IGIB team enhances the efficiency of DNA delivery into the skin for treating skin disorders

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Dr. Munia Ganguli (left) and Dr. Manik Vij have improved DNA penetration into skin by pretreating the skin with silicone oil. – Photo: Lavanya Lokhande.

By pretreating the skin with silicone oil, a team of researchers led by Dr. Munia Ganguli from the Delhi-based Institute of Genomics and Integrative Biology (CSIR-IGIB) has been successful in delivering plasmid DNA into the skin with greater efficiency and without destroying the integrity of the skin. Unlike other enhancers currently being used, preliminary studies show that silicone oil did not get into the skin nor cause any harm. Enhancing the ability of the plasmid DNA, packaged as a nanometer-sized complex with a peptide, to penetrate the skin will go a long way in efficiently delivering drugs for skin disorders. The results were published in the journal Molecular Therapy.

“Topical application of silicone oil on the skin prior to applying the DNA-peptide (which acts as a carrier of DNA) complex allows the DNA to reach the lower part of the epidermal layer of the skin; a little bit of DNA gets into the dermis as well,” says Dr. Ganguli, the corresponding author of the paper.

The skin with its three layers — stratum corneum (top layer), the epidermis (middle layer) and dermis (inner layer) — acts as a tough barrier for the entry of any foreign substance. Since the top layer of the skin is rich in lipids it becomes particularly difficult for the DNA (which is water-loving or hydrophilic) to penetrate it.

Only 30% of cells have the DNA complex when the skin is not pretreated with silicone oil. It increases to 45% once the skin is pretreated. “Silicone oil forms an occlusive layer which prevents water loss from the skin and keeps it well hydrated. The rise in hydration pressure, in turn, opens up many porous pathways for entry of the DNA complexes into the skin,” says Dr. Manika Vij from CSIR-IGIB and the first author of the paper. Besides increased hydration, there are also minor changes in the lipid and protein organisation in the skin.

The use of another enhancer (sodium laureth sulfate-phenyl piperazine — SLA-PP) in place of silicone oil also improves DNA penetration but it was found to damage the skin and was highly toxic to the skin cells; when applied on cell lines, plenty of cells died after 24 hours.

The researchers used hairless mice (the absence of hair follicles makes the skin more closely comparable to human skin) to test the penetration of DNA into the skin. Since the DNA is labelled with fluorescein, it was possible to measure the amount of nanocomplexes that got into the skin by measuring the fluorescence. Other tests revealed that topical application of silicone oil does not damage the integrity of the skin or damage the tissues.

Other potential applications

Talking about potential use of the DNA nanocomplexes along with silicone oil, Dr. Vij says: “In the DNA we can put any gene that encodes for any specific therapeutic protein. This way we can address several skin diseases.”

The researchers are planning to test the ability of the peptide-DNA complexes to cross the skin and enter the blood. “If it does, then it increases the potential to address diseases of other organs,” Dr. Vij says. “We are yet to carry out studies to see if the DNA gets into the blood circulation or gets locally degraded in the skin cells.”

Published in The Hindu on April 14, 2017

Indian researchers find a new bacterial target for drug development

Anshika Andaleeb Richa-Optimized

(From left) The study by Anshika Singhal, Andaleeb Sajid and Richa Misra helped understand how bacteria form biofilm.

Indian researchers have found a new target that can potentially be used for developing new antibiotics that will be effective against many bacteria. The new target is made of two proteins — which form a complex that is responsible for the formation of biofilm — that perform very important functions and are critical for bacterial ability to successfully infect humans. The results were published in the journal Biofilms and Microbiomes.

Bacteria form biofilms, a kind of matrix, during infection in plants and animals. Biofilm shields the bacteria from antibiotics and help bacteria to survive harsh conditions such as extreme temperature or stress. Now a study by Indian researchers has found the molecular signaling events that play a crucial role in biofilm formation in Bacillus anthracis, the causative agent of anthrax.

Till now, all attention has been on developing antibiotics that target disease-causing bacteria and not the biofilm itself.
One of the basic questions that scientists have been trying to answer is how and when bacteria decide to form biofilm. “One possibility is that bacteria has sensors on the surface which senses some signal and helps in biofilm formation,” says Andaleeb Sajid from the Institute of Genomics and Integrative Biology (IGIB), Delhi and one of the authors of the paper.

“It was serendipity. Our lab was working on signaling in bacteria and we were studying PrkC and similar proteins. When PrkC protein is deleted, Bacillus bacteria are unable to form biofilm. So we started studying the mechanism by which PrkC protein controls biofilm formation,” she says.

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Gunjan Arora says the GroEL-PrkC complex could be a target for developing new drugs.

“Our hypothesis is that PrkC senses some signal and transmits it from outside to inside the cell. This signal goes to other proteins like GroEL. PrkC adds phosphate group (phosphorylate) to different proteins. The mystery to biofilm formation lies with one chaperone protein called GroEL. The addition of phosphate to this tiny machine initiates a course of events within bacterial cells leading to complex biofilm formation,” Dr. Sajid says.

The team found several proteins receive signals from PrkC protein. Using cutting edge genetics, molecular biology and proteomics techniques, they confirmed that GroEL was regulated by PrkC.

“From other unrelated bacteria we already had a clue that GroEL has a role in biofilm formation. We looked at the molecular level and found six amino acid residues where phosphate was getting added to the GroEL protein. Through a series of steps, we ascertained how important phosphorylation was for proper functioning of GroEL,” says Gunjan Arora from IGIB and the first author of the paper.

“We wanted to know if the bacteria has any other compensation mechanism to form biofilm in the absence of PrkC. So we made PrkC mutant bacteria to produce more of GroEL. The bacteria were able to form biofilm even in the absence of PrkC. This experiment helped us understand that PrkC is the influencer and GroEL is key to biofilm formation,” Dr. Arora says.

Both PrkC and GroEL perform very important functions and are critical for bacterial ability to successfully infect humans. “We think GroEL-PrkC complex could be a target for developing new antibiotic that will be effective against many bacterial pathogens such as the ones that cause MRSA, TB and pneumonia. One strategy to tackle drug resistant bacteria will be to develop multi-drug regimen that combines traditional antibiotics with candidate drugs that can block bacterial signaling and prevent biofilm formation,” Dr. Arora says.

Published in The Hindu on March 26, 2017

Researchers from Indian national institutes publish in predatory journals

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The paper published in Current Science exposes the practice of researchers from CSIR, ICAR, ICMR, IITs and NITs publishing in predatory journals.

India not only publishes the most number of predatory journals in the world but researchers based in India are one of the biggest contributors to such bogus journals; an earlier study found that researchers in India accounted for 35 per cent of publication in bogus journals.

Predatory journals very often trick authors into submitting papers, rarely peer-review manuscripts thus allowing sub-standard papers and even those that contain plagiarised content and falsified and fabricated data to be published, rarely index papers with standard indexing bodies and are more focussed on article processing fees.

Based on 3,300 papers published between September 2015 and mid-February 2016 and randomly chosen from 350 predatory journals, researchers found that 51 per cent of papers in predatory journals were published by researchers from colleges affiliated to universities and autonomous colleges. It was followed by private universities/institutes (18 per cent), State universities (15 per cent) and national institutes (11 per cent).  The results were published in the journal Current Science.

What is more surprising is that researchers from ICAR, CSIR, and ICMR labs, and national institutes such as IITs and NITs too published papers in such junk journals.  Of the 11 per cent publication from national institutes, ICAR labs had the most number of publications (17 per cent). It was closely followed by CSIR at 15 per cent, NITs at 11 per cent, IITs at 9 per cent and ICMR at 6 per cent.

“Among the State universities, Annamalai University in Tamil Nadu has the most number of papers (70) published in predatory journals, while at seven papers Banaras Hindu University (BHU) Varanasi has the highest number among the central universities,” says G.S. Seethapathy from the University of Oslo, Norway and the corresponding author of the paper. “The National Chemical Laboratory (NCL), Pune has 15 papers while the Delhi-based Institute of Genomics and Integrative Biology (IGIB) has 12 papers. Among the ICAR institutes, the Indian Institute of Horticultural Research, Bangalore (17) and the Indian Agricultural Research Institute, New Delhi (12) have the most papers in such journals. In the case of IITs, IIT Bhubaneswar has eight papers.”

Of the 480 researchers who responded to a questionnaire, 20 per cent claimed that they were unaware that their paper was published in a predatory journal. While 10 per cent said they knowingly published papers in these journals, the remaining 70 per cent were not willing to answer the question.

How well the funding bodies that provide research grants monitor the quality of publication comes under cross hairs as 112 research grants were documented in the papers published in these journals. Nodal bodies such as DST, DBT, UGC besides AYUSH had provided most of the 112 research grants.

The publish or perish situation has now turned grave with pressure to publish becoming enormous for researchers both at the stage of appointment and promotion. In the study, 75 per cent of the respondents admitted that they were under “pressure to publish research articles”.

Due to this pressure, quantity and not the quality has become the focal point both for both researchers and for institutions. Predatory journals, which by default publish even the most ludicrous manuscript for a huge fee, have therefore come handy. It’s a win-win situation for researchers who are unable to do quality research and those publishing predatory journals. Little wonder that the number of predatory journals published from India is ever increasing. It is true that some researchers are genuinely cheated into submitting papers to such bogus journals but that number seems to be quite small.

“The introduction of academic performance indicator (API) by the University Grants Commission (UGC), lack of clarity in identifying and evaluating journals, the focus on ‘quantity’ over ‘quality’, unhealthy competition between peers, and overall, a favourable non-scientific publishing environment have led Indian researchers to publish in mediocre journals wherein most manuscripts are published without any peer review. Perhaps it is also the fear of peer review that has nourished predatory journals, making India one of the world’s largest base for predatory open-access publishing,” notes a September 2014 Editorial in Current Science.

“Universities need to re-examine the way they perform academic evaluation. They need to stop counting the number of one’s publications as a method for academic evaluation. This counting leads people to pay for easy publishing in predatory journals, and this, in turn, leads to a proliferation of predatory journals. The same would apply to government laboratories as well,” Prof. Jeffrey Beall, Scholarly Communications Librarian at Auraria Library, University of Colorado, Denver had told me earlier. Prof. Beall coined the term “predatory journal” and has prepared a long list of such journals which any serious researcher can refer to to avoid getting trapped.

Published in The Hindu on December 15, 2016

Zebrafish provide insights into a rare human disease

CHARGE Syndrome

Zainab Asad (foreground) the first author of the paper from IGIB and Dr. Chetana Sachidanandan discovered that CHD7 protein causes CHARGE syndrome by modifying a second gene — sox10.

Scientists from the Delhi-based CSIR-Institute of Genomics and Integrative Biology are a step closer to bringing hope to children born with a rare disorder — CHARGE syndrome — if the results seen in zebrafish are reproducible in humans. The results of a study were published on July 13 in the journal Human Molecular Genetics.

About 1 in 20,000 people in the world, and an estimated 50,000 in India alone, are born with CHARGE syndrome — multiple life-threatening problems such as deafness and blindness, heart defects, genital problems and growth retardation and facial bone and nerve defects that cause breathing and swallowing difficulties. There is a high death rate in the very first year in children born with CHARGE.

A mutation in the CHD7 gene is responsible for 60-70 per cent of all CHARGE defects. The expression of the gene peaks in the early stages of embryo development, starting from 2-4 cells.

The team led by Dr. Chetana Sachidanandan from IGIB studied the fertilised eggs of zebrafish to better understand the CHARGE syndrome. Following fertilization, zebrafish embryos are transparent. This allows scientists to observe the inside of the embryo and watch in real time how various organs develop. Since most organs begin forming in the first 24-36 hours and are fully formed within five days, it allows researchers to study the development of an organism from egg to maturity.

An RNA injected into a one-cell embryo interferes with the making of the CHD7 protein, thus producing zebrafish embryos with very similar problems as human babies with CHARGE syndrome.

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The researchers were able to find defects taking place at an early stage of embryo development as the zebrafish embryos are transparent. – Photo: Dr. Chetana Sachidanandan

“Looking inside the transparent embryos we found defects taking place at an early stage of embryo development. We discovered that zebrafish embryos had defects in the nerves of the digestive tract and myelination of peripheral nerves. These defects have not been previously noticed by doctors in CHARGE patients, most likely because such deep lying defects are not apparent on gross examination of patients. These findings mean that the doctors can now look specifically for these defects and thus provide better support to the patients,” Dr. Sachidanandan says.

But more importantly, the researchers found that the CHD7 protein causes CHARGE syndrome by modifying a second gene — sox10. “We found more sox10 protein. So if the CHD7 mutation was producing more sox10 protein, we wanted to know if we can reduce the defects by reducing the amount of sox10 protein,” she said.

And that is precisely what they achieved by reducing the amount of sox10 protein using RNA interference. “There was a dramatic reduction in the intensity of the defects,” she recalls. The defects in the facial bones and myelination (the process of surrounding the axon of some nerve cells with a fatty white substance that improves signal transmission/conduction) were reduced. “Since facial bone defects contribute to breathing and swallowing difficulties, we think reducing the facial bone defects could help a baby breathe and swallow better” she says. Similarly, an improvement in myelination will, in turn, lead to more efficient neuron functioning.

Though CHARGE syndrome is extremely complex with multiple defects, reducing the sox10 protein in CHARGE patients may go a long way in reducing their suffering and improve their chances of survival.

To that end, Dr. Sachidanandan and her team have already begun screening various drugs that can reduce the sox10 protein.

Published in The Hindu on July 23, 2016

Screening for rare genetic disorders becomes easier

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The Institute of Genomics and Integrative Biology has the data of all disease-causing mitochondrial mutations and the frequency of every single disease-causing mutation in the global population.

Fast and accurate diagnosis of disease-causing mitochondrial genetic mutations is now possible thanks to automation of the entire process of data analysis and interpretation by a team of researchers at the Delhi-based CSIR Institute of Genomics and Integrative Biology.

The comprehensive pipeline developed by a team led by Dr. Sridhar Sivasubbu and Dr. Vinod Scaria of IGIB includes methodologies to sequence mitochondrial genome using next generation sequencing and a software to appropriately analyse and interpret the data at a point-of-click.

Mitochondria, the powerhouses of the cell, are unique in several ways. A cell can have multiple mitochondriae, with each mitochondriae differing from one another by a few variations.  This phenomenon is known as heteroplasmy. Heteroplasmy can vary between cells of the same tissue, organ, individual or even between individuals of the same family.

Though heteroplasmic mutations implicated in mitochondrial diseases are seen even in healthy individuals, the reason why they do not manifest as disease is due to low-frequency of heteroplasmy, usually less than 10 per cent. “Nearly 20 per cent of normal individuals harbour heteroplasmic mutations reported to be implicated in mitochondrial diseases but the frequency is less than 10 per cent,” says Dr. Scaria.

The mutations could also be acquired during cell division. While all cells will carry the mutations associated with disease when it is inherited, cells in different tissues may have different mutations when it is acquired.

Also, compared with nuclear genome, the mitochondrial DNA has a 5-10 times greater rate of mutation than nuclear DNA.  “So it is not surprising that mitochondrial disorders are one of the commonest rare genetic disorders, with an incidence of approximately 1 in every 5,000 births globally,” says Dr. Scaria.

The entire process of data analysis and interpretation has been automated.

Though many hospitals now have the Next Generation Sequencing, doctors do have the expertise to do the entire process of seeing where the mutations are and interpret if the mutations so seen cause disease or not. Also, the frequency of heteroplasmic mutations needs to be known.  Many of the so-called mutations are common in the population but at a lower heteroplasmic frequency. “If the frequency is more in the population then the mutation is probably not a disease-causing one,” Dr. Scaria says.

“We have attempted to close this gap by automation of the entire process of data analysis and interpretation,” he says. First the mitochondrial genome is sequenced using next generation sequencing. To make interpretation possible, the raw data is overlaid on the mitochondria genome and the positions where the variations are present are noted and the frequency of the variation in the sample is also recorded. The disease caused by each mutation is checked with the data bank and compared with the population frequency. Since IGIB has the data of all disease-causing mutations and the frequency of every single disease-causing mutation in the global population, comparing the frequency of the sample with the population can be carried out automatically.

“The commercial application of the knowledge base would enable fast and accurate diagnosis of mitochondrial genetic mutations with implications in clinical diagnosis, prenatal testing and carrier screening,” he says.

This technology has been licensed to the Bengaluru-based Eurofins Clinical Genetics India Pvt Ltd. to enable fast and accurate diagnosis, screening at clinical turnaround times and research into mitochondrial diseases. The service is presently available in India as MitoSure.

The company has been testing samples since mid-April. “We have so far tested 25 families,” says Dr. Surendra Chikara, Executive Director of Eurofins. The cost per test ranges from Rs.15,000-20,000 and has a turnaround time of two weeks. “We will soon be starting screening campaigns for families with known history of disease-causing mitochondrial genetic mutations. This will help the family to know the female members’ carrier status,” Dr. Chikara says. In this case, the identified mutation alone is screened and cost Rs.5,000 per person.

Why Indians, SE Asian Malays respond differently to some drugs

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In general, unlike the Caucasians, the Asian population requires a lower warfarin dose to achieve stable anticoagulation, says Ambily Sivadas of the Institute of Genomics and Integrative Biology, Delhi

A couple of years after successfully mapping the genetic variants associated with differential responses to two widely used drugs — warfarin (an anti-coagulant drug) and clopidogrel (an antiplatelet drug) — in 2,000 people from Delhi, Harayana, Uttar Pradesh, Bihar and Punjab, Dr. Vinod Scaria and Ambily Sivadas studied the pharmacogenetic markers in a cosmopolitan population of Malays (southeast Asian Malays).

Dr. Scaria is from the Delhi-based Institute of Genomics and Integrative Biology and Sivadas is a Research Scholar at IGIB. The results of the study were published recently in The Pharmacogenomics Journal.

The duo used the recently released whole genome sequences of 100 South East Asian Malay individuals from Singapore Sequencing Malay Project for the study.  Using this data, they checked if the pharmacogenetic markers in the Malay population were similar or different from those seen in the rest of the world and looked for percentage of people who had these markers. Differences in the markers and how frequently they were seen in a population will result in differences in drug response in the population.

Genetic variation in absorption and metabolism of the drug can affect the concentration of the drug and in turn the effect of the drug. Also, genetic variation in the drug target can change the effect of the drug. For instance, they found potential deleterious effects in the gene VKORC1, which is the enzymatic target of the commonly used anticoagulant, warfarin. The genetic variation in the gene meant that in the SE Asian Malay population the amount of warfarin required for the desired effect is lower than the rest of the world.

“In the case of India, different populations have different frequencies of the marker connected with warfarin metabolism. Therefore, it is important to capture the sub-population data within India to optimise drug dosing,” said Sivadas. “In general, the Asian population requires a lower warfarin dose to achieve stable anticoagulation.”

Similarly, as a result of predominance of polymorphism in the gene GRIK4, the response to antidepressants was found to be very good. “We can predict higher success in treatment outcomes with antidepressant medications in SE Asian Malays,” he said. “But additional validations would be required for this to be considered definitive.”

Compared with other East Asian populations, the SE Asian Malays were found to be poor metabolisers of an antihypertensive drug debrisoquine. So the drug dosage should be lower to avoid toxicity.

IGIB

The Institute of Genomic and Integrative Biology, Delhi

“The real impact of the study is that this information could lead to a change in dosage of a certain drug for a particular population to achieve the same effect.  And in future, the dosages can be modified before undertaking any clinical trial in this population,” they noted.

This information is particularly useful as dosages of most of the drugs in the market are based on information derived from clinical trials carried out on Caucasians.  “Asian subpopulations including Indians and Malays are still not sufficiently represented in comprehensive pharmacogenomic research and drug development and so the efficacy of the drugs in these minority populations is not known,” he said. The varied response to drugs both by Indians and SE Asian Malays compared with Caucasians would mean that future trials have to necessarily include a few volunteers from these countries to know the precise dosing.

The earlier study carried out in India revealed significant differences in the percentage of people in the five States who had the markers for the drug warfarin and clopidogrel. “Given the ethno-linguistic diversity represented by India, these studies further emphasize the need to profile more Indian subpopulations in order to build a comprehensive pharmacogenetic map for the entire Indian subcontinent,” Dr. Scaria stressed. “We are very keen on creating such comprehensive pharmacogenetic maps for all known drugs in use for the Indian populations which will immensely benefit safe drug dosing in our populations, provided we have adequate funding.”

While the Indian studies were limited by the availability of low-resolution genotype microarray datasets which allows one to profile only a set of known common variants, the latest study on Malays used the more powerful whole genome sequencing. Whole genome sequencing helps in identifying the common genetic variants when performed for a population as well as the very rare and personal variants that are found unique to an individual.

The SE Asian Malay study has helped build one of the most comprehensive pharmacogenetic maps including 227 common and 466 rare potentially functional variants in 437 genes in the population.

Published in The Hindu on June 12, 2016