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

Taking help from ageing cells to suppress tumours

December 8, 2014

As cells grow older, their DNA gets damaged. Depending on the extent of damage, the cell can repair the DNA and continue its life, or self destruct and die. A molecule called ATM kinase is involved in this decision making process.

Deepak Saini’s lab at IISc has delineated the role of ATM kinase in this important process. The extent of DNA damage either triggers activation of cancer causing genes, or deactivation of tumour suppressor genes. Both these processes can initiate uncontrollable multiplication of cells, leading to cancer. The other possible outcome of DNA damage, especially if very severe, is cell death. The decision of the cell’s fate lies in the hands of the genetic errors accumulated. If the errors cannot be repaired, or can be detrimental if left unrepaired, the cells enter cellular senescence, which is basically ageing. Cell function deteriorates and ageing of the organism is the inevitable result.

The senescent condition of the cells depend on their respective abilities to maintain a persistent DNA damage state without inducing death or repair. There are a number of molecules like ATM kinase and ROS (reactive oxygen species) that play a critical role in regulating cell fate after the genomic damage.

Cellular senescence can be divided into two distinct phases – initiation or early senescence and the maintenance of senescence. The present research delineates the roles of ATM kinase in the initiation of senescence and importance of ROS in maintaining senescence. ATM kinase is one of the key proteins which decides the fate of a cell; it also acts as a quantitative sensor for DNA damage. When DNA damage is not so severe, the cell repairs its DNA and continues growth; in severe damaged states, the cell dies.

In the intermediate stages of damage, the cell enters the senescent stage activated by ATM kinase. “Our studies show that senescence or aging is one of the cell fates in response to DNA damage and the decision is dependent on the dose of damage and ATM kinase protein. Aged cells generate free radicals which is critical in maintaining their status quo”, said Dr. Saini.

Since the other two alternatives after DNA damage – death and cancer – are obviously harmful, a possible way to push a cell toward senescence instead of the other options can have possible therapeutic value. Cell senescence can be induced in tumour and cancer cells by using a sub-lethal dose of stress, by agents like gamma rays, hydrogen peroxide etc. which triggers the DNA damage response leading to senescence. Further research on this could help us devise a very simple yet attractive tumour suppressing mechanism.

The paper will be published in the Journal of Cell Science and appeared online on 21st November. http://jcs.biologists.org/content/early/2014/11/20/jcs.159517.abstract

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A potential therapeutic for septic shock

January 5, 2015

We sometimes hear of post-surgery infections, which can even result in untimely death. The life-saving surgery at times leads to a life threatening recovery. In the intensive care units of hospitals, microbial contamination induces massive inflammation leading to sepsis or septic shock. This has been a rising cause of mortality worldwide in the hospital intensive care unit admissions.

As the famous saying goes “the more the merrier” does not necessarily hold true with new drugs because “less is always more”. All we need is a single efficient drug to combat the sudden and rapid spread of sepsis in the intensive care units of hospitals.

Sepsis is caused by the uncontrolled expression of several inflammatory genes in the host, leading to irreparable damages. The sudden onset and excessive expression of these genes leads to accumulation of harmful metabolic end products, resulting in multiple organ failure. During such cellular stress, some proteins are activated. Development of inhibitors to these stress activated proteins can help devise treatment of such disorders.

The stress activated proteins are comprised of two main subsets- c-Jun N-terminal Kinase (JNK) and p38 Mitogen Activated Protein Kinase (MAPK). It is interesting to note that this work stems out of an extensive collaborative work by three groups from IISc, K. Durga Prasad and T. N. Guru Row from SSCU, J. Trinath and K. N. Balaji from MCBL and Anshuman Biswas and K. Sekar from Bioinformatics. Carefully planned chemical modifications on the commercially available and expensive JNK inhibitor SP600125 improve its ability to bind and inhibit JNK at very low concentrations. The inhibitor also reduces the expression of the inflammatory genes, which in turn cascade into septic shock.“Our study is among the first reports of the description and meticulous biochemical characterisation of selective JNK inhibitors” says Professor Balaji K. N.

This selective and more efficient inhibition activity of JNK inhibitors could facilitate the generation of novel therapeutics to treat sepsis and other inflammatory disorders. It can also pave the way to understand the essential biological function of signalling pathways related to JNK.

The paper appeared in the journal Scientific Reports in end November 2015. http://www.nature.com/srep/2014/141127/srep07214/full/srep07214.html

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An “antioxidant-like” protein to fight free radical damage in the body

September 22, 2014

A protein found in high levels in some cancer cells can be used for treating diseases caused by oxygen free radicals in the body, a recent study has found.

Oxygen free radicals such as hydrogen peroxides and superoxides, called Reactive Oxygen Species (ROS), are found in the cells as byproducts of cellular metabolism. Uncontrolled levels of ROS in the cell can lead to oxidative stress. Diabetes, atherosclerosis and neurodegenerative diseases like Parkinson’s and Alzheimer’s disease find their roots at the damage caused by oxidative stress in the cells.

Patrick D’Silva’s group at the Indian Institute of Science have found that Magmas, a mitochondrial protein also regulates the level of ROS in cells, apart from its already known function.

Magmas is involved in protein transport in cells, and is found in elevated levels in certain cancer types. There is very little information already available about regulation of ROS in the body, and this paper brings forth a lot of missing links in this research area.

The team found that the levels of Magmas in the cell are dependent on the cellular ROS levels. Elevated levels of Magmas help in lowering the concentration of ROS and vice versa. It not only plays an important role in controlling the production of free radicals, but maintains the ROS homeostasis by efficient scavenging. This protects the cell viability and also increases cellular stress tolerance.

“By maintaining a free-radical balance in cell, this protein prevents stress mediated cellular damage to biomolecules such as DNA, proteins and lipids. Hence, overproduction of Magmas protein provides unique advantages to the cells against free radical stress”, said Prof D’Silva.

Higher levels of Magmas are typically found in metabolically active tissues, cancer cells and tissues at different developmental stages. In cancer cells, Magmas prevents cell death, and hence helps in the proliferation of cancer cells. Even in non-cancerous cells, Magmas shows controlled levels of ROS and much lesser oxidative stress.

Such molecules that regulate the number of free radicals can be used while designing possible therapies for oxidative stress related disorders. “The inhibitors or stimulators against Magmas can be used as a therapeutic intervention against cancer as well as multiple free-radical induced stress related diseases”, said Prof D’Silva.

Further research is required to elucidate the mechanism of ROS regulation by Magmas and to discover the other proteins involved in the regulatory circuit.

The paper appeared in the journal Cell Death and Disease on 28th August 2014.

Link: http://www.nature.com/cddis/journal/v5/n8/full/cddis2014355a.html

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