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NOBEL PRIZES 2020

PHYSICS · PHYSIOLOGY OR MEDICINE · CHEMISTRY

Black Holes – Exotic Bottomless Pits in the Universe

Smiles Mascarenhas

PHYSICS

Roger Penrose Reinhard Genzel Andrea Ghez

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COVER STORY

December 2020 | Science Reporter | 11

THREE Laureates share this year’s Nobel Prize in Physics for contributing to our understanding of one of the most enigmatic and tantalizing phenomena in the Universe, the Black hole. The work of Roger Penrose is purely theoretical – he devised mathematical models that showed how the General theory of relativity leads to the formation of Black holes. Reinhard Genzel and Andrea Ghez led two independent teams of observational astronomers who discovered that an invisible and extremely heavy object govern the orbits of stars at the centre of our Galaxy. A supermassive Black Hole is the only object that explains their observation.

One half of the prize money goes to Penrose and the other half is shared by Genzel and Ghez. Roger Penrose is from the University of Oxford, U.K., Reinhard Genzel is from the Max Planck Institute for Extraterrestrial Physics, Garching, Germany and Andrea Ghez is from the University of California, Los Angeles, USA.

Black holes are super dense objects with a phenomenal gravitational field from which not even light can escape. Because of this, they are expected to be dark but matter in the neighbourhood can form an accretion disc around it emitting X-rays in the process. Observational astronomers were actually looking for such objects in the Universe. Little did they realize that Black holes would be the foundation for all the galaxies in the Universe.

It is speculated that Black holes can range in size from tiny micro Black holes to supermassive Black holes. It is generally believed that the concept of Black hole sprung up after Albert Einstein published his General Theory of Relativity in the year 1915. The first theoretical description of what we now call a Black hole came just a few weeks after the publication of the General Theory of Relativity. The German astrophysicist Karl Schwarzschild was able to provide Einstein with a solution that described how heavy masses can bend space and time.

Later studies showed that once a Black hole has formed, its boundary is defined by an event horizon. Nothing within the event horizon can escape and the Black hole remains forever hidden inside it. The greater the mass, the larger the black hole and its horizon. For a mass equivalent to the Sun, the event horizon has a diameter of almost three kilometres and, for a mass like that of the Earth, its diameter is just nine millimetres.

The first calculation of the collapse of a massive star was made at the end of the 1930s, by physicist Robert Oppenheimer. When giant stars, more than ten Solar masses, run out of fuel, they first explode as supernovas and then collapse into extremely densely packed remnants, so heavy that gravity pulls everything inside. Not even light can escape from it. Einstein himself suspected if such objects could really exist or can be objects of mathematical curiosity. Only after his death in 1955, a series of milestone discoveries on General relativity opened up the possibility of the existence of Black holes. These came in the form of ‘singularity theorems’ starting with the work of the Indian physicist A.K. Raychaudhuri and culminating with the classic 1965 paper of Roger Penrose.

The question of the existence of Black holes was again revived in 1963, with the discovery of quasars, the brightest compact objects in the far corners of the universe. For almost a decade, radio astronomers were puzzled by radio waves from mysterious star-like sources, such as 3C273 in the constellation of Virgo. Doppler measurements in visible light finally revealed that 3C273 is over a billion light years from the Earth. If the light source is so far away, it must have an intensity equal to the light of several hundred galaxies. It was given the name ‘Quasar’ (from Quasi Stellar Radio object, since it resembled a star).

Astronomers soon found quasars that were so distant they had emitted their radiation during the infancy of the universe. How did quasars produce such incredible energy in a compact dimension? There is only one way to obtain that much energy within the limited volume of a quasar – from matter falling into a massive black hole. To explain the effect, Penrose toyed with the idea of trapped surface. Trapped surface forces all rays to point towards a centre, regardless of whether the surface curves outwards or inwards. Using trapped surfaces, Penrose was able to prove that a black hole always hides a singularity, a boundary where time and space end. Its density is infinite and, as yet, there is no theory on how to approach this strangest phenomenon in physics. Trapped surfaces became a central concept in the completion of Penrose’s proof of the singularity theorem. He introduced differential geometry and topological methods in the study of our curved universe.

Coming to the observational part of this year’s Nobel Prize, even though we cannot see the Black hole, it is possible to establish its properties by observing how its colossal gravity directs the motions of the surrounding stars.

One hundred years ago, the American astronomer Harlow Shapley was the first to identify the centre of the Milky Way, in the direction of the constellation of Sagittarius. A serendipitous discovery by Karl Jansky in 1931 showed a strong source of radio waves in that direction, opening up the exciting field of Radio Astronomy. This source was given the name Sagittarius A*. Towards the end of the 1960s, it became clear that Sagittarius A* occupies the centre of the Milky Way, around which all stars in the galaxy orbit.

For more than fifty years, physicists have suspected that there may be a black hole at the centre of the Milky Way. Ever since quasars were discovered in the early 1960s, physicists reasoned that supermassive black holes might be found inside most large galaxies, including the Milky Way. However, no one can currently explain how the galaxies and their Black holes, between a few million and many billion solar masses, were formed.

German astronomer Reinhard Genzel and US astronomer Andrea Ghez each led separate research groups that explore the centre of our galaxy, the Milky Way. Shaped like a flat disc about 100,000 light years across, it consists of gas and dust and a few hundred billion stars including our Sun. From our vantage point on Earth, enormous clouds of interstellar gas and dust obscure most of the visible light coming from the centre of the galaxy. Infrared and Radio telescopes first allowed astronomers to see through the galaxy’s disc and image the stars at the centre. Using the orbits of the stars as guides, Genzel and Ghez have produced the most convincing evidence that there is an invisible supermassive object hiding there. A black hole is the only possible explanation.

It was not until the 1990s that bigger telescopes and better equipment allowed more systematic studies of Sagittarius A*. Genzel and Ghez each started independent projects to attempt to see through the dust clouds to the heart of the Milky Way. Along with their research groups, they developed and refined their techniques, building sensitive instruments and committing themselves to long-term research.

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Only the world’s biggest telescopes will suffice for gazing at distant stars. Genzel and his group initially used NTT, the New Technology Telescope on La Silla Mountain in Chile. They eventually moved their observations to the Very Large Telescope facility on Paranal Mountain, also in Chile. With four giant telescopes twice the size of NTT, the VLT has the world’s biggest monolithic mirrors, each with a diameter of more than 8 metres.

Andrea Ghez and her research team use the Keck Observatory, located on the Hawaiian mountain of Mauna Kea. Its mirrors are almost 10 metres in diameter and are currently among the largest in the world. Each mirror is like a honeycomb, consisting of 36 hexagonal segments that use adaptive optics to better focus the starlight.

With large telescopes we suffer from the problem of scintillation of starlight. Large bubbles of air above the telescope, which are hotter or colder than their surroundings, act like lenses and refract the light on its way to the telescope’s mirror, distorting the light waves. This is why the stars twinkle and also why their images are blurred. The advent of adaptive optics

was crucial in improving observations. The telescopes are now equipped with a thin extra mirror that compensates for the air’s turbulence and corrects the distorted image.

For almost thirty years, Genzel and Ghez have followed their stars in the distant stellar jumble at the centre of our galaxy. They continue to develop and refine the technology, with more sensitive digital light sensors and better adaptive optics, so that image resolution has improved a lot. They are now able to more precisely determine the stars’ positions, following them night after night.

The researchers track some thirty of the brightest stars in the multitude. The stars move most rapidly within a radius of one light-month from the centre, inside which they perform a busy dance like that of a swarm of bees. The stars that are outside this area, on the other hand, follow their elliptical orbits in a more orderly manner. One star, called S2 or S-O2 completes an orbit of the centre of the galaxy in less than 16 years. This is an extremely short time, so the astronomers were able to map its entire orbit. We can compare this to the Sun, which takes more

than 200 million years to complete one lap around the Milky Way’s centre. This duration is called a Cosmic year. During the last lap of the Cosmic year, dinosaurs were dominating the surface of the Earth.

The agreement between the measurements of the two teams was excellent, leading to the conclusion that the Black hole at the centre of our galaxy should be equivalent to around 4 million solar masses, packed into a region the size of our solar system.

We may soon get an image of Sagittarius A* mapped. This is next on the list because, just over a year ago, the Event Horizon Telescope astronomy network succeeded in imaging a supermassive black hole thousand times more massive than Sagittarius A*. This is at the core of the galaxy known as M87 which is at a distance of 55 million light years from us. I am proud to add that one of the members of that team was my former student.

Prof. K. Smiles Mascarenhas was formerly a Scientist at the Millimeter wave Astronomy Lab of the Raman Research Institute, Bangalore. Email: smiles51@rediffmail.com.

PHYSIOLOGY OR MEDICINE

Nobel Recognition for Solving a Mystery

Biju Dharmapalan

Harvey J. Alter Charles M. RiceMichael Houghton

December 2020 | Science Reporter | 13

IN a year when the whole world has been held hostage by a minute virus, the Nobel Prize for Physiology or Medicine going to scientists who discovered the hepatitis C virus seems to be a morale booster for researchers working on the COVID-19 virus.

The 2020 Nobel Prize in Physiology or Medicine has been awarded to Harvey J. Alter from the US National Institutes of Health in Maryland, Charles M. Rice from Rockefeller University in New York, and Michael Houghton, a British virologist at the University of Alberta in Canada, for discovering the liver-ravaging hepatitis C virus, a breakthrough that led to cures for the deadly disease and tests to keep the scourge out of the blood supply.

“For the first time in history, the disease can now be cured, raising hopes of eradicating hepatitis C virus from the world population,” the Nobel committee said in a statement.

In the 1940s, it became clear that there are two main types of infectious hepatitis. Hepatitis A is transmitted by polluted water or food and generally has little long-term impact on the patient. Hepatitis B virus virus is transmitted through blood and bodily fluids and represents a much more serious threat since it can lead to a chronic condition, with the development of cirrhosis and liver cancer.

In the 1960s, Baruch Blumberg determined that one form of blood-borne hepatitis was caused by a virus that became known as Hepatitis B virus, and the discovery led to the development of diagnostic tests and an effective vaccine. Blumberg was awarded the Nobel Prize in Physiology or Medicine in 1976 for this discovery.

Even after the discovery of Hepatitis B, scientists were intrigued by the existence of another deadly form of Hepatitis. This intriguing deadly virus, later identified by this year’s Noble laureates as Hepatitis C, has affected around 150 million people globally.

Harvey J. Alter was working in a blood bank at the US National Institutes of Health during the 1960s when hepatitis B was discovered. He was studying the occurrence of hepatitis in patients who had received blood transfusions. Although blood tests for the newly-discovered Hepatitis B virus reduced the number of cases of transfusion-related hepatitis, a large number of cases still remained. Tests for Hepatitis A virus infection were also developed around this time, and it became clear that Hepatitis

A was not the cause of these unexplained cases.

In 1978, Alter showed that plasma from patients with unexplained hepatitis could cause disease when transferred to chimpanzees, indicating it was caused by an infectious agent. In additional experiments using chimpanzees, Alter and his colleagues showed the disease was likely caused by one or more viruses. Alter’s methodical investigations had in this way defined a new, distinct form of chronic viral hepatitis. The mysterious illness became known as “non-A, non-B” hepatitis.

Michael Houghton was a young researcher at Chiron Corporation, an American pharmaceutical company (now part of Novartis), when he began searching for the Hepatitis C Virus (HCV)

in 1982. Houghton, along with colleagues Qui-Lim Choo and George Kuo collected RNA from the serum of infected chimps and used the RNA to make a new cDNA collection. They put that collection into bacteria that could produce the proteins encoded by the DNA snippets. Finally, they used serum from an infected patient — which they assumed would carry antibodies to the virus — to identify any bacterial colonies that might produce a viral protein. Out of 1 million bacterial colonies they screened, one coded for a protein from a virus. The researchers described their seminal work in 1989 in a paper published in Science in which they named the disease hepatitis C.

Houghton’s work led to the development of a diagnostic test to identify the virus in blood, enabling doctors and

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researchers for the first time to screen patients and blood donors. This allowed blood donations around the world to be screened, which dramatically reduced the number of newly infected people.

The University of Alberta recruited Houghton in 2010 and he moved to the Edmonton area shortly thereafter where he became the director of the Li Ka Shing Applied Virology Institute.

The discovery of Hepatitis C virus was pivotal; but one essential piece of

the puzzle was missing: could the virus alone cause hepatitis? To answer this question the scientists had to investigate if the cloned virus was able to replicate and cause disease. Charles M. Rice, a researcher at Washington University in St. Louis, along with other groups working with RNA viruses, noted a previously uncharacterized region in the end of the Hepatitis C virus genome that they suspected could be important for virus replication. Rice also observed genetic

variations in isolated virus samples and hypothesized that some of them might hinder virus replication.

Through genetic engineering, Rice generated an RNA variant of the Hepatitis C virus that included the newly defined region of the viral genome and was devoid of the inactivating genetic variations. When this RNA was injected into the liver of chimpanzees, virus was detected in the blood and pathological changes resembling those seen in humans with the chronic disease were observed. This was the final proof that Hepatitis C virus alone could cause the unexplained cases of transfusion-mediated hepatitis. This work was also published in the journal Science in 1997. It also set the stage for work that would lead to the development of drugs, which can now cure most people who are infected with hepatitis CProf. Satyajit Mayor, Director, NCBS

Prof. Sudhir Krishna, NCBS

Dr Ranabir Das

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In the midst of the current pandemic, Professor Rice’s work reminds us of how challenging viral diseases are to eradicate and how revealing basic biological mechanisms of disease is critical to designing treatment. The 68-year-old currently works at Rockefeller University in New York.

The prize announcement has also created heated debate in India. India contributes a large proportion of this HCV burden. The prevalence of HCV infection in India is estimated at between 0.5% and 1.5%. “In the medicine Nobel, it’s always related to some fundamental questions and impact on amelioration of a deadly disease. How this choice is made is an interesting question? Putting money into research on

emerging viral diseases is paramount (as is putting money into science in general).” says Prof. Satyajit Mayor, Director, National Centre for Biological Sciences (NCBS).

“India is weakly positioned in virus research,” says Dr Ranabir Das, also from NCBS. “The financial support for these kinds of research work is minimal. Unless India commits to improve the basic infrastructure and give financial long-term commitment on virus research, we will be found wanting in a future situation when a virus specific to the Indian sub-continent hits us.”

According to Prof. Sudhir Krishna from NCBS, “India needs to build a virology network encompassing

CHEMISTRY

Chemistry Nobel Prize for Molecular Genome Editing Tool

M.S.S. Murthy

Emmanuelle Charpentier Jennifer A. Doudna

ON October 7, 2020, The Royal Swedish Academy of Sciences awarded the Nobel Prize in Chemistry 2020 jointly to Dr Emmanuelle Charpentier of Max Planck Unit for the Science of Pathogens, Berlin, Germany and Dr Jennifer A. Doudna, University of California, Berkeley, USA, “for their

veterinary medicine, human medicine and even plant research and should provide training to build capacity building. The Nobel Committee often recognizes basic science that has laid the foundations for practical applications in common use today. All these Nobel prizes stress on the need to focus on basic science research.”

Mr Biju Dharmapalan is Assistant Professor & Head, School of BiosciencesMar Athanasios College for Advanced Studies Tiruvalla (MACFAST), Kerala-689101. E-mail: bijudharmapalan@gmail.com biju_dharmapalan@yahoo.co.uk

development of a method for genome editing”. The method is known as CRISPR-Cas9.

Why edit genes? Genes regulate every function in our body and determine the basic characteristics like height, weight, skin colour, intelligence, etc. This complete set of instructions known as

genome is present in the form of 24 pairs of chromosomes in every cell of our body, while the germ cells, the sperms and eggs have one chromosome of each pair.

We inherit them from our parents, one set from the father and one from the mother, and pass them on to our children. Any changes in these genes, known as

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mutations, generally alter their functions which, many a times cause diseases. If such changes are produced in the germ cells, they are passed on to our offspring as inherited diseases. The only possible way to handle such problems is to edit such genes – either to inactivate or replace them with unmodified ones. This approach, known as gene therapy, which has been a dream all these years is becoming possible because of the tool created by the awardees for editing the genome. There are many more applications for gene editing.

CRISPR-Cas9 has been adapted from the natural immune system of certain types of bacteria. In the 1980s, bacteriologists discovered that widely different bacteria contain in their genetic material “DNA sequences that are surprisingly well preserved; the same code appears over and over again.” These repeat sequences of constant length, about 30 nucleotide long, were partially palindromic. They read the same way backwards and forwards like the words “mom”, “race car”, etc.

Their observations also revealed that the repeat sequences were interspersed with unique, non-repetitive nucleotide sequences that differ from each other. They called this Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR).

Another interesting observation that came later was that the unique non-repetitive sequences in the CRISPR

showed homology, that is they were matched with parts of genetic codes of various viruses that generally invade bacteria. Subsequently, bacteriologists identified another group of genes adjacent to the CRISPR region in the bacterial genome, which they called CRISPR associated genes (Cas genes). These genes code for proteins called nucleases, which specialize in unwinding and cutting DNA strand. So, the thinking at that time was that CRISPR, together with Cas genes was part of an ancient immune system, that protected bacterial from the invading viruses.

Putting all this information together, bacteriologists proposed a scheme for bacterial defence system. If a bacterium survived a virus infection, it grabs a piece of viral DNA and adds it to its own genome at the CRISPR region as a memory. When the virus attacks again, the bacterium copies the genetic material in the CRISPR region into an RNA, which now contains a sequence that matches with the viral DNA as well as the repeat sequences. Cas proteins attach themselves to the CRISPR RNA. As the RNA seeks and binds to the viral DNA, the Cas proteins cleave the two strands of the viral DNA at corresponding positions, creating a double strand break, which is enough to destroy the virus. In fact, these spacers appeared to evolve in time, adding new spacers.

Each infection which the bacteria survive is like a vaccine shot for future protection. While mapping the entire CRISPR-Cas system in the bacteria Streptococcus thermophilus, Dr Jennifer Doudna found that it is a complex machinery requiring many different Cas proteins to disarm a virus.

In 2009, Emmanuelle Charpentier, working at Umea University at Sweden, was interested in gene-regulating RNA molecules in the bacteria Streptococcus pyogenes. During her investigations she found one type of small RNA molecules that existed in large numbers with genetic code partially complementary to the CRISPR RNA. Intrigued by this finding her team mapped the CRISPR region in S. pyogenes and found out two important aspects.

First, she showed that the small unknown RNA molecules she had identified earlier, which she called trans-acting crispr RNA (tracrRNA), had a decisive role in the bacterial defence system. When the bacteria produced the CRISPR RNA in response to a viral attack, it would be a long molecule containing all the repetitive as well as the unique viral sequences. This was precursor CRISPR RNA (pre-CRISPR RNA).

To function as a part of the defence mechanism, it was necessary to convert it to an active form by cleaving it into smaller units. This is brought about by

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the tracrRNA. When tracrRNA pairs with this precursor CRISPR RNA, the long molecule is cleaved by an RNase III enzyme into smaller units, each containing a single spacer and a partial repeat sequence, constituting what is called crRNA. She called it a process of maturation. It was also found that Cas9, the Cas protein found in S. pyogenes, was more versatile than the Cas proteins worked out by Doudna and the system required just one Cas9 protein to cleave any viral DNA.

At this stage Charpentier realised that she had hit upon something extraordinary and decided to collaborate with Doudna for further work. In its natural form, the CRISPR-Cas9 system works on viral DNA. Charpentier and Doudna wanted to design a programmable gene scissor that could cut any DNA at predetermined locations even in other types of cells, enabling genome editing.

To test their concept, they picked up a DNA sample stored in the freezer and selected five locations where it should be cleaved. Based on these locations, they synthesized tracrRNA and crRNA in the laboratory and fused them into a single module called ‘guide RNA’ and complexed it with Cas-9 protein. When the system was

mixed with the DNA molecule in a test tube, it was spliced in exactly the intended locations. Thus, was born the molecular genetic scissor, a simple two component system that could be easily programmed for sequence specific cleavage of target DNA in any type of cell.

How does gene splicing lead to gene editing? When a cell undergoes DNA damage like a double strand break, there are two ways in which the cell can repair the break. The broken ends of the DNA strands can be ligated, in a process called non-homogenous end joining, to form a continuous strand. However, this process is prone to produce errors by either deleting certain bases (Deletions) or adding new bases (Additions) that are originally not part of the gene. In either case, the reconstituted gene sequence is altered and this may turn off the gene. In such cases, the protein product is not formed.

Alternatively, the repair system can use a sequence in the genome that is homologous or highly similar to the cut sequence as a template to repair the break (Homology directed repair). A researcher can hijack this system by introducing copies of DNA that are homologous to the target sequence and also containing a stretch of new sequence or modifications that he desires. During repair, this modified sequence can get precisely incorporated to the genome at the spliced region.

CRISPR-Cas9 is not the first gene editing tool. There are others like Zinc finger and TELENE which are in use for some years now. However, these are highly cumbersome to program, time consuming and expensive. In all these aspects CRISPR-Cas9 beats them. Hence, soon after Charpentier and Doudna published their results in 2012, several research groups adapted the system to edit genes in plants, insects, animals and humans. It has revolutionised agriculture by producing drought resistant, salt resistant, nutrition rich crops; modified insects like mosquitoes from spreading malaria, dengue. Animal breeders have adopted this system to breed animals with desirable characteristics.

More recently, in the face of the COVID-19 pandemic researchers have adapted the CRISPR-Cas9 gene editing tool for rapid coronavirus diagnostic tests. The Council of Scientific and Industrial Research in India which has also developed such a test known as Feluda test, says it can deliver results in 45 minutes to one hour.

Health researchers have been trying to develop new therapies for cancer, which are due to mutations that lead to uncontrolled cell division in tissues. They have already been using this technique to make the dream of curing inherited diseases come true by experimenting on sickle cell anaemia and beta thalassemia. These diseases arise from a single mutation in the beta globin gene that encodes for the protein haemoglobin, the oxygen carrying molecules in the red blood cells. The defective haemoglobin turns the red blood cells from their usual doughnut shape to sickle shape. They not only have reduced oxygen carrying capacity, but also get jammed in blood vessels causing pain, organ damage and often premature death. In the experimental treatment the doctor removes the patient’s bone marrow cells, treats them with CRISPR-Cas9 system, repairs the defect and transfuses the cells back to the patient. There are more than 6000 genetic diseases for which specific causal mutations in humans have been identified.

These therapeutic efforts involve editing somatic cells. The tool can also be used to edit germ cells – the egg and sperm cells or the cells in the early embryo to fix disease-causing mutations, which otherwise will be inherited by the offspring. However, it is also possible to attempt to create what geneticists call the “Designer babies”, wherein selected characteristics such as sight, skin colour, intelligence, etc. can be introduced in yet unborn babies. Since these attempts alter the genome of future generations, they raise ethical considerations and social issues.

Though CRISPR-Cas9 has been found to be a versatile gene editing tool, there are a few safety aspects in its widespread use. Some genes have multiple functions and editing them to correct one problem may affect the other functions of the gene. This is particularly important in while editing germ cells and embryos. Furthermore, some studies have shown that the system may induce off-target cuts, at locations other than intended. These clearly will have undesirable consequences. Researchers are working out to improve the system and make it safer.

Mr M.S.S. Murthy, I-Block, #304, Mantri Alpyne Apartments, Vishnuvardhana Road, BSK 5th Stage, Bengaluru-61. Email: mssmurthyb104@gmail.com