic base-code sequence have appeared repeatedly on this blog in respect to the battle against Covid-19. Sanger was one of the great pioneers of biochemistry, whose groundwork has already led to the saving and betterment of countless lives, and will undoubtedly continue to do so for many years to come. There is an institute in Cambridge named after him, the Wellcome Sanger Institute where the Nobel Laurette, John Sulston did so much on the Human Genome Project. The Wellcome Sanger Institute continues its work in Fred Sanger’s name to this day, including sequencing viral genetic codes.

Just before I met Fred Sanger at a ceremony in London in the mid 1990s, I visited the National Portrait Gallery and discovered a painting of the scientist by Paula MacArthur. I mentioned this to him and he wasn’t keen on it because he thought the way his eyes were painted made him look like a stereotypical mad scientist.  Judge for yourself, if you think we was right. 

He died in 2013. There were obituaries in some newspapers and BBC Radio-4 did a piece on him in the Last Word. But compare the outpouring of hero worship adorned on a household name because he was a cocaine addicted footballer and someone who saved the lives of thousands, if not millions and few have ever heard of. I admit I’m biased but I’m left with a feeling that much of humankind has its priorities rather confused.

Pfizer-BioNTech are making a mRNA vaccine, but what is that?

Covid-19 blog for the non-expert

Screenshot 2020-11-12 at 14.27.24The Pfizer-BioNTech vaccine for Covid-19 is all over the media. What hasn’t been in the headlines so much, is that this is a mRNA vaccine and if successful, it’ll be the first of its type. Some say they will not take it because it’s “rushed” but this misses the point that we are not making vaccines in the same way we did even a few years ago, and in fact mRNA vaccines have been in development for over three decades.

But what is a mRNA vaccine and why are they so important? In this blog post I try to explain.

The general principle behind a vaccine is that it contains something called an antigen – a piece of the target pathogen (or something resembling the pathogen) that the body recognises as being foreign. The antigen is such that it’s able to trigger an immune response, but without causing the disease itself. In a way, it fools the immune system an infection has occurred, thus sounding the bugle for attack. The attack in the case of the immune system is to produce antibodies that latch onto the antigen acting as beacons to white blood cells (called T-cells) which come along and destroy the pathogen. Key to vaccination is the fact that the immune system bares a grudge even bigger than in a Mafia war. The immune system, like the Corleone family, “goes to the mattresses” and patiently waits. If the antigen, this time in the form of the genuine pathogen, should reappear, then the immune system comes out of hiding and attacks before the disease has had a chance to fire a shot.

To understand how a mRNA vaccine brings the immune system into play against a pathogen, we first need a little biochemistry. As I’m sure you know, DNA contains a code in the form of base-pairs which our biochemistry translates into proteins. There is however, an intermediate step whereby the code in DNA is first translated and carried to the protein-making mechanisms within cells by messenger-RNA (mRNA). This is happening inside the cells of your body all the time, making new protein from enzymes to muscle to haemoglobin. A mRNA vaccine works on the principle that part of the viral DNA (or RNA in the case of SARS-CoV-2) is translated to mRNA in the laboratory. Following modification, this mRNA gets placed into a lipid nanoparticle. Before any Bill Gates conspiracy theorists get too excited, the lipid nanoparticle isn’t a microchip, it’s simply a lipid (fat) particle about a billionth of a meter in diameter (hence “nano”) that helps the mRNA cross biological membranes to enter cells. Once in the cell, the protein-making machinery translates mRNA into the viral antigen. In many ways, this is similar to what the virus does when it takes over a cell to make more copies of itself – biotherapeutic irony perhaps. Once the viral antigen is present, then the immune system triggers in the same way I’ve described above. It sounds simple, but there are complexities. The viral antigen, made from mRNA, is part of the spike protein on SARS-CoV-2, which latches onto the human cell to gain entry. It’s important to select mRNA for an appropriate part of the spike protein because the cunning virus coats much of it in sugar molecules to hide it from antibodies. Biochemistry is never that straightforward.

The likely starting point for mRNA vaccines appears to be 1989 when a San Diego biotech company called Viral Inc published the first paper*. In the early days, it was all done in test tubes (in vitro) or with animal models and the first human trials took place in the early 2000s. Despite some efforts, a successful mRNA vaccine has eluded researchers up until now, and so if the Pfizer-BioNTech vaccine is successful, it will be first in its class. Of course, no therapy is entirely risk free and some side effects have been reported over the years, but I’ll leave this to another time, if the vaccine gets approval. I should add however, that in respect to the current Covid-19 clinical trials with over 43,000 participants, the vaccine does look very safe.  The other issue with this type of vaccine, which the media has widely publicised, is the need for storage at -70℃ because of the inherently unstable nature of mRNA. That, however, is a logistical question and so I’ll not tackle that one here. Personally, I would say there’s some benefit in a more gradual roll-out, if for nothing else to allay the fears of some of the public on its safety. (However, -70℃ storage does cause problems in the developing world).

If this does turn out to be the first mRNA vaccine the implications are indeed profound. This is what’s known in the biotech industry jargon as a platform technology. As time goes on and we acquire more experience, then its perfectly possible a mRNA vaccine could be made within a few months to combat future pandemics – which will surely come sooner or later (let’s hope later). In fact, it’s not necessary to even isolate the virus in order to make the vaccine, it can be done from just knowing the sequence of the genetic code, which is routine these days. In the case of SARS-COV-2, the disease (Covid-19) was first reported in December 2019, and by February 2020 the 26,000 – 32,000 RNA code sequence went round the world via the internet. The rate limiting step therefore, will likely not be the vaccine itself but the safety tests and clinical trials.

And finally a note of extra optimism. If Pfizer-BioNTech stalls then other vaccines are coming through apace and I suspect announcements will appear soon. Some of these will be other mRNA vaccines and others will be vector types. And, as some have asked, would I take it? You bet – I’d be first in line if I could be.

We have likened the pandemic to wartime and in many ways that analogy holds true, because it’s times of the greatest threat to humankind that we seem to make our most profound advances.

* R.W. Malone, P.L. Felgner, I.M. Verma, Proc. Natl. Acad. Sci. U.S.A., 86 (1989), pp. 6077-6081. 

90% effective Covid-19 vaccine

The headlines are jubilant with “Covid-19” vaccine 90% effective. As one of those skeptical scientists, I’m in a difficult position because I don’t want to dampen any hope, but at the same time it’s worth questioning the headline to see what’s really behind it.

The announcement was from Pfizer and BioNTech, and the media are pretty much echoing the contents of their press statement. I’ve blogged previously on science by press statement, rather than relying on peer reviewed literature and so this should should be the first warning bell. 

Many might assume that 90% effective means that on average in a population of 100 people, 10 will get full blown Covid-19 and 90 will be symptomless and not be carriers. This is however, a simplistic interpretation because effectiveness can be calculated based upon (1) ability to prevent infection (2) ability to prevent the disease, although individuals are still infected, and (3) ability to prevent serious disease. There is also the issue of how effective a vaccine reduces the infection rate to others, generally known as herd immunity. (This is, incidentally, the genuine type of herd immunity associated with vaccines, not the idea that we go out and get the disease, which has pretty much now been debunked).

The Pfizer-BioNTech vaccine is of the messenger-RNA type, which if effective, will be the first of its type. Although the science is sound, this class of vaccine does not have a track record, as yet. Looking any deeper at present is difficult without the full peer reviewed publication and all we have to go on is a press release. I’ll nevertheless, end by saying a vaccine even with a low rate of effectiveness could make a huge difference, and so we should very optimistic. Nevertheless, let’s temper the optimism with a little realism and wait and see how things develop. 

New Covid-19 tests

A Covid-19 blog post for the non-expert

Screenshot 2020-11-04 at 11.34.43We’ve been hearing how the UK government wants to return to normal life under its £100bn Operation Moonshot Covid-19 testing programme. Headlines such as those in the Daily Mail have proclaimed “Prospect’ of 10-minute ‘rapid turnaround’ Covid tests” but others including the BMJ are not so sure. I thought, therefore, the time was right to look at the emerging Covid-19 tests Operation Moonshot is pinning its hopes on, and see how they work. 

In a previous blog post I looked at two Covid-19 test techniques, one which detected the presence of the virus itself and another which looked for the presence of an antibody to the virus in a blood sample. Scientists don’t hang around during a pandemic and two new tests are coming into play, known as the Lateral Flow Test (LFT) and Loop-Mediated Isothermal Amplification, or a LAMP. In this post, I will look at what LFT and LAMP are, how they work, and what are their advantages and potential shortcomings.

Some media reports have given confusing accounts of these new tests, probably because they are based on existing technology and the technical terms describing them are very similar. In an attempt to avoid confusion here, I will first provide definitions of the four most important technical terms.

Antigen – this is what’s being tested. With Covid-19 the antigen is a protein on the SARS-CoV-2 virus.

Antibody – is a protein associated with the immune system which locks on to an antigen such one on the SARS-CoV-2 virus. Since the virus has many proteins, the body will raise a range of different antibodies, each sticking onto its own viral antigen.

Monoclonal antibody – this is an antibody made in the laboratory that targets one specific antigen. All the antibody molecules are clones of one another, with essentially the same molecular structure, which is where “monoclonal” gets its name.

RNA – Ribonucleic acid is the genetic material inside SARS-CoV-2. (Some viruses have DNA and others, such as SARS-CoV-2, have RNA). SARS-CoV-2 RNA comprises a chain of around 30,000 nucleic acids (known as bases) which codes for viral proteins.

The current gold standard test for Covid-19 is the Polymerase Chain Reaction, or PCR test (see previous blog post). In a nutshell, the method replicates SARS-CoV-2 RNA many times over, generating enough material for it to be reliably detected. PCR is used routinely in molecular biology and once we know the code-sequence of the genetic material, scientists can usually generate a PCR test within a few weeks. Running a PCR test however, is not straightforward and requires laboratory conditions and skilled analysts. In particular, PCR has multiple heating and cooling cycles to promote the required chemical reactions, and the reagents, being relatively complex, demand continuous preparation. Loop-Mediated Isothermal Amplification (LAMP) is still a PCR method, but operates at a single temperature (60-65 ºC – hence “isothermal”) and does not require the same fresh reagents. These, and other modifications, reduces the time to carry out a LAMP test to as little as 15-minutes, or an hour in the case of Covid-19.  The test also has the potential to be run on a bench-top type device rather than requiring sophisticated laboratory conditions. While LAMP sounds like a great leap forward, it’s nevertheless a recent development in Covid-19 testing and so we have to remain cautious to its reliability. It no doubt has great potential but the traditional PCR will remain the gold standard, at least for the time being.

While LAMP detects viral RNA, LFT detects viral protein. It does this using monoclonal antibodies, which is probably the source of confusion between LFT and an “antibody test”. So to be clear, there is a test which looks for the presence of an antibody to SARS-CoV-2 in blood that shows if you’ve contracted the disease sometime in the recent past. LFT does not do this, it detects the presence of the virus itself.  LFT technology has been around for some time and it’s used in home pregnancy testing kits. LFT is divided into two types known as sandwich and competitive. In the sandwich type, lines appear showing a positive test, in the competitive type the absence of lines indicate a positive test. The LFT for Covid-19 is the sandwich variety. The device comprises a flat plastic plate (see image), with a well at one end and a panel where lines appear if the test is positive. A sample from the nose or throat is placed into the well, where it migrates along the plate by capillary action. It reaches a line of monoclonal antibodies (made to recognise a specific SARS-CoV-2 antigen) which are attached onto a tiny particle, typically made of gold or carbon (the antibody-particle complex is known as a conjugate). We classify the particles as nanoparticles because they are about a billionth of a meter in diameter.  The conjugate, along with the bound SARS-CoV-2 antigen, continues to migrate along the plate by capillary action until it reaches another line of monoclonal antibodies. These monoclonal antibodies don’t recognise the SARS-CoV-2 antigen however, but the monoclonal antibody conjugate. (You can see where things get confusing because the antibody conjugate is now essentially an antigen to the second monoclonal antibody). As the conjugate binds to the second line of monoclonal antibodies, they hold the particles upwards forming a visible coloured band – that is a positive test. Depending upon the test, there may be more than one line indicating a positive test and an indicator line showing the device is working correctly.

LFTs are tricky things to develop, which is why it’s taken until now for them to appear. Appropriate monoclonal antibody production is not straightforward and even variables such as capillary flow-rate within the device can affect reliability. The first LFTs have however, passed their assessment and should be available soon. They have the advantage that the test is complete in about 10-15 minutes and do not need a sophisticated laboratory to carry them out. Don’t get too excited however, because the announcement by the Prime Minister may have been a little over optimistic, in that it still requires some expertise and you are unlikely to be able to do them at home. LFTs have a higher rate of false positives than RNA-based methods and so a positive result may necessitate confirmation via PCR or LAMP. Nevertheless, with appropriate training LFTs should have a major impact on test and trace.

For both LAMP and LFT the one huge advantage is not so much the tests themselves, rather than the impact on logistics. The biggest problem we’ve had in the UK has been getting test results back within an appropriate time frame, which sparked arguments over government statistics on testing capacity versus actual tests returned. Both LAMP and LFT however, have the potential of testing while-U-wait. And of course, tests continue to evolve and perhaps the next big step might be a simple saliva test.

And I will leave this post with some optimism flavoured with realism. Despite politicians saying no one could have predicted Covid-19, in truth it was inevitable sooner or later.  Equally so, is the occurrence of another pandemic sometime in the future. I hope that the words of Friedrich Hegal don’t come true, “we learn from history that we do not learn from history” and we retain Covid-19 testing capability ready to rapidly adapt to a new virus. Fingers crossed.

Atomic Butterflies

Red AdmiralWhen I first moved to my current house some 20-years ago, my next-door neighbour had a buddleia growing in their garden which overhung part of my driveway. In the summer, the shrub was a resting spot for a multitude of red admiral butterflies, which exploded into a swarm if I got too close. Sadly, that buddleia was a casualty of renovation work a few years ago but even if the shrub had survived, I suspect the butterfly swarm might still be a thing of the past. Every year the UK organises the big butterfly count where over 100,000 members of the public score the number and species of butterflies in a particular area over a given time. This year they reported the lowest average number of butterflies since recording began in 2010.

The red admiral is a migratory butterfly, happy to inhabit that buddleia in the summer but then heads south for the winter. If conservationists are to identify the causes of the falling population, then they need to understand the red admiral’s migratory behaviour, and that’s not such an easy thing to do. The traditional approach is to capture butterflies, place labels on their wings before releasing them back into the wild, then record the labels as butterflies turn up in other geographic locations. This is a hit and miss method because information on migration routes rely on butterfly labels turning up in the expected location. Moreover, insect migration, known as migration phenology, can be deceptively complex, sometimes involving journeys of thousands of kilometres over more than one generation. Wing labelling therefore provides a snapshot in time, rather than overall and longer-term migration phenology data. Instead of relying on wing labels, recently scientists have turned to isotope analysis to attain more nuanced migration phenology data.

I have mentioned isotopes in previous blog posts, and they are essentially different versions of the same chemical element, distinguished only by the atomic mass. Hydrogen, for example, has an atomic mass of one because its nucleus has one proton. An isotope of hydrogen called deuterium on the other had has an atomic mass of two, because its nucleus has one proton and one neutron. Scientists distinguish isotopes by given the total number of protons and neutrons as a prefixed superscript to the elemental symbol. Hydrogen is 1H and deuterium is 2H for example. All the chemical elements have a range of isotopes. For instance, the most common isotopes of oxygen are 16O, 17O and 18O (all oxygen atoms have 8 protons, 16O all has 8 neutrons, 17O has 9 neutrons and 18O has10 neutrons). The relative combinations of isotopes in molecules depends upon their surrounding physical conditions. The relative amounts of hydrogen and oxygen isotopes in water, for example, reflect the conditions of evaporation from oceans and precipitation as rain. Pour a glass of water from your tap and the blend of 1H216O, 1H217O, 1H218O, 2H216O, 2H217O and 2H218O depends upon where on planet Earth your tap resides. 

Butterflies incorporate hydrogen isotopes into wing keratin, which is a fibrous protein found in hair (and butterfly wings). Once formed, keratin is stable, locking in the isotopic pattern. The 2H to 1H isotope ratios vary according to rainfall and local temperatures and so can be used as regional markers. To compare isotopes in butterfly wings to specific regions requires data on geographical isotope patterns. This is achieved through painstaking sample collection and analysis over wide landscapes, to build what’s called an isoscape.

Traditionally considered to have regular migration patterns, red admirals go to northern Europe in the spring where they reproduce with the next generation heading south for the winter. Although hydrogen isotope analysis confirmed this general concept, it also revealed complexities not previously realised. Analysis of 2H showed red admirals migrate from the south probably as far away as north African locations in the spring, but in August they originated further from the north in what are two distinct migration patterns. In spring, butterflies migrating from the south carried eggs but we find no eggs in August on butterflies coming from the north. Differences in 2H patterns measured in central Europe in spring compared to winter, are consistent with the butterflies reproducing in the Mediterranean during winter. In contrast to the migratory patterns of spring and winter, in autumn 2H isotope patterns indicated butterflies are of a more local origin. Surprisingly, they may be two populations of red admiral because the 2H isotope patterns in those from western Europe rarely correspond with those of north-eastern Europe migrating in the autumn. Based upon isotopic patterns therefore, the migration phenology of the red admiral is far more complex than first realised.

The reason why some butterflies seem to be on the decline remains obscure, but the red admiral is not alone in this respect. In North America, monarch butterflies that overwinter in the Oyamel forests of Mexico have suffered an 80% decline and the possibility of extinction looms. Phenology studies on monarchs, prior to 1997, was carried out using traditional wing labelling techniques but despite hundreds of thousands of butterflies being tagged, they only ever found 125 in Mexico. In 1998 a Canadian research group turned to using isotopes of hydrogen and carbon to trace the origins of monarch butterflies. They measured 2H and 13C in the wings of monarchs captured in Mexico and compared the isotope abundances to isoscapes across North America. Data for 2H reflected local rainfall and 13C data correlated to milkweed, the principle food source for monarch caterpillars. They determined that around half of the monarchs that overwintered in Mexico originated from the Midwest corn and soybean belt of the United States, Nebraska, Kansas and Texas on the western edge and the coastline to the east. Once these butterflies migrated to the Oyamel forests they bred and the next generation returned to its origins in the Midwest. Some conservationists believe that pesticides and genetically modified crops are responsible for the butterfly decline but others point to limiting factors on the migratory routes (now there’s a better understanding of those routes). Either explanation is possible but isotope analysis turned up another possible explanation.

In 2018, 2H and 13C isotope analysis on captured monarchs in south Florida showed there were two populations, one resident to Florida and the other migratory. Not all monarchs are migratory therefore, and it seemed Darwinian evolution was at work because the resident population had a smaller wing size. The migratory population mostly originated from the Midwest and the Texas-Oklahoma border, which correlates to the origins of the Mexican population. This raises the intriguing possibility monarchs are not so much on the decline but perhaps relocating away from Mexico and towards Florida. Given the complexity of red admiral migration so elegantly revealed by isotopic analysis, I’d like to think perhaps those butterflies which once rested on the buddleia overhanging my garden have just moved on to more suitable locations as our climate warms. Without evidence perhaps this is wishful thinking but I’m sure the answer is hidden somewhere within the isotopes.

Death by Liquorice

A blog post for the non-expert

Liquorice
The media is reporting a 54-year-old construction worker from Massachusetts has died of liquorice poisoning. Not for the first time when dealing with such issues, the press reports are so similar that they were likely all cloned from the original source, a paper in the New England Journal of Medicine. I thought, therefore, a blog post for the non-expert might put the toxicology of liquorice into perspective.

They make liquorice from the root of the flowering plant Glycyrrhiza glabra, largely native to western Asia. Media reports will tell you a substance called glycyrrhizic acid contained within the root, is responsible for its toxicity. This is correct, to a point. I blogged on the terpene pathway in plants before, pointing out the biochemical connection between turpentine and rubber, and even cannabidiol.  Glycyrrhizic acid is yet another byproduct of this pathway, but in this case it also has two sugar molecules attached. Glycyrrhizic acid itself is not particularly toxic because the sugars inhibit its absorption through the gastrointestinal tract. Bacteria in the digestive system however, remove the sugars to make glycyrrhetinic acid, which is absorbed into the bloodstream and that’s where the trouble starts.

There’s an enzyme with the somewhat complicated name of 11-β-hydroxysteroid dehydrogenase, but those nice biochemists have shortened it to 11β-HSD which is less of a mouthful. 11β-HSD converts a sterol called cortisone into cortisol, which, amongst other things, affects how the kidneys regulate sodium and potassium transport. Messing around with the body’s sodium and potassium levels can lead to a variety of problems. In fact there’s a disease called Cushing’s syndrome, where too much cortisol in the bloodstream leads to a decrease in potassium and a condition known as hypokalemia. Glycyrrhetinic acid is a sterol-like compound* which 11β-HSD mistakes for cortisone, throwing a metaphorical spanner in the biochemical works. This leads to excretion of potassium and sodium retention and in turn causes cardiac arrhythmias and renal failure, which is what seems to have happened to the Massachusetts construction worker. 

Some quarters of the press seem amused there’s such a thing as liquorice poisoning, but we should remember the victim had family and friends who might not see it as a joke. I suspect some will call for a liquorice ban, and warnings have been around for some yearsParacelcus. In truth it’s not as simple as that because it’s not only sold widely as a confection, we use glycyrrhetinic acid and its related compounds in a range of foods as a natural sweetener and also in some cosmetics from lipstick to suntan lotion. Some believe liquorice has curative properties for anything from cancer to being an anti-viral –  claims which I believe we should all take with a pinch of salt. The trouble is that anything is toxic if taken in sufficient amounts. This was realised back in the 15th century by a German-Swiss physician, alchemist, astrologer and occultist with the horrendous name of Philippus Aureolus Theophrastus Bombastus von Hohenheim. Perhaps even in his own time, his name may have been a bit of a mouth-full, because he was just known as Paracelsus. Amongst a lot of mumbo-jumbo of his age, he got at least one thing right. He famously said, “what is it that is not poison? All things are poison and nothing is without poison. It is the dose only that makes a thing not a poison.” Over time this became abbreviated to, “the dose makes the poison.”  Anything taken in excess can be toxic, even the most benign of substances, like water for example. Water intoxication, or hyperhydration, is rare but there are occasional cases. In 2007 for example, a Californian woman died after taking part in a water-drinking contest where she drank up to 4 L in an hour. At the other end of the scale, all vegetables contain tiny amounts of more potent toxins. Potatoes, for example, have substances related to glycoalkaloid poisons found in deadly nightshade called solanines. You might believe celery is the most beguine ingredient in a healthy salad but it has a carcinogenic chemical known as psoralen. Cabbage, broccoli and cauliflower all contain a group of chemicals called glucosinolates, which can impair thyroid function. Your body is more than capable of coping with these small of potentially toxic substances in your everyday diet. In fact we have evolved some exquisite detoxification biochemistry to do so with no need for so called detox diets. Talking of detox, grapefruit, so popular with that fad, contains furanocoumarins that inhibit certain enzymes otherwise involved in everyday natural elimination of toxins from the body – biochemical irony!

The Massachusetts construction worker supposedly ate a bag and a half of liquorice a day – that’s around 400-500 grammes. In 2004 a Yorkshire woman in the UK, suffered serious muscle failure requiring hospitalisation after eating less than half that amount of liquorice per day. So the lesson is, everything in moderation and remember Paracelsus, anything in large enough doses can kill you. Now where did I put those Bassetts Liquorice Allsorts?

 

  • – to be more precise it’s a triterpenoid but I’m not getting into that level of detail