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The new protein was able to correct sickle cell anaemia mutation in patient-derived stem cells

Improving precision: The newly discovered protein can replace the currently used one which has a propensity to cause unnecessary alterations in the DNA.Getty Imagesvchal

Researchers at the Delhi-based Institute of Genomics and Integrative Biology (CSIR-IGIB) have discovered a protein variant from a different species of bacteria that can edit the DNA with very high precision. In the tool now commonly used for editing disease-causing mutations in DNA (CRISPR-Cas9), the Cas9 protein behaves like a molecular scissors that cuts the DNA at a specific location and inserts a foreign piece of DNA to correct the mutation that causes the disease.

In addition to binding to the intended target on the DNA, the commonly used Cas9 protein from Strepotococcus pyogenes bacteria (SpCas9) and its engineered derivative tend to potentially bind to DNA at multiple unintended sites thereby leading to unnecessary alterations in the DNA.

The researchers found their new Cas9 protein, which binds and cuts the DNA, was able to correct sickle cell anaemia mutation in patient-derived stem cells. The protein (FnCas9) used by the researchers to edit the DNA is derived from a bacterium — Francisella novicida.

The Cas9 protein is supposed to bind to the DNA only when there is a perfect match between the DNA and the protein, thus reducing the chances of the protein binding at non-target sites on the DNA. But even when three mismatches exist between the protein and the DNA, the currently used SpCas9 protein binds and cleaves the DNA. In contrast, the team led by Debojyoti Chakraborty from IGIB found the new FnCas9 protein showed negligible binding when there exists more than one mismatch in the target DNA. The results were published in the journal Proceedings of the National Academy of Sciences (PNAS).

High specificity

“The high specificity of the new FnCas9 protein arises due to reduced affinity to bind to DNA when there is even a single mismatch. And when there is more than one mismatch, complete absence of binding of the protein to the DNA is seen in many cases,” says Dr. Chakraborty.

“If the Cas9 protein remains bound to DNA at mismatched locations for a long time, there is a possibility that it might cut the DNA at these locations. Also, if it remains bound to DNA, the protein might block the transcription (which is the first step in gene expression) at that location. And if Cas9 is bound at multiple unintended sites then the transcription machinery gets stalled and the expression of genes at these locations might be altered,” Dr. Chakraborty explains.

In nature, DNA often gets damaged and is routinely repaired through one of the two pathways. In the case of the homology-directed repair (HDR) pathway, which is relatively less error-prone, matching sequences are used to repair the DNA. “The FnCas9 protein was found to increase the HDR repair rate fourfold compared to the widely used SpCas9,” says Deepanjan Paul from CSIR-IGIB and one of the first authors of the paper.

Sickle cell anaemia

The researchers tested the precision of binding and cleavage at the desired sites on the DNA using mouse cell lines (embryonic stem cells and brain cells), human kidney cell lines and induced pluripotent stem cells (iPSc). In the case of human iPS cells, the FnCas9 protein was found to bind to the DNA at the specific site, cut and repair the sickle cell anaemia mutation.

“The correction process is the same for any disease-causing mutation and so our FnCas9 protein should theoretically correct any mutation in the DNA. The efficiency might vary, so we must test it for each disorder,” says Dr. Chakraborty.

The efficiency of any Cas9 protein delivery as well the ability to correct mutations is generally low in the case of iPS cells. The efficiency of correction is about 1.6%. Though the efficiency to correct mutations is low in iPS cells, the corrected cells can be isolated, multiplied and converted (differentiated) into haematopoietic stem cells. Once differentiated into haematopoietic stem cells, they can be transfused into patients.

“Differentiating iPS cells into haematopoietic stem cells is not trivial. Plenty of experimental work is under way to make it efficient for clinical translation,” says Dr. Chakraborty.

Recalling how he started working on FnCas9 protein for genome editing, Dr. Chakraborty recalls that he was looking for a Cas9 protein which can target RNA instead of DNA. There was one study that reported that FnCas9 could potentially target viral RNA. “We were not able to target RNA using FnCas9 proteins. So we started to investigate whether it can target DNA as well since it was not known if FnCas9 can be used for precise gene correction. We found that not only does it target the DNA but does so with very high specificity,” he says.

“We are now proceeding for preclinical studies to establish the efficacy of FnCas9 protein for genome-wide binding and targeting using patient-derived cells and mouse models,” he says.

This requires complex cognitive capacity

Watch my mouth: Bonnet macaques have a goal in mind and use gestural communication to fulfil that goal.Shreejata Gupta Shreejata Gupta

Gestures are an important aspect of human communication. Until now, gestures in apes were believed to lie at the evolutionary roots of human language. However, a recent study on wild bonnet macaques implies that gestural communication — the basic tools for language — is observed even in that species. Given that monkeys diverged from apes (including humans) much earlier than humans diverged from other apes (such as chimpanzees and gorillas) this finding has an evolutionary significance as well. The study has been published in the journal Behavioural Processes.

Species ethogram

Anindya Sinha from National Institute of Advanced Studies, Bengaluru, who has been studying the behaviour of bonnet macaques in the Bandipur-Mudumalai region for close to 25 years, had developed an ethogram of the species. In essence, a list of objectively-defined behaviours — an ethogram — becomes the reference document for subsequent studies.

Developing on this, the researchers recorded 32 independent gestures made by the bonnet macaques. Of these, they could not identify the context for eight gestures. For the remaining 24 gestures, they were able to identify the context of use. “It would have been perfect if we could get all the behaviours videographed,” says Shreejata Gupta who worked on this problem, in an email to The Hindu. Now a postdoc at York University’s Department of Psychology, she explains how this made it difficult to address some features of intentionality, such as changing body orientation according to the receiver’s orientation.

Differential grooming

“When I first saw the bonnet macaques using their grooming gestures possibly referring to body parts that they intended to be groomed, I was absolutely elated…” she recalls. After accumulating more of these observations, the team published a paper on referential grooming in bonnet macaques, a cognitive capacity that was earlier believed to be restricted to human and non-human apes. This was published in the journal Animal Cognition.

“Bonnet macaques have voluntary control over their gestural communicative signals. They have a goal in mind, and they communicate with others to fulfil that goal. The entire process requires complex cognitive capacity, different from an involuntary action such as an alarm call at the sight of a predator,” says Dr Gupta. It has been known earlier that apes such as humans, chimpanzees, bonobos, gorillas and orangutans communicate intentionally. This study extends this behavior to monkeys.

“This continuity of similar cognitive processes underlying communication systems of monkeys and apes imply that our minds have had the building blocks of producing complex language for millions of years,” says Dr Gupta.

Two fossils dating back 25 million years were found in Makum coalfield in Assam

Spread from India: This finding strengthens the theory that bamboo spread to Asia from India and not from Europe.

With over 49,000 plant species reported as of 2018, India holds about 11.5% of all flora in the world. Now, a new fossil record has shown that India is the birthplace of Asian bamboo, and they were formed about 25 million years ago in the north-eastern part of the country.

Ancient fossils

An international team of researchers found two fossil compressions or impressions of bamboo culms (stems) and after further study noted them to be new species. They were named Bambusiculmus tirapensis and B. makumensis - as they were found in the Tirap mine of Makum Coalfield in Assam. These belonged to the late Oligocene period of about 25 million years ago.

They also found two impressions of bamboo leaves belonging to new species Bambusium deomarense, and B. arunachalense, named after the Doimara region of Arunachal Pradesh where it was discovered. These leaves were found in the late Miocene to Pliocene sediments, indicating that they were between 11 and three million years old.

Yunnan Province in China now has the highest diversity of bamboo, but the oldest fossil in that region is less than 20 million years old, clearly indicating that Asian bamboo was born in India and then migrated there. This finding further strengthens the theory that bamboo came to Asia from India and not from Europe.

Wide niche

“Today, we can see bamboo in a wide range of climatic conditions from as cold as 5 degree C to even 30 degrees C. And at sea level to heights of about 4,000 metres. They can survive in varying rainfall conditions too. We wanted to know what made it develop such a wide environmental niche,” explains Gaurav Srivastava, from the Birbal Sahni Institute of Palaeosciences, Lucknow. He is the first and corresponding author of the paper published recently in the journal Review of Palaeobotany and Palynology.

Role of plate tectonics

In fact, the European bamboo fossil is about 50 million years old. Dr Srivastava explains that the Indian plate collided with the Eurasian plate about 50 million years ago. However, the suturing between the two plates were not completed until 23 million years, meaning the plates were not completely joined, restricting migration of plants and animals. And also as the Himalayas were not formed yet, the temperature was also warm and humid in the Northeastern region, with not many seasonal variations.

The present climate in the region is cold with strong winter and summer conditions. Bamboo braved these climatic and geographical changes making it the fittest in the survival race.

“Bamboo fossils are not very common in India as they are known only from the Siwalik sediments. We have planned more studies in this region and search for its fossils in older rocks too,” adds Rakesh Chandra Mehrotra, the study head from the Birbal Sahni Institute of Palaeosciences.

Dr. Mehrotra adds that paleobotany is studied only in very few pockets in India. This study has shown that India is a treasure trove of plant fossils and more importance needs to be given to its study.

The highly unstable perovskite mineral was converted into a robust form capable of decomposing toxic organic pollutants commonly present in water

Cleansing water: The catalyst becomes active when exposed to sunlight and degrades the pollutants.Getty ImagesKrimKate

Researchers at the Indian Institute of Science Education and Research (IISER) Pune have successfully converted the highly unstable perovskite into a highly stable photocatalyst capable of decomposing toxic organic pollutants commonly present in water. The catalyst that becomes active when exposed to sunlight was synthesised by encapsulating nanocrystals of organic-inorganic perovskite inside a metal-organic framework (MOF).

The team led by Sujit K. Ghosh from the Department of Chemistry at IISER Pune utilised the hydrophobic nature of the MOF material to render greater chemical stability to perovskite nanocrystals that form inside the MOF cavities. The perovskite-MOF composites displayed “outstanding” stability when immersed inside water and alcoholic solvents for as long as 90 days.

Stable composite

The composites remained stable in water even when at boiling temperature for 20 days. While perovskite encapsulated by MOF showed 70% similar photoluminescence intensity before and after heat treatment at the end of 20 days, the photoluminescence intensity of naked perovskite decreased by 95% in just five hours of heat treatment. Likewise, the photoluminescence intensity of the composite remained almost intact even after being exposed to UV light for 20 days.

It is the hydrophobic nature of MOF that renders chemical, heat and photostability to perovskite.

The researchers found less than 1 ppb of lead metal leached from the composite at the end of 90 days of being exposed to different solvents, including water. “It is possible to replace lead metal with other nontoxic elements to make the composite more efficient and much safer to degrade organic pollutants,” says Dr. Ghosh. The results were published in the journal ACS Applied Nano Materials.

“This is the first time perovskite-based composite material as a photocatalyst has been used for the degradation of toxic organic pollutants such as antibiotics, dyes etc. It will be a cost-effective method to produce clean water,” says Dr. Ghosh.

The researchers tested the composite’s photocatalytic property to degrade organic pollutants in water. They tested three organic commonly seen pollutants — methyl orange, methyl red and nitorfurazone antibiotic.

Activated by sunlight

“When the composite was exposed to sunlight it was able to degrade the organic pollutants,” says Samraj Mollick from IISER Pune and one of the first authors of the paper. “When exposed to sunlight, the perovskite nanocrystals release electrons into water thus producing hydroxyl radicals. The hydroxyl radicals are highly active species that decomposes the organic pollutants.”

But the rate of degradation of organic pollutants is not high compared with other standard materials. “Compared with other materials, perovskite is inexpensive. It is also possible to scale up its production easily,” says Tarak Nath Mandal, the other first author of the paper.

“This is only a proof-of-concept study. It is possible to increase the degradation rate by using different perovskite and MOF materials,” says Dr. Ghosh.

The researchers were able to recycle the composite thrice and even on the third cycle the composite displayed over 90% degradation capacity.

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