Friday, 27 October 2017

Fly Stem Cells May Help In Understanding Muscle Disorders

A new population of stem cells in the Drosophila muscles involved in repair of injured tissue.

BY Ratneshwar Thakur    Published in India Science Wire   Journal Ref. : eLIFE
Also appeared in The Hindu BusinessLine BioTechTimes NetIndian BioVoice NewsNow Scroll

(Left- Rajesh Gunage, From Top- K. VijayRaghavan, Heinrich Reichert, Dhananjay Chaturvedi)

Indian researchers are trying to understand the mystery of injured muscles recovery. Stem cell studies are a start of the big quest of understanding muscles related problems like Muscle dystrophy, a progressive muscle disorder where muscles fail to recover after damage. Adult Stem cells repair damaged tissue and are the secret behind why our organs can function without stopping. Stem cells are the hot topic of research with a huge potential for therapeutics.

At the National Center for Biological Sciences-TIFR, Bangalore, the team members (Rajesh Gunage, Dhananjay Chaturvedi, Heinrich Reichert (Biozentrum, Switzerland) and K. VijayRaghavan- have discovered a new population of stem cells in the Drosophila muscles involved in repair of injured tissue.

Previous work from the same group laid the foundation for the project and was continued by Dhananjay Chaturvedi. ‘Excellent team efforts are required to understand complex biology and this study would not have been possible without Dhananjay’s efforts, believes Dr. Gunage. 

Exercise such as running and lifting weights causes muscles damage, but they recover and are suitable for work again. How is this achieved? “We thought we can study this in the most easily accessible model i.e. Fly. These are small flying insects, we see hovering on the ripened banana. We were puzzled about how an insect can fly for so long, much like we, humans run and walk throughout our life. It is a simple question with vast implication to understand about muscles in general. Fruit fly came as an easy model,” says Rajesh Gunage (current affiliation-Harvard Medical School), a senior author on this study who also co-authors with Dhananjay Chaturvedi.

In the study done with core support from NCBS, the team designed a simple Pin Prick Assay as an experimental strategy. The ‘pin prick' assay involves a tiny metal pin with dimensions close to an adult’s eyebrow hair. “Using this we could induce non-life threating damage to tiny flight muscles of fly and observe for recovery of flight,” says Dr. Gunage. 

Flight muscles as the name suggests are involved in flying, and they are equated to our muscles of leg or arm. Much like the recovery of damaged muscles in case of a runner who recovers from it and starts to run again, researchers were surprised to see fruit fly flying once again after a brief period of recovery. Detailed analysis using best microscope possible- led to the discovery of novel stem cells. Team members observed that pin injury activates stem cells and they multiply in number. These newly formed cells then become part of injured muscles to help them recover from the injury. These results were published in the journal eLife Sciences.

“Anybody can do this experiment even at home. All you need is a small pin and a banana. Banana attracts small flying insects named Drosophila. All you need to do is prick them in the thorax with a pin to injure their muscles. Initially, they fail to fly due to injury but soon due to stem cells, the damage is reversed, are set into action again. Within no time you can see them resume their normal flight,” says Dr. Gunage.

Researchers said, “Just imagine small flying insects with stem cells. Hard to believe, isn’t it? Our reaction was the same and we were skeptical for a very long time. Insect muscles are very small and to confirm the presence of stem cells, we used electron microscopy. Under this microscope, a tiny marble looks like a giant football! Using this we could clearly see these small stem cells next to muscles, much like a number of satellites around the planets.”

“Using the knowledge gained from the current findings, one can understand how in old animals, muscles fail to work or show less recovery following accidental damage. Questions such as how different diet, regular exercise affect muscles or even a possible drug expected to enhance muscle function affect can now be tested. It just makes many difficult experiments easy to perform with much less effort,” said Gunage and Chaturvedi.

Monday, 16 October 2017

Nanoparticles Designed For Sustained And Controlled Drug Delivery

Nanoparticles can be designed to carry and deliver drugs to very precisely diseased cells. This way drugs can be delivered in the small dosage as well as in controlled release, thereby minimising side effects.

BY Ratneshwar Thakur      Published in India Science Wire Journal Ref. : ACS Omega

(Dr. Neetu Singh with her student Sonal Deshpande at IIT Delhi.)

Indian researchers are trying to engineer effective drug delivery system through nanoparticles. The use of nanotechnology enables researchers to encapsulate medicine in tiny particles which are the size of viruses. Nanoparticles can be designed to carry and deliver drugs to very precisely diseased cells. This way drugs can be delivered in the small dosage as well as in controlled release, thereby minimising side effects.

A group of researchers led by Dr. Neetu Singh from Centre for Biomedical Engineering at Indian Institute of Technology (IIT), Delhi has demonstrated a simple concept for achieving controlled and sustained release of drugs using the nanoparticle system.

Conventional drugs administered to patients get easily cleared from the body, which makes frequent administration of the drugs necessary. This makes patient compliance an important factor that determines the efficiency of treatment. Nanoparticles have been investigated as drug carriers as they can increase blood circulation time of a drug as well as help in targeting it to the disease site. Thus it can reduce its side effects on healthy tissues and improve the treatment efficiency.

Previous studies had shown that nanoparticle systems developed are designed to have components that respond to a stimulus like pH, different enzymes, temperature etc. When exposed to the stimulus, they release the drug either slowly over a period of time or quickly all at once.

“Our system shows a combination of release profiles, where there will be an initial release of the drug, which on demand, can be accelerated using radiofrequency (RF). Thus, the system can control when the drug has to be released. It is similar to loading few tablets in a reservoir and triggering when and how many are to be released as per the requirement,” explained Dr. Singh.

Researchers said that since external triggers provide a better control, they had options for selecting nanoparticle responsive to the magnetic field, near infra-red radiations (NIR), ultrasound and radio-frequency field (RF). They opted for gold nanoparticles because of their bio-inertness, ease of synthesis and heat generation on exposure to NIR and RF. As the tissue penetration depth is better for RF, they optimized the gold nanoparticle accordingly.

The next important component was the one that will respond to the temperature at which healthy cells are unharmed. The gold nanoparticles were therefore covered with a shell of a polymer which shrinks when exposed to temperatures close to the body temperature, thereby releasing the drug.

“Our system can be used to deliver a drug on demand for the longer period of time, thereby improving the drug efficiency and reducing the frequency of drug administration. Additionally, one can load different types of drugs used in combinatorial therapy and control the sequence of their release in the body using the radio-frequency trigger,” said Dr. Singh.

“Sustained release of drugs on demand from nanoparticles is still a challenge in the field of drug delivery that motivated us to work towards designing a simple system that can help to achieve a sustained, triggered release of drug from nanoparticles,” said Sonal Deshpande, a member of the research team. The results of the study have been published in journal American Chemical Society (ACS) Omega. The research team also included Sapna Sharma and Veena Koul. 

Friday, 13 October 2017

Epigenetic Manipulation May Inhibit Cancer Spread

The study suggests that DNA methylation can be targeted to switch the cancer-specific splicing isoform to normal isoform and thereby inhibit the growth of cancer cells.

BY Ratneshwar Thakur      Published in IndiaScienceWire     Journal Ref. : PNAS

(Dr. Sanjeev Shukla and his team members)

Manipulating tags on DNA without changing the underlying sequence triggers a chain of molecular events that may help prevent the spread of cancer, a new study by Indian scientists has revealed.

Development of cancer is a multistep process involving changes in several genes. Although the blueprint for cell’s life is kept in DNA for all events like growth, maturation, division, and even death, it is proteins that essentially do all the required work.

The new study shows epigenetics (changing activity of a DNA segment without changing sequence) anomalies could alter DNA instruction manuals for splicing that may lead to the synthesis of a particular type of mRNA and eventually proteins, in cancer cells. 

In the study published in journal Proceedings of the National Academy of Sciences, Dr. Sanjeev Shukla's group at Indian Institute of Science Education and Research (IISER), Bhopal- has successfully identified a novel way to target glucose metabolism and thereby breast cancer cells by switching the alternative splicing of PKM gene. 

According to the study, most of the cancer cells derive their energy from an energy production process called aerobic glycolysis that involves the transformation of glucose to lactate, when limited amounts of oxygen are available. Pyruvate kinase, an enzyme that catalyzes the last step of glycolysis seems to be an important regulator of this switch in the metabolism of glucose. Out of four isozymes of pyruvate kinase, isozymes M1 and M2 result from alternative splicing of transcripts coded by the PKM gene. Interestingly, the PKM2 alternative isoform is expressed by highly proliferating cells including cancer cells suggesting that regulation of PKM1/PKM2 ratio may hold the key to regulate cancer proliferation.

Researchers have discovered that the PKM2 specific exon inclusion is regulated by DNA methylation and a CTCF related protein BORIS (Brother of Regulator of Imprinted Sites) which preferentially binds to the PKM2-specific-methylated exon. As a result of this, splicing process gets altered in the case of cancer cells in such a way that instead of exon 9, exon 10 gets included when the pre-mRNA is spliced into mRNA.

“Our study suggests, we can potentially target DNA methylation to switch the cancer-specific splicing isoform to normal isoform and thereby inhibit the growth of cancer cells,” explained Dr. Shukla, who is a Wellcome Trust/DBT India Alliance Fellow. “DNA methylation or BORIS may serve as a potential target to inhibit the growth of cancer cells. As the epigenetic marks are reversible, they are an attractive target for cancer therapy,” he added.

Collaborators in this study, Dr. Rajendra K. Panday and Dr. Atul Samaiya from Bansal Hospital, Bhopal say “the new findings will help in the development of potential target for cancer therapy and will be useful in patient management in future.”

“As a therapeutic strategy, inhibiting the growth and accelerating the death rate of breast cancer cells may be possible by starving the cancer cells of glucose or by inhibiting or reversing aerobic glycolysis. Aerobic glycolysis confers a proliferative advantage to the cancer cells. The work of Dr. Shukla's group connects DNA methylation to alternative splicing and cell proliferation suggesting that epigenetic manipulation may provide a novel approach to dysregulate RNA processing and inhibit cancer proliferation,” says Dr. Sanjeev Galande, Scientist at IISER-Pune, who is not related to this study.

Friday, 6 October 2017

New Method To Tinker Cell Surface Proteins Can Help Design New Drugs

A group of Indian scientists have found a new way to modulate specific cellular functions by tinkering with proteins that play a role in cells response to external chemicals such as drugs.

(Dr. Shukla and his team members at IIT-Kanpur, India)

The new finding, described in a paper published this week in journal Nature Nanotechnology, makes it convenient for researchers to study the workings of a large family of sensor proteins called G-protein-coupled receptors (GPCRs) and to develop potential synthetic-antibody-based drugs.

Research teams led by Dr. Arun K. Shukla at IIT Kanpur have engineered synthetic Antibody fragments that target beta-arrestin (a class of proteins inside the cell). GPCRs are mobile proteins that sit in the cell membranes and support cells to respond to chemical signals from the body parts or the outside world. GPCRs are the most sought-after drug targets in modern medical research. Major Pharma companies are investing their pockets out to identify new drug molecule which can target key GPCRs implicated across wide-ranging pathophysiological conditions.

GPCRs are involved in almost every physiological and pathophysiological process in the human body such as cardiovascular regulation, immune response, neurotransmission, behavior and mood regulation. Beta-arrestin comes to play-- to regulate the actions of GPCRs by attenuating its signaling.

“There are more than 800 GPCRs and only limited beta-arrestin which controls its functioning, therefore, targeting Beta-arrestin will enable us with a greater handle over the desired signaling across all GPCR types. So, we developed synthetic Fabs (Fragment antigen-binding) that targets β-arrestin and have potential to modulate their functioning,” said Dr. Arun K. Shukla.

According to the study, small molecule-based drugs, which either bind to stimulate or inhibit GPCR, remains a traditional way of tweaking the GPCRs. Dr. Shukla says that beta-arrestin functions are usually studied by either knocking the gene out or turning their expression down by siRNA based approaches (tool for inducing short-term silencing of protein-coding genes). But the synthetic Fab molecules can specifically target a particular part of the protein and selectively offset its function without interfering with its any other function, attributed to its other domains. “Our finding, therefore, is the first demonstration of targeting a specific function of beta-arrestins without altering its other functions,” said Eshan Ghosh, first author of this paper.

This study was an ambitious project to design a generic tool to study GPCRs. “Initially, in vitro studies, we found very encouraging results but the real deal was to replicate the study in cellular systems, where we can really see the molecule in action by monitoring its cellular readouts,” said Dr. Shukla. Interestingly, during microscopic experiments, Dr. Shukla’s team members could observe that this fab in form of an intrabody (an antibody that works within the cell) was clearly blocking the endocytosis (pulling in of a GPCR from the surface to inside of a living cell).

“Synthetic intrabody we have identified has the potential to treat many diseases implicating GPCRs if its delivery vehicle is well devised,” said Mithu Baidya, one of the authors of this study.

“Our approach has the potential to take antibody-based drug discovery to the next level, where we can dissect to compartmentalize the functioning of a protein and thereby modulate it. This study will flag off a new direction for future drug discovery,” said the study leader.

Research team included Eshan Ghosh, Ashish Srivastava, Mithu Baidya, Punita Kumari, Hemlata Dwivedi, Kumari Nidhi, Ravi Ranjan and Arun K. Shukla (Indian Institute of Technology, Kanpur), Shalini Dogra Prem N. Yadav  (CSIR-Central Drug Research Institute, Lucknow), Akiko Koide, Shohei Koide (Laura and Isaac Perlmutter Cancer Center, USA), Sachdev S. Sidhu (University of Toronto, Canada).

Tuesday, 3 October 2017

A Novel Antifungal Molecule Discovered In Bacteria

The present study not only showcases high-end basic science research, but it has opened up various translational applications in controlling fungal diseases.

BY Ratneshwar Thakur, Published in Singapore based APBN

A novel broad-spectrum antifungal protein has been identified by a research group led by Dr. Gopaljee Jha at the New Delhi- based National Institute of Plant Genome Research (NIPGR) -An autonomous Institute of Department of Biotechnology, Govt. of India. The results were published in the journal Nature Communications.

Fungal pathogens have been a challenge for sustainable agriculture and they have been behind death and disability in humans, wildlife extinctions, and population declines. By and large, it is very difficult to control fungal diseases and globally there have been urgent thrust to devise newer strategies to control them.

The breakthrough was heralded by Dr. Jha’s team when they isolated a novel bacterium Burkholderia gladioli strain NGJ1 from healthy rice seedling which exhibits broad spectrum fungal eating property (the phenomenon known as mycophagy).The researchers observed that bacterium was killing fungal cells to utilize their metabolites for its own growth and survival.

It is very interesting how a bacterium (smaller in size) can feed on fungi, which is relatively much bigger in size.

“Considering phenotype associated with mycophagy, we expected that NGJ1 would perform as a better biocontrol agent than other antifungal bacteria. Due to its mycophagous property, the bacterium can not only prevent fungal growth but can eradicate fungal biomass as it utilizes them as a source of nutrients. Indeed treatment of NGJ1 was found to prevent the disease-causing ability of Rhizoctonia solani, the causal agent of sheath blight disease of rice,” said Dr. Jha. 

Scientists have always believed that phages are bacterial predators and upon induction, they can lyse bacterial cells. However, Dr. Jha’s group observed that the NGJ1 has made its phage inactive (prophage) and deputes one of the prophage tail-like proteins (Bg_9562) to forage over fungi.Through series of experimentation, the group could demonstrate that the NGJ1 utilizes one of its phage proteins to eat fungi.

The researchers tested the purified Bg_9562 protein to check the broad spectrum antifungal activity. Strikingly, the antifungal activity was observed against several economically important phytopathogens, such as Rhizoctonia solani (rice sheath blight pathogen), Magnaportheoryzae (rice blast pathogen), Fusarium oxysporum (pathogenic to various plants), Aschocytarabiei (chickpea blight pathogen), Venturiainaequalis (apple scab pathogen) and Candida albicans (causes Candidiasis in humans).

“This is the first report stating that a potential Type-3 secretion is required during the Bacterial-Fungal interaction. This opens up a new area of basic science research,” said Dr.Durga Madhab Swain,(One of the first authors in this paper).

“Considering broad-spectrum antifungal activity, it is being proposed that the protein could be utilized to control fungal diseases of plants as well as humans/animals. For example, a formulation using this protein can be sprayed over the agricultural field to control various fungal diseases at one go or an ointment based on this protein can be used to treat fungal infections of animals/humans. Furthermore, the Bg_9562 gene can itself be used as a transgene to develop broad-spectrum fungal disease resistant plants, which is need of the hour,” said the study leader Dr. Jha.

The present study not only showcases high-end basic science research, but it has opened up various translational applications in controlling fungal diseases.

Dr. Jha says that it has been a gigantic task and various researchers (Durga, Sunil, Isha, Rahul, Rajeev, Srayan, Joyati) have worked day and night to identify the molecular basis of bacterial mycophagy.

Study Identifies Genetic Link To Heart Disease In Indian Population

About 35 to 40 percent Indians carry a set of genetic variations which puts them at higher risk of heart disease, finds a new study.

By Ratneshwar Thakur Published in India Science Wire 
Also appeared in The Hindu, APN News,, BioTech Times, Bioinformatics, NetIndian

(Research team at IIT-Madras)

A team of Indian researchers has discovered that carriers of a set of genetic variants in the Chromogranin A (CHGA) gene called ‘CHGA promoter haplotype2’ may be at higher risk for cardiovascular and metabolic disorders. An estimated 35 to 40 percent of Indian population may be carrying this genetic variant.

The study is based on analysis of genomic DNA samples from over 750 individuals from Indian population. The research findings have been published in Journal of Biological Chemistry.

Though the CHGA promoter haplotype is present in other ethnic populations, it occurs more frequently in populations of South Asian ancestry. “It is a protein of neuroendocrine origin and is secreted along with hormones like catecholamines. 

Earlier studies had suggested its role in the regulation of cardiovascular and metabolic diseases but there was no data about it in South Asian populations,” said Lakshmi Subramanian, first author of this paper.

“We studied genomic DNA of Indians and discovered a specific set of changes in the CHGA gene sequence called Haplotype2 which contributed to increased CHGA gene expression, and ultimately increased CHGA protein levels in plasma. When the clinical parameters of those in the study were compared, Haplotype2 carriers displayed higher levels of metabolic and cardiovascular traits like plasma glucose, blood pressure, and body mass index,” explained Dr. Nitish R. Mahapatra, Professor at IIT-Madras.

However, he said, these results need further validation in animal models as well as large-scale studies in individuals with metabolic syndrome. “We hope these findings would help unravel biological pathways and mechanisms underlying these complex diseases and would help in the development of therapeutic, preventive strategies,” said Dr. Mahapatra.

“Basic research in cardiovascular biology is in a nascent stage in India with only a handful of researchers working in this area. This new study significantly contributed towards understanding the molecular basis of cardiovascular and metabolic diseases. This is a fast emerging area with tremendous therapeutic and diagnostic potential,” commented Dr. Shyamal K. Goswami, a Professor at School of Life Science, JNU, who is not connected with the study.

The study was led by Dr. Nitish R. Mahapatra at Indian Institute of Technology -Madras, Chennai, in a collaborative effort with Dr. Ajit S. Mullasari at Madras Medical Mission, Chennai, and Dr. Madhu Khullar at PGIMER, Chandigarh. (India Science Wire)