Nobel Vesicles

Nobel season is here once more. Has it really been a year?
Last year, The Nobel Prize in Physiology or Medicine was shared by Sir John Gurdon at Cambridge, and Shinya Yamanaka, a Japanese researcher with labs in Kyoto and San Francisco, for their work on cell reprogramming.
This year, The Karolinska Institute in Stockholm announced the winners: James E. Rothman,; Randy W. Schekman, and Dr. Thomas C. Südhof, on 07 October 2013 "for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells". Their basic research solved the mystery of how cells, which are factories producing molecules, organize a system to transport the molecules within cells and export them outside.
In laymans term, each cell is a factory that produces and exports molecules. Cells move molecules around using tiny membrane-enclosed packages called vesicles. In a large and busy port, systems are required to ensure that the correct cargo is shipped to the correct destination at the right time. The cell, with its different compartments called organelles, faces a similar problem: cells produce molecules such as hormones, neurotransmitters, cytokines and enzymes that have to be delivered to other places inside the cell, or exported out of the cell, at exactly the right moment. Timing and location are crucial. Miniature bubble-like vesicles, surrounded by membranes, shuttle the cargo between organelles or fuse with the outer membrane of the cell and release their cargo to the outside. This is of major importance, as it triggers nerve activation in the case of transmitter substances, or controls metabolism in the case of hormones. For instance, insulin is manufactured and released into the blood and signaling molecules called neurotransmitters are sent from one nerve cell to another. These molecules are transported around the cell in small packages called vesicles.  How do these vesicles know where and when to deliver their cargo?
This year’s Nobel Laureates, who will share the $1.2 million prize, discovered how cells get those vesicles to their intended destination at the intended time.
Randy Schekman, Cell biologist at the University of California, Berkeley was fascinated by how the cell organizes its transport system and in the 1970s decided to study its genetic basis by using yeast as a model system. In a genetic screen, he identified yeast Saccharomyces cerevisiae cells with defective transport machinery, giving rise to a situation resembling a poorly planned public transport system. Vesicles piled up in certain parts of the cell. He found that the cause of this congestion was genetic and went on to identify the mutated genes. Schekman  developed a genetic screen of the yeast to determine the genes that regulate vesicle trafficking. With this information, he identified 23 key genes, which can be divided into three classes that control vesicles at the Golgi complex, the endoplasmic reticulum, or the cell surface.

James Rothman a cell biologist at Yale University was also intrigued by the nature of the cell´s transport system. When studying vesicle transport in mammalian cells in the 1980s and 1990s, Rothman discovered that a protein complex known as  SNARE (soluble N-ethylmaleimide-sensitive factor-activating protein receptor) enables vesicles to dock and fuse with their target membranes. In the fusion process, proteins on the vesicles and target membranes bind to each other like the two sides of a zipper.  These proteins had already been discovered by others, but their function was unknown. Rothman determined that these proteins interact with high specificity: The SNARE protein on a particular target membrane is able to interact with only one or a few vesicle SNARE proteins. The fact that there are many such proteins and that they bind only in specific combinations ensures that cargo is delivered to a precise location. The same principle operates inside the cell and when a vesicle binds to the cell´s outer membrane to release its contents. Thus, Rothman unravelled protein machinery that allows vesicles to fuse with their targets to permit transfer of cargo.
It turned out that some of the genes Schekman had discovered in yeast coded for proteins corresponding to those Rothman identified in mammals, revealing an ancient evolutionary origin of the transport system. Collectively, they mapped critical components of the cell´s transport machinery

Dr. Thomas Südhof, a biochemist at Stanford University, was interested in how nerve cells communicate with one another in the brain. The signalling molecules, neurotransmitters, are released from vesicles that fuse with the outer membrane of nerve cells by using the machinery discovered by Rothman and Schekman. But these vesicles are only allowed to release their contents when the nerve cell signals to its neighbours. How is this release controlled in such a precise manner? Calcium  ions were known to be involved in this process and in the 1990s, Südhof searched for calcium sensitive proteins in nerve cells. He identified molecular machinery that responds to an influx of calcium ions and directs neighbour proteins rapidly to bind vesicles to the outer membrane of the nerve cell. He discovered how calcium regulates neurotransmitter release and that two proteins—complexin and synaptotagmin-1—are key players in calcium-mediated vesicle fusion. Synaptotagmin-1 acts as a calcium sensor during synaptic fusion. Complexin acts as a clamp during synaptic fusion to make sure that regulated exocytosis occurs instead of the vesicle simply being incorporated into the cell membrane. The zipper opens up and signal substances are released. Südhof´s discovery explained how temporal precision is achieved and how vesicles´ contents can be released on command. Südhof also identified the genes that are responsible for controlling the timing of vesicle fusion, particularly those involved in the release of neurotransmitters
The three Nobel Laureates have discovered a fundamental process in cell physiology. Through their discoveries, Rothman, Schekman and Südhof have revealed the exquisitely precise control system for the transport and delivery of cellular cargo. Vesicle transport and fusion operate, with the same general principles, in organisms as different as yeast and man. The system is critical for a variety of physiological processes in which vesicle fusion must be controlled, ranging from signalling in the brain to release of hormones and immune cytokines. Glitches in vesicle transport occur in a variety of diseases including a number of neurological and immunological disorders, as well as in diabetes. . Mutations in genes associated with the protein machinery are involved in specific diseases. For example, mutations in one of the genes are involved in certain forms of epilepsy. Thus, they have revolutionised understanding of how cells are organised which is fundamental to huge number of diseases. Without this wonderfully precise organization, the cell would lapse into chaos.

TP53 Mutation screening

Hello!
My first batch of results and one novel mutation!
It fascinates me, how single base switch in DNA of a person have so much effect on the person chances of getting the diseases..
Check out my presentation below..
TP53 Mutation screening

Ataxia telangiectasia

How did the condition arise?

Ataxia means no coordination in the simple movements such as walking and Telangiectasias are enlarged blood vessels (spider-like veins) just below the surface of the skin. Ataxia-telangiectasia (A-T) is genetically inherited disease, which means it is passed down from generation to the next.

Every living organism possesses a genome that contains the biological information needed to construct and maintain that organism. The chromosome is a discrete unit of genome carrying many genes. These genes are units of genetic information expressed in the form of proteins. Each of them consisting of a very long molecule of duplex DNA(deoxyribonucleic acid), where DNA is a molecule made up of chains of subunits called nucleotides.

Human Genome is comprised of 23 pairs of Chromosomes, of which 22 pairs are autosomal and 1 pair is sex chromosome. These chromosomes have a long q arm and a short p arm. Each of these pairs has one chromosome from mother and one from father. They carry a copy of a number of duplicate genes known as alleles.

A-T is caused by autosomal recessive mutations in the ATM (Ataxia Telangiectasia Mutated) gene located on chromosome 11 section 11q22-23. Mutation is a change in the nucleotide sequence of the gene. A-T is an autosomal recessive trait,that means mutation is in a gene present on autosomal chromosomes and both parents must provide a defective alleles of the gene to the child to have symptoms of the disorder. More than 500 unique ATM gene mutations are known. Most mutations are null mutations which results in total absence of ATM protein.

For the organism to grow, it’s cells need to divide.For that cells need to undergo sequence of activities called the cell cycle. In the cell cycle, there is multiplication of cell along with its DNA content. There is a control mechanism in this process called Cell cycle checkpoints which ensures whether the processes at each phase of the cell cycle have been accurately completed before progression into the next phase.Few of them are DNA damage checkpoints where the damage in DNA to be replicated is detected and repaired.DNA repair is the process by which a cell identifies and corrects damage to it’s DNA molecules.

The ATM protein is thought to play a role in regulating cell cycle checkpoints and in repair of double stranded DNA. As the result, the mutated ATM gene leads to a high frequency of mutations resulting in various complications and abnormalities seen in A-T patients.

What is the prognosis likely to be?

Ataxia-telangiectasia is a complex genetic disorder. Generally, in all the cases telangiectasia is followed by ataxia, after a gap of few years. A-T patients usually show uncontrolled infection in adolescence which may prove fatal, though some show variability in severity, age of onset, and rate of progression.

The primary features of classic A-T include progressive ataxia with onset between ages one and four years, progressively slurred speech, oculomotor apraxia that is inability to follow an object across visual fields, choreoathetosis which means writhing movements, oculocutaneous telangiectasia that is enlarged blood vessels in skin of nose, ears, and inside of the elbow , knee , whites of the eyes etc. A-T pateints also show hypersensitivity to ionizing radiation with increased susceptibility to cancer and susceptibility to frequent infections due to immunodeficiencies.Immunodeficiency (or immune deficiency) is a state in which the immune system's ability to fight infectious disease is compromised or entirely absent.

Ataxia is progressive and show various other neurological manifestations, including mask-like face, excessive drooling, abnormal eye movements and loss of tendon reflexes. Where tendon reflex are the one elicited by a sharp tap on the appropriate tendon or muscle to induce brief stretch of the muscle, followed by contraction. There are other complications like chromosomal instability,poor development of secondary sexual characteristics, premature aging with strands of gray hair and abnormalities such as diabetes.

A distinct feature of A-T pateints is that they have extreme sensitivity to ionizing radiation. For e.g., X rays used in the therapeutic treament of the pateints with AT often produce devastating death of normal tissues. X ray induced DNA breaks in one or both strands of the double helix may go unrepaired, leading to a vareity of chromosomal breaks and rearrangements.Thus the patients have a high frequency of mutations resulting into a marked increased risk for various types of cancer, particularly Leukaemia (Blood Cancer) and Lymphoma (cancer of cells of Immune systems).

A-T heterozygote is the one who have different alleles of ATM gene, that is one defective and one normal ATM gene on chromosome 11 pair. These heterozygote for ATM gene defects are common and appear to be particularly vulnerable to the induction of cancer by radiations. Female heterozygote for AT mutation have overall two fold increased risk of breast cancer and a five folds higher risk for the breast cancer before the age of 50 years. Epidemiologic studies suggest that A-T carriers are at an increased risk for heart disease. Eventually, it can be seen that the prognosis for individuals with A-T is poor, where death occurs in their teens or early 20s.

What treatments are available?

There is no specific treatment for ataxia-telangiectasia. Treatment is directed at specific symptoms such as Cancer, Diabetes etc. Immunodeficiencies are present in 60% to 80% of individuals with classic A-T.Blood concentration of the immunoglobulin’s like IgA, IgE, and IgG are reduced where immunoglobulins are the protein produced by immune cells which play an essential role in body’s Immune system by fighting infection. IVIG (Intravenous immunoglobulin) replacement therapy is given to the A-T patients were blood product containing immunoglobulin (IgG) is administered intravenously in them.

Brain atrophies (shrinkage) are observed early in the course of classic A-T, leading to neurological symptoms. Although steroids can temporarily improve in condition of A-T in children, the neurological symptoms reappear within days of their discontinuation.

Speech therapy may also be needed. It can significantly improve diction, especially in the second decade of the disorder. Early and continued physical therapy is helpful to minimize contractures (continuous muscle contraction) and scoliosis (curved spine) which appear in almost all A-T patients with time. Orthopaedic assessment like corrective procedures can be helpful for joint or postural problems, particularly in the lower limbs or spine. Supportive therapy involving usage of drugs, surgeries, and retraining of movements are tried out but individual responses to such therapies vary.

What are common complications and how are they managed?

The infections observed in classic A-T does not include common opportunistic infections, but lung infections are frequent. These patients require aggressive pulmonary hygiene.In A-T patients the frequency and severity of infections correlates more with general nutritional status than with the immune status. So, good health and hygiene should be observed by the patients and their family.

The cells from A-T patients are 30% more sensitive to ionizing radiation than cells from normal .They should avoid exposure to excessive ionizing radiation including those from diagnostic procedures such as CT scans and some chemotherapeutic agents used in treatments.

Parents should regularly monitor their children for early signs of malignancy (e.g., weight loss, bruising, localized pain or swelling) and report it to the Physician. Monitoring of immune status should also be done of the children with severe recurrent infections or undergoing IVIG therapy .They should look out for the more subtle beginnings in their young children for early recognition of A-T. This would also encourage more vigorous treatment for the respiratory infections to which the A-T affected children are prone.

Would gene therapy be appropriate to treat this condition?

Researchers are exploring therapeutic strategies that target the underlying causes of A-T, rather than just the symptoms. Gene Therapy is one of the strategies, in which there is an attempt to replace the defective ATM gene with the normal one. Gene therapy by definition involves the direct genetic modification of cells of the patient to achieve a therapeutic goal.

In one of the approach of gene therapy, gene addition is carried out, where aim is to supply a functioning gene copy that will supplement defective gene. It is generally carried out to treat diseases that are the result of a gene not functioning (Strachan and Read, 2004).It was carried out by transfection of full-length DNA of ATM gene where transfection is the process of deliberately introducing DNA into cells. It was observed that ,expression of ATM DNA in A-T cells enhanced the survival of these cells in response to radiation exposure, decreased radiation-induced chromosome breakage and partially corrected defective cell cycle checkpoints. This correction of the defects in A-T cells suggested future possible approaches in gene therapy against A-T. It is seen that gene augmentation would not be suitable for loss-of-function conditions in which irreversible damage has already been done but still it can be used to mitigate the symptoms of the disease as seen in above example.

The efficiency of various gene therapy protocols are being tested by the scientist in mice and simultaneously new gene therapy protocols are being formulated for A-T which would hopefully allow for stable, long-term production of the ATM protein in the body. However, gene therapy faces many challenges from ensuring that the therapeutic gene inserts safely into the genome to regulating when, where, and how much it is expressed.

Will future children be similarly affected?

A-T occurs at a frequency of about 1 in 40,000 children in the world population but note that the disease may be much more common, since some children with A-T are misdiagnosed or die before they are accurately diagnosed. It is inherited in an autosomal recessive manner. Parents of a A-T patient are both obligate heterozygote carriers of an ATM gene mutation. The carrier is an individual that carries one gene for a particular recessive trait, such that he does not express the trait but, when mated with another carrier, can produce offspring that do. The risk of each sibling of an affected individual being affected is ¼, risk of being an asymptomatic carrier is 1/2, and risk of being unaffected and not a carrier is ¼. Once an at-risk sibling is known to be unaffected, the risk of his or her being a carrier is 2/3.Each sibling of the patient’s parents is at a 50% risk of being a carrier. Most individuals with A-T do not reproduce. So there are very few chances of inheritance of the disease from the patients to his or her future generation.

Genetic counselling and genetic testing are particularly useful in patients with recessive diseases. In case of A-T, if one parent has ataxia and the status of the other parent is not known, genetic testing will determine if the unaffected parent is a carrier of the mutated gene. If the unaffected parent is not a carrier, then there is no chance of transmitting the disease to his or her children. Since results of the genetic tests may affect important life decisions such as whether to have children, Genetic counselling should always be done before the testing.

There are list of mutations that are routinely tested and which help to known which mutations are present in the person. But, genetic tests are available for only few types of hereditary ataxia. For example, in North African Jews, a direct test can be performed to identify the mutation in the ATM gene - C103T - by testing for this mutation alone.

In short, genetic testing can be helpful for people who do not yet have ataxia but are at risk due to family history.

Synthetic Life ... not yet but close

What is life or can we go other way round how you will describe a synthetic life. Creating artificial life is something that every fan of science fiction ponders as a terrifying curtain to be parted—terrifying and at the same time exciting for the potential of such science. If you’ve followed this story at all, or if you are only aware of it in passing, you might enjoy hearing more about the scientist himself, Craig Ventor.

He is called by various names Bad boy of science, Frankenstein Doc, brilliant biologist and shrewd entrepreneur etc. He had the audacity to challenge the US scientific establishment by engaging it in a much-hyped race to map the human genome. And he actually wins the contest time and again. J Craig Venter, Vietnam War veteran, can't seem to stay away from the limelight for long.

Previously, scientists have altered and manipulated DNA piecemeal to produce a variety of genetically engineered plants and animals. But the ability to artificially design an entire genome - the `book of life' that controls an organism's functions - puts a different spin on the meaning of terms such as creation, evolution and life. J Craig Ventor has delivered on a promise he made 15 years ago: to create the world's first synthetic life, a cell controlled by DNA built from scratch in the laboratory. But has he really created a so called synthetic life?

First, the form of life that was created was not new. What was essentially done was the re-creation of an existing bacterial form of life, except that it was given a prosthetic genome (synthesized in the laboratory), and except that the genome was put into the cytoplasm of a slightly different species. It should be emphasized that it will probably be very difficult to make very new forms of life. This is because even the simplest form of life is very complex, so it is very difficult to predict what will happen when you substantially change their genomes. Biologists are nowhere close to imitating what a living cell accomplishes with apparent ease. Venter’s lab essentially copied the code, borrowed existing parts, and depended completely on cell machinery. In effect, they plagiarized living cells.

Second, even if the synthetic genome was substantially different from any existing form of life, one might still object to calling this the creation of new life, because the synthetic cell was made by modifying an existing form of life. Almost all of the material in the synthetic cell comes from a previously existing form of life; only the genome is synthesized. In this respect, one might say that a synthetic cell qualifies as “new” life only if the whole cell is synthesized. If one copies the neighbor’s house, is it an artificial house?

Evolution created life – Venter made an extra copy.But I am not the only one getting worked up about semantics and usage here. Even back home, India's premier geneticist Dr P M Bhargava, who set up the Centre for Cellular and Molecular Biology (CCMB) in Hyderabad, was categorical that Venter, though a fine scientist, had this time around overstated his work's significance. "Venter's team took a cell, removed its DNA and replaced it with a host of other material. This can at best be called a massive bit of genetic engineering.

His words find an echo in the reaction of Dr Steen Rasmussen from the University of Southern Denmark. In a published statement, he said, "Implementing a synthetic genome in a modern cell is a significant milestone in understanding life today. However, the radical 'top down' genetic engineering that Venter's team has done, does not quite constitute a synthetic cell by my definition. Bottom-up researchers, like myself, aim to assemble life — including hardware and the programme — as simply as possible, even if the result is different from what we think of as life. Constructing life using different material and blueprints will teach us more about the nature of life than reproducing life as we know it."

But, one must, for all the sneering, congratulate the team that did this. It was difficult, industrial-scale synthesis. The effort plans to move on to much more purely invented genomes, stripped down and assignable to no specific natural species but equipped for novel jobs. This is a huge accomplishment because it has tremendous implications in genetic engineering. Scientists are already talking about introducing man-made DNA into a certain bacteria so that it be programmed to feed on algae to create biofuels The advance - reported in the journal Science - is, literally, living proof that designer microbes can be built for special jobs, such as producing pharmaceuticals or removing pollutants from water or air. It will undoubtedly take years before a profitable and beneficial organism can be created for widespread use, but I don’t think it’s an exaggeration to say that this technology has the capability to profoundly change the world for the better in the near future.