Next Generation Sequencing Meets Traditional medicine

June 4th, 2015

Traditional knowledge in combination with modern scientific techniques could help unravel deep hidden mysteries. Scientists from NCBS, Bangalore, have revisited the age old knowledge of “Tulsi and its medicinal effects” in their labs, only to be overwhelmed by their scientific findings. Ocimum tenuiflorum or commonly known as Tulsi has been mentioned in ancient Indian scriptures and has found wide usage in the Indian traditional system of medicine, Ayurveda. Known for producing many aromatic compounds, Tulsi gained an informal name as the “Queen of Herbs”. It is considered sacred in Hindu households and mostly used for spiritual and religious purposes in India.

Tulsi grows extensively in tropical climate, hence found in most parts of Asia, Africa, Central and South America. It consists of a wide range of bioactive compounds which are known for their anti-bacterial, anti-fungal, anti-pyretic and anti-cancer properties. These compounds or plant metabolites are very poorly understood because of absolute lack of genomic information. Prof. Ramanathan Sowdhamini and team have produced the first draft genome of O. tenuiflorum Krishna subtype which is a huge leap in understanding and identifying the genes responsible for production of metabolites with medicinal properties. Focussing on the important metabolite genes, the team used five different types of Tulsi, (Ocimum tenuflorium subtype Rama, O. tenuflorium subtype Krishna, O. gratissimum, O. saccharicum and O. kilmund) to collect the genomic data and compare it with the nearest genetically related species. “The genome sequencing projects involved generation of huge quantity of data. The genes were identified from this enormous amount of data using complex prediction models and then they were numbered for easy identification. This assembled genome and the set of genes served as a start point for all downstream analysis”, said Adwait Joshi, one of the team members.

Like every other plant, Tulsi also produces specialized metabolites as a part of its defence mechanism. Linalool, Linalyl, Geraniol, Camphor, Thymol, Safrol, Apigenin, Citral, Eugenol, Taxol and Urosolic acid are few examples among the important secondary metabolites of Ocimum species. “Apigenin, Taxol and Urosolic acid are implicated in anti-cancer properties of the plant, Citral for its anti-septic nature and Eugenol for its anti-infective properties and so on”, says Prof. Sowdhamini. Few metabolites have been used in the perfume and cosmetic industries. While others have been exploited in curing human ailments like malaria, bronchitis, diarrhea and dysentery, etc. The metabolic pathway concerning the synthesis of Ursolic acid was investigated as a case study. Studying mature roots, leaves, flowers, seeds and other parts of the plant, the team found that the precursors of these metabolites are synthesized in young tissues and retain their specific medicinal properties in their mature counterparts.

Owing to the 3000 years of cultivation of Krishna Tulsi and extensive descriptions in the Vedas and Puranas, it is assumed to be of Indian origin. The findings of the experiments at CCAMP, NCBS, reinstate the household knowledge passed on by grandma, even when prodded by the modern scientific techniques. Prof. Sowdhamini said, “This is the first report of draft genome sequencing of a plant species from NCBS and we hope to do more”. Convinced of the huge array of genes and their respective downstream compounds yet to be unraveled in further research, the team looks forward to working in collaboration with an independent parallel initiative by CAMP, Lucknow, to provide the next version of the draft of Tulsi genome.

Perfectionism on part of proteins in cargo delivery could save lives

August 2015

A minor fault in any member of the team of proteins carrying structural elements for melanin pigment maturation could deprive us of not just our colour, but could be fatal when combined with few other factors.

Trucks and lorries rule the world of cargo delivery. Any malfunction in them affects the timely delivery of the cargo at the destination and where and how they are used in the further processes. This chain of events is not very different at a cellular level. Our cells also have their own transport pathways responsible for the cargo delivery at the right destination at the right time. Any variations to that system shows up as symptoms to fatal diseases. Dr. Subba Rao and team from the Indian Institute of Science, Bangalore, unravel the nitty gritties of one such transport pathway in animal cells where failure to deliver the cargo, in this case melanin synthesizing enzymes, could result in fatalities.

Melanin pigments are responsible for the colour of our skin and also play an important role in protection against radiations and any other damage from light. Melanin pigment is produced in cellular organelles called melanosomes which need melanin synthesizing enzymes transported from other organelles. The enzymes transported into premature melanosomes facilitate the maturation into fully pigmented melanosomes. The transport pathway is completed with the help of four multi subunit protein complexes, BLOC 1, 2, 3 and Adaptor protein 3 complex.

BLOC 1 consists of 8 subunits, functioning in the upstream of the pathway while BLOC 2, a 3-subunit protein complex, functions towards the end of the pathway in directing the transfer of molecules towards maturing melanosomes for subsequent reactions. It does so by the specific method of “tethering” or by stabilizing the intermediate molecules that need to be transported.

Mutations in BLOC 1 or BLOC 2 proteins result in inefficient delivery of melanin synthesizing proteins to melanosomes and thus failure in full expression of the melanin pigment. This malfunction manifests in the form of albinism of skin, ear and eye, also referred to as oculocutaneous albinism. This is one of the primary symptoms in Hermansky-Pudlak Syndrome (HPS). The other symptoms are lung infections, which are mistaken as Tuberculosis in most cases in India. Both the lung pathology and albinism put together result in HPS but the confirmatory diagnosis is genetic sequencing of the patient and the parents. HPS generally shows up in children within the age group of 4-6. Out of the 16 possible genetic mutations that can result in HPS, only 9 are known so far. Three out of those nine subtypes are a result of mutations to the BLOC 2 protein.

Even though BLOC 1 and 2 play their respective roles in the overall transport pathway, their molecular functions are not yet clear. There are additional proteins that are responsible for membrane trafficking throughout the cell in most transport pathways. These proteins are called Soluble NSF (N-ethylmaleimide sensitive fusion proteins) Attachment Protein REceptor (SNARE). SNARE proteins, a family of about 60 proteins has been known for their role in membrane fusion during transfer of information. For the first time, the team from IISc has identified two members from the SNARE family that are involved in the transport pathway to melanosome. Immortal melanocyte cell lines from mice, both wild type and mutated, were used for the experiments. The expression of these cell lines were estimated by their absorbance at certain wavelengths and compared with levels of protein expressions found in healthy cells. The team concluded that not only do SNAREs play a vital role in the endosome and melanosome membrane trafficking but are also responsible for maintaining the melanosomal proteins in their stable states until delivered to the maturing melanosomes. Very strong interactions between the SNARE proteins and BLOC 1 has been reported in the initial steps of the transport pathway.

Dr. Subba Rao and team intends to further work on uncovering the details of the interactions between the SNAREs and BLOC 1 and 2 complexes. It is important to understand the specific roles that BLOC 2 plays in the cell and would help in filling the gaps in the transport pathway. How and what delivers the cargo at the destination is yet to be understood. Whether the membranes actually fuse for the transport of the proteins or only the proximity of molecules with the opposite membrane to the surface completes the transport? What are the guiding proteins? If the SNAREs go back to their respective states after the transfer is completed? These are few questions the team is looking forward to resolve in their future research.

 

A biosensor to peer into the insides of a HIV infected cell

December 8, 2014

One of the unique features of the AIDS virus, HIV-1, is that it can exist inside human cells for years without causing any harm. It then reactivates to cause infection when conditions are suitable. Researchers from IISc, Bangalore, the International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi and Jamia Millia Islamia, New Delhi have exploited a non-invasive biosensor that can measure what is going on within HIV-1 infected cells in real-time.

This technology can offer insights which can help in controlling the AIDS infection and also provide insight on the interactions between HIV-1 and the tuberculosis causing bacteria, Mycobacterium tuberculosis (Mtb),within the cells.

Acquired Immune Deficiency Syndrome or AIDS is a devastating disease, which is unfortunately quite common. Since its discovery, AIDS has caused an estimated 36 million deaths worldwide (as of 2012). Its causative agent, the Human Immunodeficiency Virus (HIV), has thus been a hot topic of research.

Our body produces oxygen free radicals called Reactive Oxygen Species or ROS, during routine cellular metabolism. When not regulated properly, accumulation of these ROS can lead to oxidative stress. Heightened oxidative stress is one of the primary causes of reactivation of HIV-1 in infected cells.

Oxidative stress also decreases proliferation of disease fighting immune cells; besides, it causes loss of memory in immune cells. These factors reduce the efficiency of the immune response toward the HIV. A major cellular antioxidant called glutathione (GSH) functions as a protective shield against the oxidative stress. GSH levels in infected cells and tissues are indicators of the level of infection.

The team has devised a non-invasive biosensor methodology for precise measurements of GSH levels within HIV-1 infected cells. Earlier methods use whole cell or tissue extracts, which destroy detailed information related to the GSH levels in different areas within an infected cell. Study discovered that a modest increase in oxidative stress is sufficient to reactivate virus from latency. This may allow researchers to adopt a “shock-and-kill” strategy in which virus could be reactivated by oxidative stress inducing compounds and subsequently killed/flushed by current anti-HIV drugs. The fluctuation of GSH levels detected by the biosensor also helps understand the expression of antioxidant genes and related pathways during latent and active stages of infection.

The sensitivity and specificity of this biosensor could be further used in understanding the physiological changes in HIV-1 infected cells and the mechanism of drug action.

“Importantly, we also discovered that Mycobacterium tuberculosis, another major human pathogen, specifically disturbs glutathione balance to increase the replication of HIV. Since TB is the major cause of HIV related deaths, our findings have major mechanistic and therapeutic potential for both TB and AIDS (among the main causes of human death)”, said Dr. Singh.

The paper appeared in The Journal of Bilogical Chemistry on 18th November. DOI: 10.1074/jbc.M114.588913