Centre for Cellular and Molecular Biology

First Cases of Omicron Variant Detected in Andhra Pradesh, Chandigarh

The countrywide tally of the new variant is at 37.

New Delhi: Andhra Pradesh and Chandigarh reported their first case of Omicron on Sunday, while Maharastra and Karnataka also recorded one more case each of the COVID-19 variant, taking the countrywide tally to 37.

According to state health officials, a 20-year-old fully vaccinated man who arrived in Chandigarh from Italy to meet his relatives and a 34-year old foreign traveller, who came from Ireland to Mumbai and then to Visakhapatnam in Andhra Pradesh, have tested positive for the variant.

A man who arrived from South Africa became the third person to test positive for the COVID-19 variant in Karnataka, while a 40-year-old man tested positive after returning to Nagpur in Maharashtra from a West African country, taking the state’s tally of Omicron cases to 18.

The man in Chandigarh had landed in India on November 22 and is currently in institutional quarantine.

His seven high-risk family contacts were put under quarantine and were tested for COVID-19 by the RT-PCR method. All of them tested negative, an official statement said.

It said the traveller has been asymptomatic throughout.

“He is fully inoculated with Pfizer vaccine which he got in Italy. He has been kept in isolation for the last 11 days.

“His report for whole genomic sequencing was received late night on December 11 and has been found positive for Omicron variant,” the statement said.

In the Andhra Pradesh case, the state health department said the person, who first landed in Mumbai, was tested and found negative for COVID-19.

Also read: A ‘Stealth’ Version of Omicron Could Challenge Surveillance Efforts

He was then allowed to travel onward to Visakhapatnam on November 27.

“On conducting a second RT-PCR test in Vizianagaram, he tested positive for COVID-19. His sample was then sent to the Centre for Cellular and Molecular Biology in Hyderabad for genome sequencing and the result came out as Omicron positive,” the Public Health Director said in a release.

The person, however, did not have any symptoms and a re-test on December 11 showed he was COVID-19 negative.

“There are no other Omicron cases in the state,” the Director said.

So far, 15 foreign travellers who came to the state were found COVID-19 positive and all the samples were sent to CCMB for genome sequencing.

“Of the 15, genome sequencing reports related to 10 cases were received and only one of them was confirmed Omicron positive,” the Director added.

Till now, Omicron has been detected in Maharashtra (18), Rajasthan (9), Karnataka (3), Karnataka (3) and Andhra Pradesh (1) and in Union Territories of Delhi (2) and Chandigarh (1).

The Omicron variant was first detected in India in Bengaluru with two people testing positive for it comprising a South African national of Indian origin and a doctor.

“Third case of #Omicron has been detected in Karnataka. A 34-year-old male returning from South Africa has tested positive. He is isolated and being treated in a govt hospital. 5 primary and 15 secondary contacts have been traced and samples sent for testing,” State Health Minister Dr K Sudhakar tweeted on Sunday.

On Saturday, Delhi had reported its second case – a 35-year-old man with travel history to Zimbabwe and South Africa – taking India’s tally to 33.

Official COVID Projections Were Toppled by Virus Variants That Genome Panel Had Warned About

Former CCMB chief Rakesh Mishra’s comments to The Wire have drawn rebukes from at least two senior science-related officials in the government.

Bengaluru: Even as scientists have acknowledged flaws in a disease transmission model that may have led the government to believe India’s second COVID-19 wouldn’t be too bad, notable members of the government’s science-oriented bodies have pushed back on reports, including by The Wire, that the powers that be ignored scientific data that would have allowed India to anticipate the brutality of the wave.

Even though it predicted a rise in infections – 1.2 lakh daily new cases by mid-May – that ought to have been acted upon by the government as it corroborated the possibility of a second wave, its numbers underestimated the problem because the disease transmission model used was suited more to explaining the past than to predicting the future, according to scientists who spoke to The Hindu.

The Department of Science & Technology had put together a committee to study the spread of the novel coronavirus in India and to recommend policy interventions to help the government close out the epidemic as quickly as possible. The committee members were M. Vidyasagar (IIT Hyderabad), who was also the chair; Manindra Agrawal (IIT Kanpur); Lt Gen Madhuri Kanitkar (Ministry of Defence); Biman Bagchi (Indian Institute of Science); Arup Bose and Sankar K. Pal (Indian Statistical Institute); and Gagandeep Kang (CMC Vellore).

The team used a data-centric ‘supermodel’ to conclude, in October 2020, that India’s wave at the time was past its peak. In late February, Agrawal said in an interview that the model suggested there would be 5 lakh more cases or so in the ten weeks to come.

But since then, especially from the second half of April 2021, India’s COVID-19 case load has accelerated to register the fastest growth rate in the world, in the pandemic’s brief but intense history. The Government of India was caught off-guard by the ferocity, so much so that the healthcare systems in many states suffered very public breakdowns, further exacerbating the second wave. In particular, many people, including experts, have blamed the government for failing to anticipate the vaccine and oxygen shortages.

There has been apprehension in scientific circles that the government seized on the supermodel and developed the impression that India’s second wave would be more manageable. However, Agrawal told The Hindu that the supermodel could make predictions but only if it assumed that the “phase” – significant characteristics of the wave – didn’t change.

Gautam Menon of Ashoka University, Sonepat, had written for The Wire Science earlier noting that a phase change had happened: there were new variants of the novel coronavirus in circulation, some of which were better at evading the immune system, and the population’s immunity also could have faded. As a result, he wrote, “The parameters that enter models of how cases might increase now need to be changed by unrealistic amounts to account for the current rise. Beyond a point, the conservative assumption of continuity from the past must be abandoned.”

Also read: COVID-19 Is Surging In India – but Will There Be Fewer Deaths This Time?

Agrawal also said that the first national seroprevalence survey’s results, which the supermodel used, could have been misleading. The survey, conducted by the Indian Council of Medical Research (ICMR), said that 0.73% of India’s population could have been exposed to the novel coronavirus by June 2020. But Agrawal said the number was likely much lower, leaving more of the population still susceptible to being infected. (Also recall that ICMR published the survey paper only 14 weeks after the survey ended.)

Science has also reported that the supermodel may have been undermined by what it didn’t use: granular data that the ICMR has been collecting from the people getting tested for COVID-19. But as it happens, ICMR has “dragged its feet” on widening access to this data, prompting over 700 researchers to write to Prime Minister Narendra Modi.

While the government may have seized on the supermodel because its conclusions fit the preconceived notions of India’s political leaders, other government functionaries have pushed back – in one instance in a nasty way – against assertions by experts that politicians wilfully ignored early warnings of potentially dangerous variants.

Indeed, the ‘phase change’ that both Menon and Agrawal have referred to is also what was on top of the Indian SARS-CoV-2 Genome Sequencing Consortium (INSACOG).

In the second half of last year, the Indian government set up INSACOG to collect samples of the virus from different parts of the country and sequence their genes to understand which strains were common where. Last week, four members of this consortium said that they had told at least the Union health secretary of a dangerous new variant in the population that could aggravate India’s crisis.

Reuters reported that it “could not determine whether the INSACOG findings were passed on to Modi himself.” However, Rakesh Mishra, until recently the director of the Centre for Cellular and Molecular Biology, (CCMB) Hyderabad, whose facilities are part of the consortium, told The Wire yesterday that it was “impossible to believe Modi wasn’t told”.

In an interview with Karan Thapar for The Wire, Mishra lambasted political leaders for the abject failure to contain India’s COVID-19 epidemic, and alleged that they had acted in defiance of information about the virus and its spread that was available.

“It was a high concern and there’s no doubt about it,” Mishra said. “We were very, very concerned”, and that INSACOG was “dreading something bad would happen”.

A little earlier, after trying for many months to interview the principal scientific adviser (PSA) K. VijayRaghavan, vaccine delivery expert group chief V.K. Paul and ICMR head Balram Bhargava, Thapar published a list of 35 questions he would like them to answer. These three men are India’s top scientists in the government, and independent experts have criticised them repeatedly for failing to ensure India’s response to COVID-19 was evidence-based.

The latter continues to be met with silence from the government – but Mishra’s comments in his interview have drawn rebukes from at least two senior science-related officials in the government: Department of Biotechnology secretary Renu Swarup and senior advisor in the PSA’s office Shailja Gupta.

In an interview with Economic Times, Renu Swarup said that Mishra retired from service on April 30 and that all decisions “are made by the core group”, implying that Mishra didn’t belong in this group. However, she ignored the fact that Mishra was in service and very much part of the core group at the time INSACOG warned the government, which was in March.

Also read: ‘India Sequenced Less Than 1% of Total COVID-19 Samples in Nearly 3 Months’

Swarup disputed the use of the word “warning”, and added, “My department is leading the INSACOG initiative and I never saw any such report which said that numbers will go very high or rise exponentially.”

After a few scientists shared the video of the Mishra-Thapar interview on Twitter and remarked that more scientists should speak up, Shailja Gupta, a senior adviser in the Office of the PSA, tweeted the following response: “Best they don’t, their internal recorded discussions at high level meetings will reveal a very different story.”

Gupta’s Twitter bio says that her views, presumably those expressed on the social media platform, are those “of a free citizen”, but she wielded her knowledge of closed-door meetings to say that Mishra and others shouldn’t speak up.

Many other scientists and other observers responded saying that instead of making veiled references to allegedly compromising information or even threatening those who are speaking up, Gupta should release the minutes of all meetings attended by government officials and scientists vis-à-vis COVID-19.

Is ‘Atmanirbhar Bharat’ Holding Up India’s Genome Sequencing Program?

India’s newly launched programme to widely and rapidly sequence genomes of the novel coronavirus has already run into major hurdles.

Bengaluru: Rakesh Mishra, the director of Hyderabad’s Centre for Cellular and Molecular Biology (CCMB), is frustrated. CCMB has been sequencing SARS-CoV-2 viral genomes since the COVID-19 pandemic began – initially as part of its own research program and since December 2020 as part of the Indian SARS-CoV-2 Genomics Consortium (INSACOG), a group of ten labs the government put together to ramp up sequencing across to India.

To do its work, Mishra’s team needs specialised plastic containers and reagents that go into sequencing machines. But buying them has become needlessly complicated in the last year, taking time away from his lab’s core jobs, according to Mishra.

The source of his troubles is a finance ministry order in May 2020 that stopped government labs from importing goods worth less than Rs 200 crore. The order was meant to boost local manufacturing, in the spirit of ‘Make in India’, but had unintended consequences for India’s fledgling genome-sequencing efforts. Several reagents and plastics used by Indian labs come from foreign manufacturers, like the US-based Illumina Incorporation, and have no Indian substitutes.

The ministry’s sudden restrictions threw these labs out of gear. By September 2020, sequencing across the country had come to a near-complete halt, as labs ran out of reagents they needed. Then, in response to their complaints, the ministry exempted reagents from its restrictions in January 2021.

But plastics are now the new thorn in his side, Mishra said. These materials still haven’t been exempted from the ministry’s order, which means his lab can’t buy them in bulk unless it can conduct a market assessment to show that no Indian alternatives exist. Such an assessment is a needless bureaucratic distraction at a time when labs desperately need to sequence more viral genomes – and faster. “It’s like asking us to run a 100-metre dash with our hands tied,” Mishra said. “I can run, but I will run very slow.”

Ill-timed government orders are just one hurdle that INSACOG labs have had to cross to sequence genomes faster across India. To pull this off, these labs need several stars to align: COVID-19 testing labs must preserve positive samples from patients, state governments must transport these samples to the ten INSACOG labs every week, and the labs must be able to buy the equipment they need to sequence quickly.

None of this has been happening two months since INSACOG was launched. To begin with, the Rs 100 crore fund that INSACOG had requested from the Union science ministry hasn’t arrived yet. Without the money, most labs can’t buy the equipment they need, and are progressing slowly with their own funds.

“We are excited to be part of INSACOG, but the money needs to come, otherwise how can we ramp up?” Uma Ramakrishnan, a molecular ecologist at the National Centre for Biological Sciences (NCBS), Bengaluru, which is part of the consortium, asked. Others say that even if labs could sequence more, states aren’t yet sending them enough samples. The reasons range from a lack of coordination between state governments and INSACOG labs to restricted availability of the right kind of RT-PCR samples needed for sequencing.

If INSACOG had followed its implementation strategy to the letter, it ought to have sequenced 5% of all positive cases in India since November 23, in addition to samples from international travellers. This adds up to at least 80,000 sequences by now. Instead, as on February 24, 2021, the consortium had completed only 3,500 sequences. And unfortunately, the labs with the most capacity, including CCMB and the Institute of Genomics and Integrative Biology (IGIB) in New Delhi, have remained heavily under-utilised.

While IGIB can sequence up to 10,000 samples a month, it has only received over a third of this number from Punjab, Haryana, Rajasthan, Kerala and Uttar Pradesh combined, said Anurag Agarwal, the institute’s director. Meanwhile, CCMB, which can sequence 5,000 samples a month, has received fewer than 200 samples from Telangana, Andhra Pradesh and Northern Karnataka, which are the areas in its purview.

Three ambitious goals

A view of CCMB Hyderabad. Photo: CSIR

For all the obstacles it faces, INSACOG’s work has never been more crucial.

Since the novel coronavirus pandemic began, several new variants of the virus have shown up worldwide. Among them are B.1.1.7, a highly contagious variant first reported from the UK, the B.1.351, a variant that first emerged in South Africa and is known to diminish the efficacy of some vaccines, and B.1.1.28, a variant from Brazil. Large outbreaks in Maharashtra and Kerala today have raised the question of whether genetic changes in the virus could be driving them.

To pick up such changes as they emerge in the viral population, and to stop them from spreading elsewhere, the government needs a coordinated and continuous surveillance program like INSACOG, said Vinod Scaria, a genomics researcher at IGIB.

Yet, until December 2020, India didn’t have such a programme. Instead, a few labs were working on their own steam to map how SARS-CoV-2 was evolving. They either used samples that came to them for testing or tied up with state governments interested in mapping the virus’s evolution. As a result, sequencing was both meagre and spotty for a country India’s size: labs got their samples from a few interested states like Kerala, Gujarat and Andhra Pradesh, but large parts of the country were flying under the radar.

In November, fears of the B.1.1.7 variant spreading through the country swung the government into action. It launched INSACOG, led by the National Centres of Disease Control (NCDC) in New Delhi. Nine other labs, including the National Institute of Virology (NIV), Pune; the National Institute of Mental Health and Neurosciences (NIMHANS), Bengaluru; NCBS, CCMB and IGIB will cover all states between them. The idea was that the NCDC would co-opt its deeply entrenched network – the Integrated Disease Surveillance Programme (IDSP) – to liaise with state governments and route samples from them to the ten labs.

The consortium has three ambitious goals. The first is to map the spread of the B.1.1.7 variant across India. The second is to investigate regional outbreaks, by collecting samples from multiple times from these regions so that new variants, if any, can be intercepted. The third is to set up a continuous surveillance program to sequence 5% of all samples across the country every month – in line with what countries like the UK have been doing.

Trouble finding old samples

An artist’s impression of a swab test. Image: United Nations/Unsplash

As of today, work has begun only on the first and second goals, and both have also quickly run into hurdles. To determine if B.1.1.7 was in India – a goal later expanded to B.1.351 and B.1.1.28 as well – INSACOG was supposed to sequence samples from every international passenger who tested positive for COVID-19 since November 23, 2020, and from 5% of all positive cases across India between November 23 and December 22. That is, as soon as INSACOG was kicked off in December, states ought to have dug into their archives, found old samples from passengers and the general populace, and dispatched them to the labs.

This didn’t really happen.

International-passenger samples were easy to collect, but states struggled to locate old samples from 5% of all cases since November 23, NCDC director Sujeet Kumar Singh said. There were many reasons. First, many of the positive samples had been tested using antigen tests, and few labs stored these samples after the test was done. Second, finding old samples from even RT-PCR tests has been an uphill task. Testing labs are required to store these samples at a temperature between -80º C and -20º C, and labs across the country are not required to do this beyond a month, given freezer-space constraints.

A third hurdle is that sequencing works best for samples that have a high viral load or with an RT-PCR cycle threshold of less than 25. “So to find B.1.1.7, we focused only on RT-PCR samples which were available with labs, and which had low cycle threshold,” said Singh.

These restrictions shrunk the pool available for sequencing to a minuscule fraction of the intended 5% (45,000 out of 9,35,251 of positive cases), according to Singh. Eventually, a total of 3,500 sequences were completed by February, of which INSACOG had detected 187 instances of B.1.1.7, four of B.1.351 and one of B.1.1.28. And of these, the B.1.1.7 variant had been detected in 18 states, among both international travellers and people who’d had no contact with such travellers.

Singh said this data meets INSACOG’s first goal because it shows that the B.1.1.7 variant is spreading in the community within India. And now that this has been established, there’s good reason to stop looking at old samples because the task has been painstaking and cost-intensive. “We have submitted to the health ministry that the first goal of INSACOG is fulfilled. Once it is done, we don’t see a rationale in pursuing it further,” he said.

Other scientists in the consortium agree. If one, some or all of the three variants are behind local outbreaks, sequencing under INSACOG’s second and third goals would pick them up anyway, they said.

The problem is that progress on the second and third goals is also happening at a crawl.

Ramping up during an outbreak

Colorised scanning electron micrograph of a cell heavily infected with SARS-CoV-2 virus particles (red), isolated from a patient sample. Image: NIAID/Flickr, CC BY 2.0

The INSACOG consortium’s second goal is to investigate outbreaks like the ones ongoing in Maharashtra and Kerala. For this, scientists typically sequence samples from people in the outbreak area at multiple time points, over weeks. If a particular variant seems to be spreading faster than others, the next step would be to figure out if it has an evolutionary advantage. B.1.1.7, for example, has mutations that allow it to bind to human cells better, which in turn makes it more transmissible. (See box: Are the E484K and N440K variants worrying?)

Are the E484K and N440K variants a worry?

After COVID-19 cases spiked in Maharashtra in late January 2021, the B.J. Medical College, Pune, reportedly identified the so-called E484K mutation in one patient sample from the Amravati district and the N440K mutation in a sample from Yavatmal. The findings sparked speculation that these mutations may be behind Maharashtra’s climbing cases. Scientists have previously found in lab studies that these genetic changes can help the virus evade some human antibody responses. Clinical trials found the performance of the Novavax and Johnson & Johnson vaccines to be reduced in South Africa, which has a high prevalence of the E484K variant.

Does this mean E484K and N440K are something to worry about? It’s too early to say. Establishing this would require a large number of samples to be sequenced from Maharashtra. By all accounts, B.J. Medical College has sequenced only a handful.

Meanwhile, INSACOG hasn’t begun sequencing the samples it asked the affected districts to send (see main story). Once it sequences samples across several weeks in Maharashtra, and if this data shows a large and growing fraction of E484K or N440K infections, that would suggest that these mutations have some evolutionary advantage – such as an increased ability to transmit or to reinfect previously sick people.

N440K is not new – it has been on Indian scientists’ radar for a while. The last time it attracted attention was when scientists at CCMB spotted it in 34% of samples they sequenced from Andhra Pradesh in August and September 2020. Because the mutation seemed to be widespread then, it was important to track its spread across India, said Divya Tej Sowpati, a scientist at CCMB. But these efforts were thwarted by a change in India’s import rules (see main story), freezing most sequencing efforts across the country. N440K then fell off the radar for a while, according to Sowpati.

There is some in vitro evidence that N440K can dodge parts of the human immune system. In one study, published in October, US researchers found that in the presence of a monoclonal antibody called C135, which is also being investigated as a drug for COVID-19, a recombinant version of the SARS-CoV-2 virus quickly acquired the N440K mutation to evade the antibody. And of the handful of cases of COVID-19 reinfection in India that scientists have sequenced thus far, N440K was present in one.

Still, this is far from strong evidence that N440K causes reinfections. The way a virus behaves in a lab doesn’t always translate to how it behaves in an epidemic. Also, even if a mutation may help a virus evade a monoclonal antibody, people may still be able to fight off the virus with other antibodies and components of their immune system, such as T-cells.

Additionally the fact that N440K was so common in Andhra Pradesh without causing any noticeable increase in reinfection cases suggests it may not be so concerning, said Anurag Agarwal, director of the Institute of Genomic and Integrative Biology, New Delhi. Vinod Scaria’s work with sequencing samples from the outbreak in Kerala has also shown N440K to be present in 10% of the samples in some districts.

This is why INSACOG’s ongoing work in Maharashtra and Scaria’s group’s work in Kerala is crucial. By observing whether these mutations are becoming more frequent over time, they will be able to determine if the mutations had anything to do with the surges. Eventually, they may find that neither mutation has any role to play – or that a combination of both does. Or they may find an entirely different combination of mutations altogether.

A final distinct possibility, said Scaria, is that it’s not the viral genomic code that has changed in a meaningful way but the behaviour of people in affected states. If susceptible people who were not infected in Kerala’s or Maharashtra’s previous outbreaks stopped taking precautions, or began organising large gatherings, that could suffice to explain a lot.

But answering the question of whether the Maharashtra outbreak is being driven by new variants is not going to be easy. INSACOG has only now begun collecting samples from the 11 worst-affected districts, including Amravati and Yavatmal. Singh said the NCDC has asked these districts to send 100 samples each, obtained after January 23, when cases began growing exponentially. These samples are currently being transported to the two INSACOG labs handling Maharashtra samples: NIV and the National Centre for Cell Science, both in Pune. Once the samples arrive, researchers at these facilities may need up to two months to sequence and analyse the data.

But according to CCMB’s Mishra, this effort may not be enough. Instead of a one-time set of 100 samples from each district, the goal should be to sequence 100 samples each week, he said. “If a variant is beginning to emerge in these districts, it has to be followed up over several weeks,” he explained. Indeed, even INSACOG’s guidelines call for sequencing 5% of all positive samples every week in outbreak regions – which adds up to over 600 samples in February in Amravati. As of now, for reasons that are unclear, the NCDC has not instructed the districts to send more samples.

Kerala, on the other hand, has had a head-start in its outbreak investigations, thanks to a state-funded sequencing programme separate from INSACOG. Under this programme, the state tied up with the IGIB to send 25 samples from each of its 14 districts every week, starting from the last week December. Soon, IGIB will have a ten-week series of samples from Kerala, Scaria said, which will help his team decide if any new variants could be driving the state’s outbreak.

This said, even Kerala isn’t sequencing 5% of its positive samples yet. A state official who didn’t wish to be named told The Wire Science that this would happen only after INSACOG kicked off.

Continuous surveillance

Tirumala Bharani K. Settypalli, a molecular biologist and senior laboratory technician at the Animal Production and Health Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, prepares samples for an RT-PCR test. Photo: iaea_imagebank/Flickr, CC BY 2.0

The last and most difficult goal of INSACOG is the continuous surveillance programme – for which 5% of all positive samples must be sequenced continuously from every single state, instead of waiting for outbreaks to occur first.

This process has barely begun as labs wait for funds and states drag their feet on sending samples. While IGIB has received only 3,500 samples since November 23, CCMB has received fewer than 200.

The situation in other labs is no different. Ravi Vasanthapuram, a virologist with NIMHANS Bengaluru, another INSACOG lab, said his team had only sequenced 163 samples since November. Murali Dharan Bashyam, a molecular oncologist at the Centre for DNA Fingerprinting, Hyderabad, said they had received no samples under the INSACOG programme at all.

Mishra worried that states with plateauing COVID-19 cases aren’t realising the importance of starting their sequencing programmes during this lull, instead of waiting for spikes. Sequencing is most helpful when it is widespread and prospective, like the UK’s programme that INSACOG is modelled after.

“That is how England was able to take some action with B.1.1.7,” Mishra said. “The more you sequence, the earlier you pick up the variant, and the more likely you are to preempt spread. It’s a simple equation.”

Several states don’t seem to have got the memo. There is also disagreement between INSACOG labs and the NCDC on who will chase after these samples. While the NCDC’s Singh said it’s up to each INSACOG lab to follow up on pending samples, officials at more than one INSACOG lab told The Wire Science that they couldn’t be held responsible for this.

Lack of motivation to sequence isn’t the only problem holding back states, however. There are also genuine logistical challenges – among them, the difficulty in ferrying samples at below -20º C across the country, and INSACOG’s exclusive reliance on RT-PCR tests at a time several states are doing too few of them.

For example, Rajesh Bhaskar, Punjab’s nodal officer for COVID-19, said his team has struggled to arrange for a courier service to transport samples at the required temperature to INSACOG labs in Delhi. Meanwhile, Utkarsh Betodkar, an official of the IDSP in Goa, said only one medical institution in the state was administering RT-PCR tests, and the remaining banked on antigen tests or a molecular test called TrueNat, which also detects viral genetic material like RT-PCR.

But since the NCDC had only called for RT-PCR samples, it was difficult to meet targets, Betodkar said. Another official from Uttar Pradesh, who didn’t want to be named, told The Wire Science that 60% of tests in the state were antigen-based, which didn’t leave enough samples for sequencing. And of the remaining, many samples didn’t have a sufficiently low cycle threshold.

These problems aren’t insurmountable, several scientists said. For one, avoiding sequencing antigen samples and RT-PCR samples with low viral loads altogether is a bad idea because it would create a selection bias, Divya Tej Sowpati, a scientist at CCMB, said. A few RT-PCR samples with low viral loads should also be sequenced even though sequencing failures would be higher, he added. Mishra said that if some towns or cities are using only antigen tests, they must be made to store samples too, for sequencing later.

But INSACOG hasn’t begun ironing out such details in earnest; its labs are foremost in need of the expected Rs 100 crore. The Wire Science asked Renu Swarup, secretary at the Department of Biotechnology, about the hold-up but received no answer.

The way forward

A view of the Institute of Genomics and Integrative Biology, New Delhi. Photo: IGIB/Facebook

Given the formidable challenges in INSACOG’s way, did the consortium over-commit itself with its 5% goal? After all, until December, India was sequencing far fewer than 1% of its cases, and even a jump up to 1% or 2% would have been a useful improvement.

But scientists across INSACOG told The Wire Science that the 5% goal was not unachievable – especially given the current size of India’s outbreak. In January 2021, India reported 4,72,317 new cases, which means the consortium would have had to sequence 23,615 samples to hit its goal. The total capacity of the ten consortium labs, according to INSACOG’s guideline document, is higher than this: 27,800 samples per month.

And even if India experienced a surge, the labs can expand their capacity further, according to IGIB’s Agarwal. As of today, IGIB can sequence roughly 1,500 samples every four days – with a process to increase it to 1,500 samples a day, by automating the lab’s sequencing machines. “The current numbers we are seeing in India are nothing for us, if we decide to go full pace. The rate-limiting factor is not our ability to sequence but whether samples can get to us in time.”

All this is possible if labs are also not hobbled by irrational rules that slow them, said Mishra. The finance ministry’s goal to encourage Indian manufacturers was an important one, in his telling: India must eventually develop local capacity to make high-quality reagents and plastics for sequencing, instead of importing forever. The problem lies with the ministry’s decision to change rules overnight, in the middle of the pandemic. “They should have taken a graded approach, such as asking labs to reduce the use of imported goods over five years. If you ban everything in a day, we will be ill-prepared.”

But he is hopeful that with public health at stake, the government will help INSACOG labs by easing restrictions – and quickly. This is crucial because while new variants pose a grave danger in the coming days, the general population may be too fatigued to comply with strict measures like lockdowns.

“We have to ensure that if a new variant emerges – and they will emerge – they are picked up early and constrained. Otherwise, with the level of fatigue all around, we won’t be able to handle them,” he warned.

The reporting for this article was supported by a grant from the Thakur Family Foundation. The foundation did not exercise any editorial control over the contents of the article.

Priyanka Pulla is a science writer.

A ‘Mysterious’ New Frog Species With Hidden Spots and an Insect-Like Call

Discovered in the Western Ghats of southern India, the species is unique enough to be placed in a newly-created genus as well.

It was after a heavy spell of rain during the 2015 monsoon that Sonali Garg walked out to a spot she had been visiting regularly, and unsuccessfully, for more than two years. A large muddy puddle.

But this time, she struck gold in the form of a new species of frog, Mysticellus franki sp. nov., which turned out to be so unique the study authors have assigned it to a newly created genus.

It all started in 2013 when Garg encountered “strange looking” tadpoles in that muddy puddle when conducting amphibian surveys for her PhD from Delhi University. It was clear the tadpoles belonged to the frog family Microhylidae, but beyond that, “we couldn’t pinpoint what species the tadpoles belonged to,” said Garg in an interview with Mongabay.

When the researchers sequenced the tadpoles and examined the DNA, it was clear that the tadpoles were of a hitherto unknown frog species.

“We have a library of sequences of frogs and other amphibians from the country and we could compare the sequence of the unknown tadpole against them,” said Garg. “To our surprise, the sequence did not match any frog species from the country; it fell into the family [Microhylidae], but beyond that, it did not match anything that was known from India.”

With no knowledge about the adult frog — where it was found, whether it was big or small. Garg and her team started visiting the puddle where they collected the tadpoles regularly. An adult specimen was needed to carry out the necessary morphological analyses to delve into the mysterious new species.

“For the next two years, we kept going back to the same spot. It was the only spot we were sure we would get it,” said Garg. “So we went back there at different seasons and different times of the year because we didn’t know when this frog would come out.”

“Eventually, after two years of this exercise of repeated searching, one monsoon, a couple of days after the monsoon hit, when there was sufficient water that collected on the ground, around the puddles … we saw the frogs. They were there in the hundreds, it was magical. It was as if the frogs were welcoming us,” she added, the excitement still palpable in her voice even after nearly four years.

The new species “was an accidental discovery,” said Garg and supervisor S.D. Biju. “We just happened to be at the right place, at the right time. And of course, often we fail to look closely. In this case, we looked closely at every tadpole, and that’s how this discovery happened!”

Male and female specimens of the newly discovered Mysticellus franki sp. Credit: S.D. Biju

A new species … and a new genus

After making careful notes from the field and recording the calls of the male frogs serenading for females in the puddles, the researchers collected specimens and brought them to the lab to carry out genetic analyses.

A combination of genetic, morphological and call data threw up the final diagnosis. The frog was definitely a new species, and sufficiently different from other members of the family Microhylidae found in India to be assigned to its own genus as well.

The new species belongs to sub family Microhylinae. The genus name Mysticellus is derived from the Latin mysticus, meaning mysterious, and ellus, which means diminutive. The name highlights “the ability of this small frog to remain out of sight despite its occurrence in wayside areas surrounding human settlements,” write the authors in the paper. The species name franki honours Franky Bossyut, a professor and amphibian biologist at Vrije Universiteit Brussel.

“I agree with assignment of the species status. The allocation of a genus status is motivated based on molecular evidence. The evidence is still tenuous because we have one member of the new genus,” said Karthikeyan Vasudevan, senior principal scientist at the Laboratory for the Conservation of Endangered Species (LACONES), Centre for Cellular and Molecular Biology.

“More than anything, it is surprising that it has not been already found and described. While very similar in shape, size, and colouration to other microhylid frogs in southeast Asia, there is nothing in the Western Ghats of India that resembles this,” said David Blackburn, the Associate Curator of Amphibians and Reptiles at the Florida Museum of Natural History. “It is only the secretive nature of these frogs that has resulted in them only now being known to science.”

The new species of frog was discovered when researchers came across a ‘strange’ looking tadpole. After continuously searching for an adult for two years, they spotted around 100 adult frogs around a roadside puddle. Credit: S.D. Biju

Hidden spots and an insect-like call

The researchers observed that the frogs started congregating in hundreds around temporary muddy puddles, two to three days after the first monsoon showers. After four to five days of intense breeding, the frogs disappeared completely, leaving the researchers mystified.

“After we first discovered this frog in 2015, we carried out several surveys in and around the region over a period of three years to study this frog. However, due to its secretive behaviour we were only able to locate it during a very short window of less than four days,” said the authors in an interview.

After the frogs disappeared, the researchers were not able to locate “even a single individual” at any other time of the year. “We don’t know where it hides, lives, and what it does for the rest of the year. The frog’s external appearance does not show morphological adaptations for burrowing. At the same time, we doubt that it simply hides under leaf litter, rocks and stone (the usual hiding places for frogs during the non-breeding time). It’s still a mystery for us,” they said.

When the males called to attract females, they raised the hind part of the body to show off “a pair of black false-eye like spots.” The frogs did the same when the researchers tried to approach them, said Garg.

“The effect is quite startling,” she said. “When the animal is sitting down, the spots are hidden. When we were close to the animal, the frog raised the hind part of its body. This movement really made the spots very visible.”

“The best guess we have is that it’s a defensive mechanism,” she said.

The call of the frog is also quite distinct, observed the researchers. “It resembles an insect chorus,” write the authors in the press release.

The frogs probably had such a unique call to attract females in the most efficient manner, said Garg. “Even if a puddle is crowded with multiple frog species and multiple individuals of the same species, even if it’s pitch dark, the female needs to be able to find her way to the male,” she said. “This is one reason to have unique calls. Also, for this species, with such a short breeding window of four or five days, the pressure to get it right is much higher,” she added.

The frog has two distinct eye-like spots on its rear-end which probably serve as a self-defence feature. Credit: S.D. Biju

A mysterious past

A phylogeny (a sort of family tree) of all known genetic data known from the family Microhylidae shows that the closest relative of the subfamily Microhylinae is the subfamily Dyscophinae, which is restricted to Madagascar.

The available molecular evidence gives us this story. The family Microhylidae would have originated on Gondwanaland, the ancient supercontinent which would eventually break up to form most of South America, Africa, the Indian subcontinent, Australia and Antarctica. When Gondwanaland broke up, the subfamily Dyscophinae took up home in Madagascar and sub family Microhylinae moved on toward Asia on the Indian subcontinent. The split between Dyscophinae and Microhylinae happened about 67 million years ago, giving Microhylinae enough time to diversify in the Indian subcontinent as it drifted along towards Eurasia. Once the Indian subcontinent docked at Eurasia, frogs that make up the Microhylinae subfamily spread all over Asia.

For M. franki in particular, the closest relative on the family tree is the genus Micryletta, also belonging to subfamily Microhylinae but found in the Indo-Burma and Sunderland biodiversity hotspots in Southeast Asia and China.

Using algorithms that can parse out evolutionary timelines by considering the rate at which DNA changes over time, the researchers were able to give tentative dates to different nodes of the phylogeny.

The secretive frogs vanished after appearing for the breeding season which lasted for around four days. Credit: S.D. Biju

“Our study shows that the common ancestors of Mysticellus and Micryletta diverged about 40 million ago. Most likely they originally inhabited the Indian Peninsula and later diverged to give rise to both these genera,” said the authors.
The authors posit that the two genera are likely to have split when the Indian landmass moved close to mainland Southeast Asia through the Myanmar-Malay Peninsula during Middle/Late Eocene.

Karthikeyan Vasudevan from LACONES said, “Recent evidence from the study of arthropods in amber suggests that prior to the final collision at around 55 million years ago with Asia, India moved close to or had land connections with Africa and Europe.” Some models of continental drift show that species could have moved between the Indian subcontinent and Southeast Asia prior to the former’s collision with Asia, he added.

“This might help explain the presence of genera that are not present in the Eastern Himalayas, but are found in South India, Sri Lanka and Southeast Asia,” he added. He gave examples of the skink genus Dasia and the pit vipers (genus Tropidolaemus).

David Blackburn from the Florida Museum of Natural History agrees. “Clearly, some lineages must have survived on India as it moved across the Indian Ocean during the late Mesozoic and early Cenozoic, but several studies, including this one, now support that colonisation of the Indian subcontinent by animals from Asia before India had fully collided with the Asian mainland,” he said.

With inputs from Sahana Ghosh.

This article was first published on Mongabay. Read the original here.

Why P.M. Bhargava Will Be Remembered as a Forthright Institution-Builder

He could often seem autocratic and dogmatic – but for all his strong opinions, he had a commitment to quality, and in the end this is essential to build anything that will last.

Pushpa Mittra Bhargava. Source: YouTube

Ram Ramaswamy teaches at the Jawaharlal Nehru University. From mid-2011 to early 2015, he was at the University of Hyderabad and interacted with PMB on committees and other forums. Somdatta Sinha, presently at IISER Mohali, was on the staff of the CCMB, Hyderabad, from 1983 to 2011. S.S. and R.R. had collaborated in the area of theoretical biology in the early 1980s while respectively working at CCMB and the Tata Institute of Fundamental Research, Mumbai.

Pushpa Mittra Bhargava – a.k.a. PMB – was larger than life. His flamboyance was multidimensional, from the striking printed bush-shirts he was very often seen wearing, to the scientific friends and colleagues he cultivated, to the remarkable institution that he built and causes he espoused. Never one to shy away from controversy, he was one of the most outspoken public scientists in the country, and one who stood his ground on political as well as scientific fronts. There have been few like him in terms of his personal courage, and fewer still who were as unafraid to be vocal on issues that challenged his personal convictions.

PMB, born in Ajmer in 1928, was educated in Lucknow. He obtained his PhD from Lucknow University in 1949 and shortly thereafter moved to Hyderabad, to work at the CSIR’s Regional Research Laboratory (RRL) as an organic chemist. Although he spent a few years in the US and in England, he remained a Hyderabadi for the rest of his life, and Hyderabad is where both of us got to know him more closely, although at different stages of our lives.

Like few other chemists in the country in the 1950s and 1960s, PMB was greatly taken up by the ‘molecular’ approach to biology. He was an evangelist, and as students we recall his efforts in the early 1970s in persuading the faculty and students of leading chemistry departments in the country to look into the then-nascent field of molecular biology. He campaigned with great energy for setting up the Centre for Cellular and Molecular Biology (CCMB), first within RRL Hyderabad (now called the Indian Institute of Chemical Technology) in 1977.

Although a separate campus was not established for the institute until the mid 1980s, he was able to attract some outstanding talent to CCMB as well as some stellar visitors – James Watson and Francis Crick among them. From its inception, the CCMB had a distinctive character, marked by a fresh and distinctly innovative approach. It was always a very special type of laboratory within the CSIR. PMB’s vision was evident on all scales, from the type of building to the art in the corridors, the groups that were formed and the problems that were studied.

In turn, two very distinctive features of PMB and his approach to institution building are worth noting. The first is the sense of aesthetics, his ability to integrate artistic sensibility into the work environment, something he shared with Homi Bhabha and C.V. Raman. Indeed, he paid close attention to the details of design and he prioritised aesthetics and functionality over all else, be it the CCMB or his own residential quarters.

The second was his belief in the need for scientists to speak up for the cause of science, and the need for public intellectuals to engage on contemporary issues in forums that were appropriate. He ensured that CCMB would have occasions to invite the common people to come see what science was being done there, but he would also make the lectures by leading scientists available to the public at large. He believed that it was the duty of scientists to fellow citizens to explain and encourage them to get excited by science, and think scientifically.

From the early 1980s to this day, thousands of school and college students and their parents, and countless others, would visit CCMB and learn from the faculty and students there as to what work was going on. Another of his passions was MARCH (Medically Aware and Responsible Citizens of Hyderabad), an organisation that he cofounded and which would meet every month to discuss some issue or other pertaining to public health. Given his sensibilities, these would be current and he would also get some of the leading experts to come and talk.

Of course, the cause of public engagement could take extreme forms. In 2015, he returned his Padma Bhushan (awarded to him in 1986) to the Government of India as a protest against the government’s attack on rationalism, reasoning and science. Years earlier, in 1994, he had resigned from the fellowship of the science academies of India for their lack of opposition to governmental plans to introduce astrology into university curricula. He spoke out against many issues, such as homoeopathy, GM crops, irrational beliefs and superstitions, pseudoscience and the lack of scientific temper, and about which there are numerous reports in the media. He responded to national issues with conviction and inevitably made enemies for his strong views and actions.

But what remain are indelible impressions of PMB the man. Both of us were associated with and influenced, in different ways, by him over a long period of time. Oddly enough, it was his intellectual curiosity that stimulated our academic collaboration, starting with an invitation to RRL to speak at the CCMB in 1983. At that time, SS had just joined CCMB as a young faculty member: fresh from JNU and working in an area of biology most people were unfamiliar with, and always ready to question the ‘administration’s decisions’.

PMB would listen patiently. Faculty meetings encouraged long discussions, dissent, arguments over institutional issues – and all this came largely from the sense of belonging that he instilled in the staff. Hugely nationalistic, in a way that was appropriate at that period of time, PMB would ask, “Why can we not do this work here?” of one or the other scientific problem. Keenly aware of trends in world science, he encouraged faculty to think of difficult and completely new problems that could have applications to society. He had the ability to find excellence and innovative ideas in people, independent of their rank or academic pedigree.

Regular group meetings with scientific literature reviews, a steady stream of national and international visitors whom the young faculty and PhD students always met, implementing a full technical group to support biologists with instrumentation issues, setting up a fully participatory and shared work atmosphere, and, most importantly, making young students and faculty feel the aura of basic science and giving the confidence of wanting to do interesting and difficult work, was PMB’s seminal contribution to the next generation of students and faculty.

There were also aspects of PMB that were difficult to deal with. Views that diverged widely from his were unsustainable. Those who could not convince him of their viewpoint either had to concur or leave. He could be arrogant, on many issues he was mistaken or inconsistent, and he could often seem autocratic and dogmatic. He had his blind spots. But for all his strong opinions, he had a commitment to quality, and in the end this is essential to build anything that will last.

And PMB was much more than even this. To people around him – students, young faculty, the lab-boys, gardeners, drivers, people who managed the instruments, air-conditioning, guesthouse and canteen, and others – he was intensely personal. Cutting across hierarchies, he was one of them, their own PMB. He made everybody feel that working together towards excellence in all spheres is the way to excel. CCMB was not only known for its science but also its cleanliness, beauty, reliably excellent facilities and the ability to have the scientific faculty, administration, engineering, stores and purchase, gardening and hospitality services work together smoothly, like a well-oiled machine.

It would not be an exaggeration to say that all this happened largely due to the contagious enthusiasm that PMB radiated. He was always there. This was a new approach to the directorial style, one that was uncommon in those days (and even now). He knew everybody by name and made it his job to know each person’s concerns. He made lab rules such that anybody could work at any time of day and night, but in a way that safety was never compromised. Support to all employees to be dropped back home at night after work, or if they were stuck in any emergency condition anywhere in India, and interactions allowing free discussions – these were all hallmarks of making one feel ‘at home’ in the workplace.

He was one of the best institution-builders in India as he could integrate the Indian culture of togetherness with the western culture of hard work. Several people who went on from CCMB to other institutions have tried to replicate such an ethos. So have several others who have seen it work so effectively at the CCMB. Beyond the flamboyance and everything else, PMB was a great inspiration. A friend and guide to many, his direct and indirect influence on Indian science and scientific culture will be lasting.

Eminent Scientist P. M. Bhargava Dies at 89

Bhargava was the founder-director of Hyderabad-based Centre for Cellular and Molecular Biology.

P. M. Bhargava. Credit: Facebook

Hyderabad: Renowned scientist Pushpa Mitra Bhargava, the founder director of CSIR-Centre for Cellular Molecular Biology (CCMB) who returned his Padma Bhushan award two years ago in protest against the NDA government’s stance towards liberal ethos in the country, died on August 1.

The scientist, popularly known as P. M. Bhargava, had been suffering from multiple health problems for some time now. He passed away around 6 p.m., CCMB sources said.

Bhargava, a reputed molecular biologist, is survived by a son and a daughter, they said.

Expressing deep sadness over Bhargava’s demise, a release issued by the staff of the CCMB said it was his vision and pioneering efforts that led to the establishment of the CCMB in 1977 as an institution for research in basic biology.

Bhargava was a pioneer in the field of biotechnology in the country and he was instrumental in setting up a separate department for biotechnology in the Ministry of Science and Technology in the 1970s, according to his portal linked to CCMB’s website.

Bhargava held various positions, including the vice-chairman of the national knowledge commission during 2005-07.

He is a recipient of over 100 national and international honours and awards, including the Padma Bhushan in 1986 and the Legion d’Honneur in 1998 from then president of France, it said.

Born in Rajasthan’s Ajmer, Bhargava studied at Theosophical College, Lucknow and Queen’s College, Varanasi and completed his BSc in 1944 in Physics, Chemistry and Mathematics.

He obtained his MSc in organic chemistry in 1946. He received his PhD in synthetic organic chemistry from the Lucknow University at the age of 21, it said.

Bhargava went to the US in 1953 and worked as a project associate at a laboratory for cancer research. He played an active part in the discovery of 5-fluorouracil, an anticancer drug, it said. He worked at different research institutions in the UK and France.

He was instrumental in pioneering research in the field of Biology and published numerous papers.

In 2005, he initiated and played a major role in drafting the Indian Council for Medical Research’s national guidelines for accreditation, supervision and regulation of Assisted Reproductive Technology clinics in India, according to the portal.

Bhargava had been a critic of the government policies. He served as a member in the National Security Advisory Board and was a nominee of the Supreme Court of India on the Genetic Engineering Appraisal Committee of the Union government.

He had opposed the approval of GM in India and called for a moratorium of at least 15 years on genetically modified crops in the country, it said.

Bhargava had returned his Padma Bhushan award in 2015 in protest against the alleged intolerance in the country thus facing flak from various quarters.

“I have decided to return the award. The reason is that the present government is moving away from the path of democracy, moving towards the path of making the country Hindu religious autocracy just like Pakistan. This is not acceptable… something I find unacceptable,” he had said then.

Last month, two other eminent scientists – Yash Pal and U. R. Rao – passed away.

How a ‘Hmm That’s Strange’ Moment Led to a Therapeutic Discovery

Jyotsna Dhawan, a scientist talks about her research on quiet proteins which could have implications for therapeutic treatment of age and disease related muscle degeneration.

Jyotsna Dhawan in her lab. Credit: Jyotsna Dhawan

“I have a friend who says that in science, what usually precedes a discovery is not ‘eureka’, but a ‘hmm that’s strange.’,” said Jyotsna Dhawan, a scientist at Centre for Cellular and Molecular Biology, a CSIR research establishment situated in Hyderabad. Some years back Dhawan and her team had a ‘hmm that’s strange’ moment while studying the role of a protein in mice. In their quest to demystify the ‘hmm’, they ended up making an important discovery that has brought science one step closer to therapy for age and disease related muscle degeneration.

“In a way, skeletal muscle is a wonderful tissue – so contractile and stretchy – but it’s also a very human tissue. It forms about 40% of our body mass and allows us to move, breathe, and even express emotions through the 72 muscles in our face,” started Dhawan, softly introducing me to her complex research. “But because it is used so often, it often needs replacement.” Luckily for us, we have a batch of reserve cells that our body kept aside exactly for this purpose. These are called muscle stem cells.

Muscle tissue sections. Credit: Jyotsna Dhawan

A crash-course in stem cells

The very first cell formed from the fusion of an egg cell and a sperm cell is called the zygote. The zygote grows and multiplies rapidly to form a hollow ball of cells called a blastocyst. From this early stage of the embryo, cells start becoming specialised or differentiated into different kinds of tissues. “What you end up with at birth is a fully functioning organism that has all these different tissues and around 200 cell types,” explained Dhawan. “Usually there’s no stopping once you set the ball of development rolling. It’s a linear process, one-way path… no returns.”

But what is fascinating is that not all cells differentiate. Take muscle development for example. “At birth, the whole muscle tissue includes thousands of muscle fibres but in between, there are also satellite cells or muscle stem cells.” Stem cells are kept aside in an undifferentiated state at some point late in embryonic development. “And it’s not a musical chairs scenario where these are the cells that failed to develop – there’s a specific programme that sets them aside and tells them they are not going to differentiate,” stressed Dhawan. These cells stay quiet until there is a need for replacement muscle cells due to wear-and-tear, injury, disease or ageing. During such times, these stem cells develop into muscle cells and cater to the demand.

Here’s a nice intro about cell differentiation and stem cells:

Quiescence is golden

Cell and molecular biologists like Dhawan are interested in the program that makes these selected cells go quiet. A ‘program’ here refers to the set of rules that guide this process. These rules are usually in the form of specific genes and proteins. Dhawan’s lab is trying to understand all the genes, proteins and pathways involved in activating cell ‘quiescence’, the technical term for the silencing of these cells.

There are philosophical implications of this too, according to Dhawan. “Normally, when you think of a program that ‘activates’ something in a cell, you think of the cell being woken up to divide or induced to differentiate – very ‘active processes’,” she mused. “The idea of something ‘actively’ becoming quiet is not something that many people have looked at, and that’s what interested us.” For Dhawan and her peers in stem cell biology, this means going away from the idea of activity and inactivity to simply asking what are the processes that dominate in the quiet state and the ones that dominate the fully proliferative state.

Buzzing with inactivity

A Hindustani Musician. Credit: Wikimedia Commons

Dhawan uses the metaphor of the khali (empty) in Hindustani music to put the role of the quiescent cell into perspective. “The taal or the beat allows you to tell where you are in the rhythmic cycle. But you always have a khali (an empty or a void sound denoted by the wave of the hand), which is like the indexing to know where in the beat cycle you are. When you think of a cell, the taal is the cell cycle or cell division with very distinct phases. The quiescent/dormant cell is the khali – here, the cell division is stalled at a specific point in the cycle called G0. When it’s time to restart, the program doesn’t restart from the beginning but from that specific point. If the cell went quiet in random phases in the cell cycle, then you can envisage that the program to re-activate the cell would be very chaotic.” The importance of the G0 (G zero) phase is clear because it turns out that all stem cells (not just muscle stem cells, but blood stem cells, liver stem cells, umbilical cord stem cells, etc) are in this phase.

What really happens in this G0 phase? “Even if you are a quiet cell, not doing a whole lot, how are you surviving? You have to be doing something actively, even though at a very low level. Are there processes which are boosted in the quiet cell which are not so active in the so-called active cell?” To answer these questions, Dhawan began looking for genes – specifically those that were more active in quiescent cells than other cells. The hypothesis was that these were likely the genes controlling quiescence.

Skeletal muscle fibres. Credit: Jyotsna Dhawan

A quietly working protein

They identified a whole bunch of such genes in mice (which is the preferred model organism for these kinds of studies) and from this bunch cherry-picked a single gene to study in more detail. “This gene, called PRDM2, produces a protein that belongs to a family known for playing different kinds of switching roles in cells – for example, switching between cell division and differentiation, switching between muscle cell to fat cell, etc. The PRDM2 protein seemed like the perfect candidate for a molecule that is going to say you are going to stay quiescent – not divide nor differentiate.”

Though their experiments caught PRDM2 in all the right places at all the right times, Jyotsna and her team wanted to be totally sure. They looked at whether PRDM2 binds to genes important in keeping a cell quiet. “The answer was an overwhelming yes. We found that the protein sat not only in cell cycle genes (important for cell division), but also on differentiation genes (important for differentiation of cells into skeletal muscle cells).”

prdm2 protein. Credit: Jyotsna Dhawan

With all these positive signs that PRDM2 is playing some crucial role in cell quiescence, the next question in front of Jyotsna was – what is this role? To probe further, Dhawan used every molecular biologists go-to toolkit – knock-out technology. She grew cells in the lab that were purposely engineered to have no PRDM2 protein and observed what happened to those cells. Such knock-out cells are used to predict what the role of a gene or protein is playing in an organism. As suspected, in the absence of PRDM2, nearly 1500 cell differentiation genes turned on in these cells. This strongly indicated that PRDM2 was inhibiting differentiation in quiescent cells. This fits, because quiescent cells do not differentiate.

‘Hmm, that’s strange’

What about cell division? This is where the puzzle began. Cell division and cell differentiation are typically antagonistic to each other – when a cell begins to differentiate, cell division shuts down. And the knock-down cells confirmed this. PRDM2-less cells that had all its differentiation genes turned on, had their cell division repressed. This suggested that PRDM2 is required for cell division. But quiescent cells can’t divide! So what is happening here? How can a protein be quieting a cell and promoting cell division at the same time? “Hmm, that’s strange,” thought Dhawan…

Further adding to this paradox was another existing feature of the PRDM2 protein. Previous studies have shown PRDM2 to be a tumour suppressor protein. Tumours involve rapidly dividing cells, hence tumour suppressors like PRDM2 should inhibit cell division. “If PRDM2 is a tumour-suppressor, then if you remove it, you should see an activation of the cell cycle, right?” she thought. But this was the opposite of what was happening in PRDM2-less cells, where cell division was repressed.

Figuring it out

But after much puzzling, Dhawan and her two graduate students realised they were looking at it all wrong. What was actually happening was this: it is true that quiescent cells do not divide. It is also true that PRDM2 sits on cell cycle genes in quiescent cells. But they had wrongly assumed that this meant that it is PRDM2 that represses the cell cycle. It turned out that repression happens by other means. PRDM2’s actual role is to keep the cell cycle alive, but on idle. Remember, a quiescent stem cell has to keep a memory of the fact that it is going to differentiate into a muscle cell at some point in the future and then begin to divide. Until then, the cell cycle has to be kept sort of idling. “The engine can’t be completely off, just on neutral. There must be something telling the cell you have to be on neutral, don’t shut off the engine (cell cycle) completely. That something is PRDM2,” explained Dhawan.

Studies like this have great potential. “Eventually, we will be able to perturb these programs and overcome some problems associated to loss of muscle stem cells during muscle dystrophies and other muscle diseases which are quite common,” informed Dhawan. That’s the long-term goal of muscle stem cell biologists, she added. Dhawan credits her two “phenomenal graduate students” Sirisha Cheedipudi and Deepika Puri for the discovery of this mechanism, which went on to be described in a paper they published in Nucleic Acids Research journal in 2015.

For Dhawan, this was the culmination of a 15-year journey that began when she submitted a proposal for funding this project. The proposal had outlined her hypothesis that such a class of proteins existed. “(Being proven right) was very pleasing from that perspective,” she said.

Science, science, everywhere

Dhawan’s foray into the sciences was not completely out-of-the-blue. She grew up in the intellectual haven of the Indian Institute of Sciences in Bengaluru; her father is the famous aerospace engineer Satish Dhawan after whom India’s rocket launch centre in Sriharikota is named; her mother Nalini Dhawan was a botanist, and her brother is an astronomer. But growing up in school Dhawan was more drawn to history and the arts. In fact, she initially had her sights set on graphic design, but she did not get into the design school she preferred. So she pursued a basic sciences degree instead.

“School was great but quite conventional so anything unconventional had to come from home. I was very lucky in that regard,” said Dhawan. Plants were a big part of her life. “My mother’s family was completely plant-mad. They dealt anyone who didn’t obsess over plants with great pity and were more likely to know the latin names of the plants around them than their own relatives,” she laughed. Dhawan believes that from such an upbringing she and her siblings began to understand that a life of ideas is a life worth living. “But at some point, it occurred to us that we need to earn a living!”

A crisis, a mistake, and finding her way

Though she went on to a Master’s in Delhi University, Dhawan was not very clear about what she wanted to do with the degree. She recalled having a crisis during one of her final exams and almost deciding to drop out. Her father, who had come by for a meeting, suggested that she write the exams anyway. “He said, ‘look, you do what you want but I think you might feel bad about quitting if you don’t do your exams. Do your exams. It doesn’t matter what the outcome is if you’ve decided to quit’.”

The decision to stick on paid off because in the subsequent year, during a summer internship with TIFR, Dhawan truly fell in love with biology. “It was 1983, and there was a huge explosion of information about molecular biology. My background until then was more oriented to ecology and botany, but in TIFR I studied yeast genetics. It was difficult for me but I was absolutely hooked – fascinated by the puzzle solving that you had to do. I decided I’m definitely doing a PhD.”

She started her PhD coursework at home ground in IISc. During a conference in her first year, Dhawan was offered a scholarship by an Indian geneticist working in Canada to join his lab. “I decided this was a huge opportunity to do science and see the world – what a great idea!” But just three days after she arrived, Dhawan realised with dread that she’d made a mistake. “It was a really dull and run-of-the-mill place compared to IISc.” She panicked a little bit but then decided she had to be accountable for her choices. “I decided that now that I’m here, I’m going to take as many courses as I can this year and then transfer.”

Luckily for her, her brother was doing his PhD in the educational hub of Boston, US. During the holidays she visited him and began scouting for universities. She got a position at Boston University and transferred there. “I had to start afresh but, as I often tell my students today, there’s no such thing as wasted time if you’ve actually reflected on what you’ve been doing. This notion of ‘oh my god, I’m 24, life has passed me by because I haven’t done ‘x’ is so mistaken.”

In Boston, Dhawan thrived. “Everybody in Boston is a student. There are nine universities, classes happening everywhere and they are very flexible. What I encountered there for the first time was real cell biology. The fact that you could culture cells, look at them under a microscope and watch them divide was just a huge revelation for me.” In her five-and-a-half years there, she began to get interested in muscle.

Dhawan went on to spend another five years in Stanford University for post-doctoral work. While on a vacation trip home during Stanford, she connected with her future husband Imran Siddiqi while on a vacation home and married him on her return. Imran is also a scientist at CCMB. “He’s a plant biologist. Naturally, my mother loved him!”

On marriage and hiring couples

Did marriage change a lot? “I had my fears, maybe that’s why I married very late, at 35-36. I was quite clear that for me, I had to be convinced that this was going to be the right person. I wasn’t interested in a relationship for its own sake. But I have to say no (not much changed) because, in many ways, we’re just pretty useless when it comes to anything family-oriented,” she laughed. “In the sense, we don’t have kids – though this was not a choice, it just so happened. Of course, home is an important domestic space for us, our lives are not focused at home.”

Dhawan feels lucky to have got a place in the same institute has her husband. “There are all kinds of structures against couples being hired. It’s an unwritten rule and I have argued against it because I think it’s not correct. Many places across the world actually hire couples specifically. It’s nobody’s argument that this should be done against merit. Of course, merit should be the primary factor but if you are trying to build places where people are happy and going to be contributing, you have to look to their personal lives as well. I was lucky to not face the issue.”

Dhawan’s current lab members. Credit: Jyotsna Dhawan

Being true with female students

Among Dhawan’s postdoc students, the gender ratio is pretty even. Though her experience with women students has been really good, she has seen them go through challenging times, most rooting from family pressures. Dhawan has seen a pattern. “Though most of their families are pleased about them going for higher education, during the third of fourth years, there comes this feeling that you are 28, when are you gonna settle down, have children… the women start feeling enormous pressure.” Dhawan has no doubt that this kind of pressure does affect them. “It affects their ability to be able to see themselves as free agents, to see that self-determination is actually an important goal. Sometimes you have to be able to say to your family – not now.”

How does she deal with these situations? With the truth. “I would be failing in my duty as a mentor if I didn’t say it’s important to see yourself as a free agent. I’m not saying you should abandon your responsibilities. if you have somebody to look after, a parent or a sibling or a cousin, you need to do that. But sometimes you need to be responsible to yourself as well. You cannot put that on the backburner. Sometimes you have to say ‘for six months, you have to leave me alone’.”

Dhawan says that the strength of some of the women in her lab has inspired even her. “One of my students has adopted a child as a single mother. There are also others who are faculty members and still single, some married.”

“One thing I learnt from when I was a postdoc was that the married postdocs with me were very very efficient. They had to work under much more constrained conditions than others who could spend the whole day in the lab not necessarily doing productive work. When you have a kid to pick up at 5.30 p.m. you’re not going to be hanging out drinking chai.” Jyotsna suspects her strong feelings on this matter may be coming from the fact that her mother did not get the opportunity to advance her career after marriage. “She had every enabling factor but still found it difficult. There were just too many responsibilities.”

Stem cells – a note of caution against stem cell therapy and banking

“One of my biggest challenges in these exciting times for stem cells is sounding a note of caution. We have way too much hype and when you try to clamp down on the hype people think you are against it. Hahaha, how can I be against it when I spend my life studying it! We need caution because we have way too many clinics operating under the radar providing what they call therapy. These are essentially unproven treatments with unproven sources of cells. They are fleecing the public. There is no gene therapy that is even approved in India. There’s no regulatory frame. Some of my colleagues are pushing for a regulatory framework because these are clinicians who see the value of gene therapy especially for conditions like haemophilia. We do need to have the biotech companies alongside us. There has been much progress, but the public needs to know that there are many people and clinics making hay while the sun shines. They are providing services like stem cell banking which is highly dubious. With private stem cell banks (which offer to store your baby’s umbilical cord stem cells as a ‘biological insurance’), what you’re paying for is liquid nitrogen. Nobody is going to use those cells. The companies cannot even guarantee that the cells will be functional at the end. They have no assays, they don’t even know how to test them. I think this is something we have to be proactive about. I’m not a clinician so i don’t like to advise anyone but I have made a set of guidelines for people seeking cord blood banking or stem cell banking to go and ask their doctors. But I think we need to do more.

This piece was originally published by The Life of Science. The Wire is happy to support this project by Aashima Dogra and Nandita Jayaraj, who are travelling across India to meet unsung women scientists.