The Science Behind DNA Analysis of Archaeological Human Remains

 DNA, even when well preserved, will not last for more than a few hundred thousand years. Therefore the description of DNA sequences in million year old dinosaur remains is due to contamination from modern ambient DNA, which is widespread in labs and elsewhere. The PCR will amplify any DNA, modern or prehistoric.

There are however certain clues to the age of DNA remains. The first is fragmentation. Ancient DNA fragments are between 50 and 500 bases long due to degradation. In fact most are no longer than 100 nucleotides.

Second, and most reliable is the observation that at both ends of DNA (5' and 3'), Cytosine (C) is deaminated to Uracil (U). This is an effect that occurs after death due to bacterial cytosine deaminase. This latter enzyme is not present in mammals (not to be confused with AID or activation induced cytidine deaminase, which is present during development and plays a major role in the affinity maturation and somatic hypermutation of B cells, in effect giving B cells their antigen specificity).

Thus if an archaeological remains has a high proportion of uracil bases in DNA- remember, this base is only present in RNA, it indicates that the specimen is thousands of years old. Archaeologists use an enzyme called uracil-DNA- glycosylase (UDG) to break the bond between deoxyribose sugar and uracil and remove the uracil bases. 

Not doing so leads to a curious phenomenon. The PCR used to amplify the DNA remains reads the uracil as thymine (T), since thymine is the native DNA base, not uracil. Hence, on the supplementary (anti-sense) strand, it puts an adenine in place of the guanine (which paired with the original cytosine). The uracil on the sense strand is now replaced with thymine to pair with adenine. The original C-G pairing has therefore now been replaced by an A-T pair. Thus there is miscoding caused by the PCR process manifesting as "transition " of C to T and G to A. (In contrast, when a purine is replaced by pyrimidine base, it is described as "transversion"). 

There can be blocking lesions caused by the formation of non-physiological chemical bonds between say DNA & protein, which can make it difficult for PCR to read through the ancient DNA. This is difficult to overcome.

Archaeologists have databases comprising thousands of single nucleotide polymorphisms (SNPs) mapped from certain lineages such as Armenian farmers or Iranian invaders which they then compare with the putative SNPs from the sequenced DNA sample to see if there is a match.

Here, mitochondrial DNA, which is of course maternal derived (the sperm does not contain mitochondria) is more useful than nuclear DNA in lineage tracing. Although mitochondrial DNA shares the same predisposition for degradation with time elapsed since death as nuclear DNA, there are far more copies of a given DNA sequence in mitochondria than in chromosomes. Thus, while the nucleus will only have two copies of a given allele (on the 2 chromosomes), each mitochondrion has 10-100 copies- a phenomenon known as heteroplasmy, which explains why mitochondrial mutations cause such variable phenotypes unlike mutations that occur in chromosomes. Mitochondrial DNA therefore is favoured by archaeologists for lineage tracing.

Are there any other ways to determine where a long dead person originated, apart from DNA sequencing? As it happens, there is.

The isotopes of Strontium which are deposited in bone and teeth (dentine and enamel) during development are a reflection of the area where the person grew up in. There are 4 isotopes of Strontium- Sr84, Sr86, Sr87 and Sr88. Sr 87 is not a natural isotope. It is formed by beta decay from Rubidium 87. The rocks and water and therefore, by extension, the remains of a person or animal growing up in a certain area will always display a specific Sr87:Sr88 ratio that is native to that area. When that person migrates to a different geographical area, his teeth, which are often well preserved thousands of years after death, will still carry the signature Sr87:Sr88 ratio of the area where he or she grew up in , as the signature is established in the developing teeth or bones, and does not change after death. 

It is thus possible to say that the woman's remains found in a Harappan valley, in fact grew up in Iran.

 

Paradoxical Effect of Caesin Kinase Inhibition in Deletion 5q Myelodysplastic Syndrome

 Caesin Kinase (CK1) is common to the Wnt-beta catenin pathway and the Hedgehog pathway. Along with other kinases, it acts as an inhibitor in both pathways to reduce the transcription of genes in response to wnt and hedgehog proteins respectively. 

Myelodysplastic syndrome (MDS) due to deletion of 5q (5q del) has an unique phenotype which can be explained by the differential action of CK1 on the wnt-beta catenin pathway. 

Subjects with 5q del have macrocytic anaemia, mild thrombocytosis with dysplastic, hypolobated megakaryocytes. MDS in 5q del is uniquely responsive to immunomodulators such as lenalidomide, although the anaemia does relapse after 2-3 years due to new mutations in other genes such as RUNX1.

Deletion of 5q leads to haploinsufficiency of the CK1A1 gene located on 5q.32. Lenalidomide inhibits the remaining CK1A1 allele and reverses the phenotype of MDS. This is admittedly non-intuitive, as there seems to be an abrogation of the dose-response effect here. If haploinsufficiency leads to a phenotype, how can suppressing the remaining normal allele reverse that phenotype (rather than worsen it)?

This apparent paradox is explained by the effect of CK1 on the wnt-beta catenin pathway. Haploinsufficiency of CK1 removes some of the inhibition exercised on the wnt-beta catenin pathway. The latter leads to enhanced survival and proliferation of the neoplastic clone, causing MDS.

However, genetic knockdown in mice or pharmacological inhibition of the remaining CK1A1 allele by lenalidomide in 5q del MDS sufferers leads to complete disinhibition of the wnt-beta catenin pathway. While this initially stimulates the haemopoetic progenitors of the neoplastic clone, continued stimulation soon leads to stem cell exhaustion and death of the progenitor cells now bereft of any CK1 activity.

Reversal of The Earth's Magnetic Axis

 The magnetic axis of celestial objects flips periodically, including a complete reversal (by 180 degrees). For example, the sun's magnetic axis reverses direction every 11 years. However, the Earth's geomagnetic axis flips much less often. The last time it reversed completely was 780,000 years ago, a phenomenon called the Matuyama-Bunhes reversal after the two scientists who described it. Since then, the Earth's magnetic field has tried to flip on 10 occasions, but on each occasion it has reverted back to its current axis.

It's fair to say therefore that such reversals happen very infrequently for the Earth. When it does happen though, it affects the polarity of magnetic material in lava flows, sea beds etc which can be detected in rocks and fossils. 

The Curie temperature is one above which magnetic substances (which includes all minerals containing iron, nickel and cobalt) lose their magnetic properties. This varies between 580 and 680 degrees Celsius for the oxides of iron -Fe2O3 and Fe3O4. Conversely, when cooled below this temperature, such objects regain their magnetism. Thus, igneous rocks have inherent magnetism dating back to the time when they were formed from cooling lava flows millions of years ago.

The reversal of Earth's magnetic axis is not instantaneous- it occurs slowly- over thousands of years. During this period, magnetic material in cooling magma will take up the polarity of the reversed magnetic polarity of Earth, and the rocks formed therefrom would reflect this reversed polarity for ever. Thus, geologists can analyse such rocks, or fossils which have enclosed magnetic material in sea beds and make a fair guess as to their age. This is one way of fossil or rock dating.

How Significant are Pfizer-Biontech's COVID-19 Vaccine Results?

 Pfizer has just announced that their COVID-19 mRNA vaccine has proven to be "90% effective" in preliminary stage 3 results. But what does this mean? 

Let's assume that Pfizer randomised the vaccine and placebo arms in a 1:1 ratio. this is not always the case- it's quite usual for trials to use a 2:1 ratio for randomisation. However, this is the most intuitive scenario, so let's use it.

From figures given, we understand that 94 subjects have developed COVID-19 across both arms of the trial so far.

Thus, there would be 22,000 subjects in each arm. Using Pfizer's own figures of 90% efficacy, it follows that 9 subjects in the vaccine arm and 85 subjects in the placebo arm developed COVID during the study.

(x/22000 divided by (94-x)/22000= 0.10, gives a value of x=9, approximately)

Plugging this into a 2x2 table:

                               Vaccine       Placebo

Disease                      9                   85

No Disease             21,991         21,915

Now you apply the chi-squared test, assuming that results would be acceptable with a confidence interval of 95%.

You thus get a p value of <0.00001.

It's very likely that the results are not due to chance, i.e. they show genuine protection against the virus.

However, there is a caveat. The Pfizer mRNA vaccine needs to be stored at -80 degree Celsius. It requires a cold chain. It must be used quickly after thawing. It might be a difficult ask particularly in countries such as India & Brazil.

Relative Nutritional Availability of Nitrogen & Carbon Shapes Evolution in Oceans

 The nitrogen/carbon content of DNA bases are inverse to each other. Thus while the pyrimidine base cytosine contains one more N than thymine, the latter contains one more C than cytosine. Similarly, the purine base guanine has the extra N , while adenine has the extra C.

G/C are therefore nitrogen rich, while A/T are carbon rich.

Evolution of life in oceans reflects the relative availability of N & C. Thus, microbes living in shallower ocean waters have access to abundant carbon due to fixation of the latter by photosynthetic plants dwelling at the surface of the ocean. However, these microbes are relatively nitrogen poor. The reverse situation applies to bacteria and archaea living at the depths, where plants are much less productive (meaning less photosynthesis), and are thus carbon poor. However, heterotrophic bacteria living in ocean depths have access to greater amounts of nitrogen from decaying plant and animal matter at the bottom.

This differential availability of nitrogen and carbon is reflected in the size and content of microbial genomes in oceans. Thus, microbes living in surface waters have smaller genomes due to the relative scarcity of nitrogen. Furthermore, surface microbes have a low GC content, compared with depth dwelling microbes, but are relatively enriched in AT, the carbon rich-nitrogen poor bases.

This of course has an effect on the absolute amount of proteins synthesised by microbes. Exons are over-represented in GC rich areas of the genome. More exons means translation of more proteins and therefore more nitrogen usage.

Nutritional constraints caused by availability of nitrogen and carbon is reflected even more vividly in the proteins produced by ocean dwelling microbes, and indeed all life forms in general. Redundancy of the genetic code means that there are multiple codons for most amino acids. The choice of a favoured codon also reflects nutritional pressures. Thus, a point mutation in the favoured codon will almost always give rise to an amino acid with a similar nitrogen or carbon content to the original amino acid rather than one with a higher nitrogen or carbon content. The non-favoured codons are not "used" by the mRNA (dictated by the genome from which the mRNA is transcribed) as point mutations here could give rise to more "expensive" amino acids, higher in either nitrogen or carbon content compared with the original. Thus, the genetic code is parsimonious in terms of nitrogen or carbon usage.

To illustrate, the amino acid threonine can be encoded by 4 triplet codes on DNA- ACC, ACA, ACG and ACT. C to G transversion (where a purine  is replaced by pyrimidine or vice versa, rather than purine to purine, etc-the latter is called transition) at the second position of the triplet code will give rise to serine for ACT and ACC, but will produce arginine if the transversion occurs in ACG or ACA. Arginine is higher in both nitrogen and carbon content than threonine while serine and threonine only differ in the position of the oxygen atom. In a ground-breaking paper published in Science, Shenav & Zeevi found that in 187 microbial oceanic species, ACT was far more likely to be favoured than ACA, while ACC was similarly preferred to ACG. This demonstrates that the genetic code has evolved to favour lower usage of nitrogen and carbon, as these two elements are likely to be in nutritional deficit in the environment. The same constraints were not found for oxygen, which is abundant.

Once again, due to the inverse relationship between nitrogen and carbon availability, mutations that lead to lower N usage are inversely related to those that lead to lower C usage. 

References:

1. L Shenav, D Zeevi. Science 370, 683 (2020).

2. JJ Gryzmski, AM Dussaq. ISME J. 6, 71 (2012).

Codon De-optimisation, Beta Turns, etc

 Why would anyone wish to design a live, attenuated vaccine for COVID-19? They cannot be administered in pregnant women or immunosuppressed subjects, but they do have one major advantage. They are the only form of vaccine that can be given intranasally. And given through this route, they stimulate the production of secretory IgA antibodies, which are the only isotype which protect the upper respiratory tract against COVID-19. (The lower respiratory tract is protected by circulating IgG). 

Unlike other types of vaccines therefore, live attenuated vaccines would be expected to stop not just illness from COVID-19 in the vaccinated subject, but also transmission of the virus to contacts.

But how is the live virus attenuated? There are various means of doing so- by growing it at a lower temperature, or in a non-human cell line, but the one method used for 3 vaccine candidates using live attenuated virus during the current pandemic use a technique called codon de-optimisation.

The genetic code is redundant. That is to say, a given amino acid can be encoded by more than one codon. Yet, amongst these multiple codons, there is one that is favoured above all others- a phenomenon called codon bias. 

Several live vaccines have attenuated the causative virus by reverse genetics (targeting the putative DNA triplet bases which codes for a certain amino acid in the peptide chain), through replacing the normally favoured codon in the viral DNA or RNA with a less favoured codon. In some cases, this goes a bit further and replaces a favoured codon pair with a less favoured codon pair. This is called codon de-optimisation.

While in theory, the replaced codon codes for exactly the same amino acid, in practice, this disrupts the tertiary structure of the peptide chain and leads to a dysfunctional viral protein being translated in the host cell. It is thought that the tRNA carrying the "non-favoured" anti-codon somehow interferes with the translation machinery.

Interestingly, when non favoured codon pairs are introduced during codon de-optimisation, it invariably introduces more CpG dinucleotides (nnCpGnn). The latter are under-represented in favoured codon pairs. It has  however been shown that this excess of CpG nucleotides is not mechanistically responsible for the disruption of translation, which is thought to be induced by the unfavourable "fit" caused by the tRNA carrying the "unfavoured" anticodon, as described above.

The corollary to this is that codon optimisation (i.e. using the favoured codon) can improve the yield of useful proteins produced for medical usage in E.coli by phages.

A related concept is stabilisation of a recombinant viral protein vaccine by introducing two proline residues around a beta turn in the peptide molecule. A beta turn is a portion of the peptide chain when there is a sudden change in direction , say from an alpha helix to a beta pleated sheet, or between two alpha-helices. The artificially introduced proline residues at the beta turn stabilises the whole protein molecule and prevents misfolding.

This technique has been used in COVID-19 by Novavax for their recombinant Spike protein vaccine. It has also been used for the mRNA vaccines produced by Moderna and Pfizer-Biontech. The mRNA molecule in these vaccines is destined to be translated into the full length S-protein inside the cells of the vaccinated person, with the difference from the wild type S-protein being the 2 stabilising proline residues.

Herd Immunity for COVID19- Who Do You Vaccinate?

 So you have access to several 100 millions of a new vaccine for COVID, But there are billions of people that need to be vaccinated. Who do you vaccinate? What is the quickest way to (a). Protect the most vulnerable? (b). Achieve herd immunity?

Let's deal with the logistics of achieving herd immunity first. For a given R0 (R-naught or Reproduction number), the proportion of population (say P) that needs to be immune in order to achieve herd immunity is given by: 

P= 1-1/R0.

Since the R0 for COVID19 is 3, the proportion of population that needs to be immune to achieve herd immunity is 2/3 or around 67%.

Yet, it is likely that for a large country of the size of say, India, a country of some 1.3 billion, you'd be struggling to lay your hands on nearly 900 million vaccines first up.

Or even if you did, which 2/3rds would you choose to vaccinate?

And here you are on the horns of a dilemma, almost game-theoryesque in its nature. Would you vaccinate the oldest third (and therefore epidemiologically the most vulnerable), the middle aged tertile, or the children?

Think before you answer!

Keep in mind that no matter which vaccine is used, the likelihood of elderly subjects developing immunity as a result is much lower than younger subjects. It's just the ageing immune system responds poorly to almost any antigen- be it natural or vaccine-carried- a phenomenon called immune senescence.

OTOH, children respond brilliantly to vaccines. National immunisation schedules are premised on this phenomenon. What's more, they then stop spreading the putative infective agent to the rest of the population, thus reducing its overall transmission to the older, vulnerable subjects.

To illustrate, when conjugate vaccines for Pneumococcus (PCV7, followed by PCV10 and PCV13) were introduced for childhood vaccination in 17 European countries, the occurrence of invasive Pneumococcal infections over the next 5 years (2011-15) fell in people over the age of 65- the most susceptible population-by over 75%, for the strains that were included in the vaccines, but increased for the strains that were not included. Overall disease burden among the elderly was at least moderately lower, simply due to childhood vaccination.

It follows therefore that if decision makers opt to vaccinate the older thirds of the population with the first few hundred millions of COVID vaccine, control of the pandemic would be by no means assured. In fact, failure is guaranteed, as these ageing group will respond poorly to the vaccine and will continue to be vulnerable to unfettered transmission from the young.

OTOH, vaccinating the group which is most likely to transmit the virus- the young- is likely to achieve the holy grail of herd immunity more quickly, due to the fact this younger population are more likely to respond to the vaccine and stop spreading it to the elderly.