Pulsating Mechanism as a Stress Response

Genomes are not merely consisting of A,T, G and C. There is so lot more to them than we know. A comparatively simpler genome of a bacteria like Bacillus subtilis can respond to a wide array of changes in its environment. Our knowledge of the exact mechanism is not complete as yet since there is a need for a better system of studying gene-protein interaction. Take, for instance the stress response that bacteria take in cold. While we resort to heaters for tackling the cold, the bacteria does the same! This is what has been reported recently by researchers at Caltech.

Fig: Florescent proteins are used to study the pulsating mechanism.

Previously we assumed that in response to stress, bacteria generally go to a dormant state-meaning that they shift from one state to another. However, new studies shows an active utilization of the existing system to tackle the stress.

What the researchers say?

Researchers at the California Institute of Technology (Caltech) are finding that cells can respond using a new kind of pulsating mechanism, instead of just shifting from one steady state to another and staying there. The principles behind this process are surprisingly simple, the researchers say, and could drive other cellular processes, revealing more about how the cells—and ultimately life—work.

The Experiment

In their experiment, the researchers studied how a bacterial species called B. subtilis responds to a stressful environment—for example, one without food. In such conditions, the single-celled organism activates a large set of genes that help it deal with hardship, by aiding cell repair for instance. Previously, biologists had thought the bacteria would handle stress by turning on the relevant genes and simply leaving them on until the stress goes away.

Instead, the researchers found that B. subtilis continuously flips these genes on and off. When faced with more stress, it increases the frequency of these pulses. The pulsating action is like switching your heater on full blast for a brief period every few minutes, and turning it on and off more frequently if you want the house to be warmer.

The Underlying Mechanism: Genetic Circuit

To make their finding, the researchers introduced a chemical to B. subtilis that inhibits the production of ATP, the energy-carrying molecules of cells. The team found that the stress induced by this chemical triggers interactions within a set of genes—collectively called a genetic circuit. This circuit, which contains a set of positive and negative feedback loops, generates sustained pulses of activity in a key regulatory protein called σB  (“sigma B”). The researchers attached fluorescent proteins to the circuit, causing the cells to glow green when σB was activated. By making movies of the flashing cells, the team could then study the dynamics of the circuit.

The key to this pulsating mechanism is the variability inherent in how proteins are made, the researchers say. The number of copies of any specific protein in a given cell fluctuates over time. The bacterial gene circuit amplifies these molecular fluctuations, also called noise, to generate discrete pulses of σB activation. The stress also activates another key protein that modulates the pulse frequencies.

More on the Genetic Circuit

By turning a steady input (the stress) into an oscillating output (the activation of σB) the genetic circuit is analogous to an electrical inverter, a device that converts direct current (DC) into alternating current (AC), explains Michael Elowitz, professor of biology and bioengineering at Caltech, Howard Hughes Medical Institute investigator, and coauthor of the paper. “You might think you need some kind of elaborate circuitry to implement that, but the cell can do it with just a few proteins, and by taking advantage of noise.”

This work provides a blueprint for how relatively simple genetic circuits can generate complex and dynamic behaviors in individual cells, the researchers say. “We’re excited to think that similar mechanisms may occur in other cellular processes,” Locke says. “It’d be interesting in the future to see which aspects of this circuit architecture also appear in more complex systems, such as mammalian cells.”

The paper has been titled “Stochastic pulse regulation in bacterial stress response” and was published in Science in October. (http://www.ncbi.nlm.nih.gov/pubmed/21979936)

-Caltech Media Relations

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Micropyramids to capture living cells

While pyramids are generally considered to be a grandeur construction, they are being used on micro level to capture live cells. The underlying principle is that since these micro pyramids have open walls, it is easier to living cells to interact-and easier for us to study them. This has been made possible through 3D Nano Fabrication.

Scientists of the research institutes MESA+ and MIRA of the University of Twente in The Netherlands present this new technology and first applications in the journal Small.

Most cell studies take place in 2-D: this is not a natural situation, because cells organize themselves in differently in the human body. If you give the cells room to move in three dimensions, the set-up is closer to what we find in nature. This is possible in the ‘open pyramids’ fabricated in the NanoLab of the MESA+ Institute for Nanotechnology of the University of Twente.

How is the pyramid being used?

If you join a number of flat silicon surfaces to form a sharp corner, it is possible to deposit another material on them. After having removed the the bulk of the material, however, a small amount of material remains in the corner. This tiny tip can be used for an Atomic Force Microscope, or, in this case, for forming a micro pyramid.

How are the cells captured?

In cooperation with UT’s MIRA Institute for Biomedical Technology and Technical Medicine, the nanoscientists have explored the possibilities of applying the pyramids as ‘cages’ for cells. First experiments with polystyrene balls worked out well. The next experiments involved capturing chondrocytes, cells forming cartilage. Moved by capillary fluid flow, these cells automatically ‘fall’ into the pyramid through a hole at the bottom. Soon after they settle in their 3-D cage, cells begin to interact with cells in adjacent pyramids. Changes in the phenotype of the cell can now be studied in a better way than in the usual 2-D situation. It is therefore a promising tool to be used in tissue regeneration research.

Possible Extension of the Idea

The Dutch scientists expect to develop extensions to this technology: the edges of the pyramid can be made hollow and function as fluid channels. Between the pyramids, it is also possible to create nanofluidic channels, which could be used to feed the cells.

Article: Erwin J. W. Berenschot, Narges Burouni, Bart Schurink, Joost W. van Honschoten, Remco G. P. Sanders, Roman Truckenmuller, Henri V. Jansen, Miko C. Elwenspoek, Aart A. van Apeldoorn, Niels R. Tas. 3D Nanofabrication of Fluidic Components by Corner LithographySmall, 2012


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The importance of invariant natural killer cells

Malaghan Institute of Medical Research has made a translational breakthrough. A study done there has revealed that boosting the activity of a rare type of immune cell could be a better method to vaccinate patients who have chronic lymphocytic leukaemia (CLL).

About CLL:

CLL is the most common blood cancer across the world and in New Zealand too. The prevalence of CLL increases with age, reaching 1 in 400 in individuals over 70 years old. Although many people with CLL never need treatment, a significant number of patients are diagnosed at a young age or have aggressive disease, exhausting conventional therapies.

Available Treatments:
Haematologist Dr Robert Weinkove says that bone marrow transplantation is the only curative treatment for CLL and involves replacing the immune system of patients with that of a matched donor.

“Part of the reason that bone marrow transplants work is that the new (donor) immune system recognises the leukaemia cells as foreign and destroys them,” says Dr Weinkove. “This is a good demonstration of how immune therapies can successfully cure established cancers in humans.”

Disadvantage with transplants:
Bone marrow transplants are not without their problems however. Not all patients find a donor; patients are prone to infections for months or even years afterwards; and the treatment itself can be so toxic that it is not suitable for many patients.

Invariant Natural Killer T Cells:
“To identify more targeted, low risk immune therapies, we focused on a rare type of immune cell called invariant natural killer T (iNKT) cells,” says Dr Weinkove.

Previous research at the Malaghan Institute and overseas has shown that iNKT cells can be activated by a compound called α-galactosylceramide (α-GalCer), which was first found in a Japanese marine sponge. This leads to significantly enhanced tumour-specific immune responses.

While iNKT cells are promising targets for immunotherapies, in many cancer patients iNKT cell numbers are either reduced, or the cells do not work properly. Since iNKT cells had never been characterized in patients with CLL before, Dr Weinkove launched a collaborative study between the Wellington Hospital Blood and Cancer Centre and the Malaghan Institute, to determine the number, phenotype and function of iNKT cells in people with this form of leukaemia.

Between 2008 and 2011, Dr Weinkove collected blood samples from 40 patients with CLL and from 30 healthy volunteers of a similar age, from the greater Wellington region. He then undertook a series of laboratory tests to compare the number and function of the iNKT cells from these individuals.

This study, which has recently been published in the open-access, international scientific journal Haematologica, constitutes the first comprehensive investigation of iNKT cell numbers and function in patients with CLL.

“We found that we could detect and isolate iNKT cells from individuals with CLL, and that these cells were able to respond to α-GalCer,” says Dr Weinkove. “This is important because it suggests that iNKT cells remain functional in these patients, and that targeting them with treatments like α-GalCer might be a way of enhancing their ability to drive anti-cancer immune responses.”

The Next Step:

Having shown such great promise in the laboratory, the next step will be to see if these results can be replicated in patients.

“Designing and running safe clinical trials is a major undertaking, but we are exploring a number of ideas, including the possibility of giving α-GalCer to patients with blood cancers to boost their immune responses,” says Dr Weinkove.

This work complements the dendritic cell cancer vaccination programme at the Malaghan Institute.

This research was supported by grants from the Leukaemia & Blood Foundation, Genesis Oncology Trust and NZ Lottery Grants Board. Dr Weinkove also received support from a Genzyme New Investigators Scholarship through the Haematology Society of Australia and New Zealand.

Download Paper:

The paper can be downloaded from here: InVariant Natural Killer Cells


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3D Device for Parkinson and Epilepsy

Nanotechnology has certainly taken a boost during the last few years and now he have a new tool for neuroscientists delivers a thousand pinpricks of light to a chunk of gray matter smaller than a sugar cube. This device is generally fiber optic based and has been created by biologists and engineers at the Massachusetts Institute of Technology (MIT) in Cambridge. The good thing about it is that it is the first tool that can deliver precise points of light to a 3-D section of living brain tissue. The work is a step forward for a relatively new but promising technique that uses gene therapy to turn individual brain cells on and off with light.

Scientists can use the new 3-D “light switch” to better understand how the brain works. It might also be used one day to create neural prostheses that could treat conditions such as Parkinson’s disease and epilepsy. The researchers describe their device in a paper published November 19 in the Optical Society’s (OSA) journalOptics Letters.

The technique of manipulating neurons with light is only a few years old, but the authors estimate that thousands of scientists are already using this technology, called optogenetics, to study the brain. In optogenetics, researchers first sensitize select cells in the brain to a particular color of light. Then, by illuminating precise areas of the brain, they are able to selectively activate or deactivate the individual neurons that have been sensitized.

Ed Boyden, a synthetic biologist at MIT and co-lead researcher on the paper, is a pioneer of this emerging field, which he says offers the ability to probe connections in the brain.

“You can see neural activity in the brain that is associated with specific behaviors,” Boyden says, “but is it important? Or is it a passive copy of important activity located elsewhere in the brain? There’s no way to know for sure if you just watch.” Optogenetics allows scientists to play a more active role in probing the brain’s connections, to fire up one type of cell or deactivate another and then observe the effect on a behavior, such as quieting a seizure.

Unlike the previous, 1-D versions of this light-emitting device, the new tool delivers light to the brain in three dimensions, opening the potential to explore entire circuits within the brain. So far, the 3-D version has been tested in mice, although Boyden and colleagues have used earlier optogenetic technologies with non-human primates as well.

Targeting neurons with light

One of the advantages of optogenetics is that this technology allows scientists to focus on one particular type of neuron without affecting other types of neurons in the same area of cortex. Probes that deliver electricity to the brain can manipulate neurons, but they cannot target individual kinds of cell, Boyden says. Drugs can turn neurons on or off as well, he continues, but not on such a quick time scale or with such a high degree of control. In contrast, the new 3-D array is precise enough to activate a single kind of neuron, at a precise location, with a single beam of light.

In an earlier incarnation, Boyden’s device looked like a needle-thin probe with light-emitting ports along its length; this setup allowed scientists to manipulate neurons along a single line. The new tool contains up to a hundred of these probes in a square grid, which makes the device look like a series of fine-toothed combs laid next to each other with their teeth pointing in the same direction.

Each probe is just 150 microns across — a little thicker than a human hair, and thin enough so that the device can be implanted at any depth in the cortex without damaging it. The brain lacks pain receptors, so the implants do not cause any discomfort to the brain itself. As in the earlier model, several light-emitting ports are located along the length of each probe. Scientists can illuminate and change the color of each light port independently from the others.

Adding a third dimension to the probe’s light-delivery capabilities has allowed researchers to make any pattern of light they want within the volume of a cubic centimeter of brain tissue, using a few hundred independently controllable illumination points.

“It’s turning out to be a very powerful and convenient tool,” says MIT professor of electrical engineering Clifton Fonstad, co-lead author of the paper.

Blue for on, yellow for off

Neurons in the brain are not naturally responsive to light, so scientists sensitize these cells with molecules called opsins, light-detecting proteins naturally found in algae and bacteria. Genes for an opsin are transferred to the neurons in a mouse’s brain using gene therapy, a process in which DNA is ferried into a cell via a carrier such as a harmless virus. The carrier can be instructed to deliver the DNA package only to certain types of cells.

Different colors of light turn different flavors of opsin on — blue might cause one opsin to activate a cell, while yellow might cause another opsin to silence it. Neurons that are sensitized with opsins gain these abilities to respond to light.

The response of an individual neuron — whether to turn on or turn off — depends on the type of opsin it was sensitized with, and the color of light used to illuminate it. In this way, the tool gives neuroscientists an unprecedented level of control over individual neurons in the brain.

Teams from around the world are currently using the technology developed by Boyden’s group to study some of the most profound questions neuroscience tries to answer, such as how memory works, the connections between memory and emotion, and the difference between being awake and being asleep.

“I’m really excited about how the brain computes — the ebb and flow of consciousness,” Boyden says. “We know so little about the brain.”

A better understanding of the brain may lead to another benefit of this technology: therapy. If a particular type of cell malfunctions in a particular disease, scientists may be able to use a modified 3-D array as a neural prosthesis that could help to treat neurological conditions. Using light to stop overactive cells from firing might alleviate the uncontrollable muscle action of Parkinson’s disease. Cells that cause seizures in the brain could be quieted optically without the side effects of anti-seizure medications. Implants that correct hearing deficiencies are also being explored with this technology.

Although the new device is effective in bringing light to the brain, other challenges remain before optogenetics can be used for medical therapy, Boyden says. Scientists do not yet know for certain whether the body will detect the opsin proteins as foreign molecules and reject them. Gene therapy will also have to prove itself if neurons are to be sensitized with opsin effectively.

“It’s a long road,” Boyden admits.

Meanwhile, he continues, the demand for the tool is currently higher than his team can supply. Boyden says his group is excited about the possibility of commercializing the new 3-D array, as one potential route that would make the devices available as quickly as possible to the neuroscience community.


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Breakthrough against Cotton Curl Leaf Virus

Breakthrough against Cotton Curl Leaf Virus

Cotton leaf curl virus (CLCV) can now be cured with the help of biotechnology as a major breakthrough has been made in transferring modified genes to local varieties, said official sources here on Friday.

A team of local scientists in collaboration with Canadian experts have developed a CLCV-resistant cotton plant through transgenic RNA interference (RNAi) technology. The new advancement would help neutralise the mutating strain of CLCV, which is also know as Burewala stain. It is a harsh reality that we do not have a remedy for the deadly strain of CLCV. This virus alone has become a potent threat to damaging cotton in several districts of Punjab.

The control of this disease can add billions of rupees to the national economy every year and help the local textile industries to attain abundant raw materials. CLCV has caused losses to almost two to three million cotton bales, which amounts to Rs200-300 billion.

RNAi is a method of blocking gene function by inserting short sequences of ribonucleic acid that match part of the target gene’s sequence. It has been hailed as ‘breakthrough of the year’ and ‘Biotech’s billion dollar breakthrough’ globally. Initially, a team of local scientists successfully used the RNAi system for curing virus-hit tobacco plants. After witnessing favorable results, they replicated this process in cotton plants and have been greatly encouraged to see virus-free cotton plants during trials.

Progress in developing CLCV plants has been verified by grafting the transgenic RNAi plants on severely CLCV infected plants. By using RNAi, the virus-affected cotton crops can be fully saved from this onslaught.

“RNAi technology has efficiently blocked viral multiplication when the virus was either agro-inoculated in transgenic plants or transmitted by whitefly, which is a vector of CLCV,” said an official.

Currently, work on this approach is being done jointly by the Institute of Agricultural Sciences and National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore and University of Toronto, Canada, under the umbrella of Punjab Agriculture Research Board (PARB). It is hoped that new virus-resistant GM cotton seeds would be available in the market in three years.

Commenting on the progress achieved so far, Dr Mubarik Ali, chief executive of PARB, said: “PARB has always done result-oriented work by undertaking need-based research projects.”

He added that the use of RNAi technology would surely increase crop productivity and save billions of rupees.

He congratulated Dr Idrees Ahmad Nasir and Dr Saleem Haider, both from Punjab University, for developing what he called miracle CLCV-resistant cotton plant.

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The intelligence of slime molds

The intelligent habits of  P. polycephalum

It is quite interesting to note that we classify molds in the category of Protists. While we may not understand much about them, they understand a whole lot better than us.

This inference comes from a knack of  P. polycephalum to be able to find the best pathway for consuming its food and can move through the most complex of mazes in order to get to what it wants.

If one were to observe the morphology of this organism, they would find that they live in a colony form-and can be generally described as cell with a million nuclei and enzymes along with its genetic material. One of the most interesting feature about this mold is that it has a cell which is termed as the “master shape shifter”. It modifies itself as per the location it is in. In the forest it might fatten itself into giant yellow globs or remain as unassuming as a smear of mustard on the underside of a leaf; in the lab, confined to a petri dish, it usually spreads itself thin across the agar, branching like coral.

In one interesting experiment that was performed by Toshiyuki Nakagaki of Hokkadio University, they were able to demonstrate that these molds were indeed intelligent. What the did was that they cut the mold in pieces and placed it in a maze. The mold began to grow and was able to complete the entire maze within no time. The most interesting thing to note was that the slime mold was able to figure out which route to the food was shortest.

While the aforementioned experiment was done in 2000, a newer set of observations have been made by Chris Reid of University of Sydney.

They findings are quite amazing and the level of sophistication of this slime mold is awe inspiring. Reid and his teammates noticed that a foraging slime mold avoids sticky areas where it has already traveled. This extracellular slime, Reid reasoned, is a kind of externalized spatial memory that reminds polycephalum to explore somewhere new.

Usage of Slime

To test this idea, Reid and his colleagues placed slime molds in a petri dish behind a U-shaped barrier that blocked a direct route to a piece of food. Because the barrier was made of dry acetate, the slime molds could not stick to it and climb over it; instead, they had to follow the contours of the U toward the food. Ultimately, 23 of 24 slime molds reached the goal. But when Reid coated the rest of the petri dish in extracellular slime before introducing the slime molds, only eight of 24 found the food. All that preexisting slime confused the slime molds, preventing them from marking different areas as explored or unexplored. Reid thinks that a polycephalum in a labyrinth is similarly dependent on its slime, using it to first map the entire maze and then to remember which corridors are dead-ends.

Recreating transportation networks

If this experiment was not enough, slime molds literally re-created the Tokyo railway network in miniature! When researchers placed oat flakes or other bits of food in the same positions as big cities and urban areas, slime molds first engulfed the entirety of the edible maps. Within a matter of days, however, the protists thinned themselves away, leaving behind interconnected branches of slime that linked the pieces of food in almost exactly the same way that man-made roads and rail lines connect major hubs in Tokyo, Europe and Canada.

Slime Molds can think spatially too!

In addition to this other experiments suggests that slime molds navigate time as well as space, using a rudimentary internal clock to anticipate and prepare for future changes in their environments. Tetsu Saigusa of Hokkaido University and his colleagues—including Nakagaki—placed a polycephalum in a kind of groove in an agar plate stored in a warm and moist environment (slime molds thrive in high humidity). The slime mold crawled along the groove. Every 30 minutes, however, the scientists suddenly dropped the temperature and decreased the humidity, subjecting the polycephalumto unfavorably dry conditions. The slime mold instinctively began to crawl more slowly, saving its energy. After a few trials, Saigusa and his colleagues stopped changing the slime mold’s environment, but every 30 minutes the amoeba’s pace slowed anyway. Eventually it stopped slowing down spontaneously. Slime molds did the same thing at intervals of 60 and 90 minutes, although, on average, only about half of the slime molds tested showed spontaneous slowing in the absence of an environmental change.

The mechanism unraveled

Because the slime mold cannot rely on its slime for this trick, Saigusa speculates that it instead depends on an internal mechanism of some kind, perhaps involving the perpetually pulsating gelatinous contents of its one cell, known as cytoplasm. The slime mold’s membrane rhythmically constricts and relaxes, keeping the cytoplasm within flowing. When the amoeba’s membrane encounters food, it pulsates more quickly and expands, allowing more cytoplasm to flow into that region; when it stumbles onto something aversive—such as bright light—its palpitations slow down and cytoplasm moves elsewhere. Somehow, the slime mold may be keeping track of its own rhythmic pulsing, creating a kind of simple clock that would allow it to anticipate future events.


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Soldier Beetle presents beneficial potential

Biotech Opportunity from Soldier Beetle

CSIRO researchers, and a colleague at Sweden’s Karolinska Institute, published details of the gene identification breakthrough and potential applications recently in the international journal Nature Communications. “For the first time, our team has been able to isolate and replicate the three genes that combine to make the potent fatty acid that soldier beetles secrete to ward off predators and infection,” said CSIRO Ecosystem Sciences research leader Dr Victoria Haritos. “This discovery is important because it opens a new way for the unusual fatty acid to be synthesized for potential antibiotic, anti-cancer, or other industrial purposes,” Dr Haritos said.


Figure: A close up view of the secreted defensive fluid containing DHMA.

Soldier beetles exude a white viscous fluid from their glands to repel potential attacks from predators, as well as in a wax form to protect against infection. The team found this fluid contains an exotic fatty acid called dihydromatricaria acid, or DHMA, which is one of a group called polyynes that have known anti-microbial and anti-cancer properties. While DHMA and similar polyyne fatty acids are found in a wide variety of plants, fungi, liverworts, mosses, marine sponges and algae, these compounds have proved very difficult to manufacture using conventional chemical processes. However, Dr Haritos and her team have developed a way to achieve this. “We have outlined a method for reproducing these polyyne chemicals in living organisms like yeast, using mild conditions” Dr Haritos said. Soldier beetles are the only animals reported to contain DHMA. This, together with the observation that the beetles forage on plants (such as daisies) which contain a lot of these types of fatty acids, led to previous incorrect conclusions that the DHMA in soldier beetles was derived from their diet. “Through our research and the gene differences we have discovered, we now know soldier beetles have evolved this same defensive compound entirely independently of its production in plants and fungi,” Dr Haritos said.

Journal reference: Nature Communications


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