Wednesday, August 3, 2011

Making blood-sucking deadly for mosquitoes

Inhibiting a molecular process cells use to direct proteins to their proper destinations causes more than 90 percent of affected mosquitoes to die within 48 hours of blood feeding, a UA team of biochemists found.


Mosquitoes die soon after a blood meal if certain are experimentally disrupted, a team of biochemists at the University of Arizona has discovered.


The approach could be used as an additional strategy in the worldwide effort to curb mosquito-borne diseases like dengue fever, and malaria.


When the researchers blocked a cellular process known as vesicle transport, on which the rely to release into the gut among other functions, it caused the affected animals to die within two days of blood feeding.


"The idea behind our research is this: If we can kill the mosquito after she bites the first person, she won't be able to bite and infect a second," said Roger Miesfeld, a professor in the UA's department of chemistry and biochemistry, who led the research project.


"We do this by blocking the mosquito's ability to digest its blood meal," said Miesfeld, also a member of the UA's BIO5 Institute.


The research team's findings were recently published in , or PNAS.


"During a blood meal, a mosquito ingests its body weight in blood. It's the equivalent of a 125-pound human consuming a 12-gallon smoothie made from 25 pounds of hamburger meat plus a half pound of butter and two tablespoons of sugar," Miesfeld said.


Miesfeld and the research team had previously shown that the blood feeding process poses a huge metabolic challenge to the female mosquito.


"By disrupting any number of needed to fully utilize the blood meal, the mosquito has a very difficult time completing the cycle," he added.


To maintain their bodily needs, the insects rely on sugary nectar from flowers, but when the time to make eggs comes, they need large amounts of protein. Only female mosquitoes bite and feed on the blood of humans or warm-blooded animals.


If a mosquito finds enough victims to bite and avoids being squashed, it can live as long as three weeks. During that time, it may lay up to five clutches of more than 100 eggs each.


For their studies, the team used mosquitoes of the species Aedes aegypti, which originally hails from the sub-tropical and tropical regions of Africa. These mosquitoes are now found in many parts of the world and are particularly abundant in towns and cities where the climate is warm and water is plentiful. A. aegypti mosquitoes buzz about at dawn and dusk in search of their next blood meal, preferably from people's ankles.


A. aegypti mosquitoes are the main vector for dengue fever, now the most common viral disease transmitted by mosquitoes. Dengue fever has made a comeback especially in subtropical and tropical regions due to the geographical spread of the mosquitoes and the virus. Four strains, or serotypes, of disease-causing dengue viruses are known.


When infected for the first time, most people suffer through a bout of high and painful fever, but usually the illness is not life threatening and renders them immune to that particular strain of dengue virus. However, a subsequent infection with any of the other dengue virus strains often triggers an all-out immune response that leads to a much more severe form, called dengue hemorrhagic fever, which can be fatal.


Miesfeld said most mosquito-borne pathogens are not passed down from the female mosquito to her offspring, but instead picked up by the mosquitoes when they bite an infected human.


In the case of A. aegypti mosquitoes, the pathogen is often dengue or yellow fever viruses, whereas the Anopheles gambiae mosquito, which is found in many parts of Africa, transmits the more deadly malaria parasite. Miesfeld further explained that malaria and dengue pathogens take about 10 to 12 days to complete their life cycle within the mosquito before they can be transmitted to humans through blood feeding.


 

"The most dangerous mosquitoes are the older ones," Miesfeld said.

His team used a technique called RNAi to specifically target genes that are required for the digestion process. The researchers homed in on a protein complex called COPI, which stands for coatomer protein 1.


COPI consists of several subunits that together make up the envelope of the vesicles on which the cell relies for internal transport and for secretion of enzymes into the gut.


When a female mosquito takes a blood meal, the cells lining its gut secrete enzymes to break down the blood proteins. The secretion process involves packaging the enzymes in small droplets called vesicles that the cells then release into the gut.


"We thought, 'Why don't we knock out the whole process that allows the proteases to be secreted?' That's where we got this amazing result," Miesfeld said. "Not only did we eliminate her ability to secrete anything, we were surprised to find that about 90 percent of those mosquitoes died within two days after feeding on blood."


The COPI RNAi does not have an adverse effect on the female mosquitoes for 10 days – unless they decide to take a blood meal.


"When she does, all hell starts breaking loose, biochemically and anatomically speaking," Miesfeld said.


"What we think is happening is that if there is protein in her gut, it induces the secretory machinery. It initiates this huge secretion process but it's defective and causes cells to disintegrate," he added. "The whole lining of the gut starts to fall apart, allowing the blood to seep into her body."


In looking at the potential causes, Miesfeld said his team found that removing any one of the COPI subunits causes the whole complex to fall apart.


"Based on what we know about the COPI system, it shouldn't have that strong of an effect," he said.


"As scientists have been knocking out COPI to learn more about its function over the past couple of years, they have achieved some interesting results," Miesfeld added. "Together with our findings they suggest that COPI does a lot more than what people thought."


Miesfeld envisions that the ultimate goal of this research is to develop a small molecule that works in place of injected RNAi and acts as a specific inhibitor of the secretion process. 


For this to be an effective mosquito-selective insecticide, it must not have any effect on humans.


The simplest use would be to soak it into mosquito nets like currently available insecticides that target the mosquito's nervous system. A slightly more complex strategy would be to include it in a pill that humans take, so the mosquitoes pick up the inhibitor drug when they bite. As part of this strategy, the researchers are looking for genes that are unique to the mosquito and could serve as targets without affecting human health.


To explain, Miesfeld said to imagine a village in the tropics during a rainy season.


"As the mosquitoes hatch in large numbers, the whole population of villagers is ready," he explained. "As soon as the insects start biting, they take up the inhibitor and before they can bite again, they die in large numbers. Over a few seasons, that can make a difference."


Miesfeld added it is unlikely there would ever be a silver bullet eliminating mosquito-transmitted diseases like malaria and dengue fever altogether.


"One potential issue with our strategy is genetic changes rendering the mosquitoes immune over time," Miesfeld said. "Many approaches from different angles will be necessary, and ours could be another tool in the toolbox."


Provided by University of Arizona (news : web)

Quick test diagnoses bacterial or viral infection

Researchers at Ben-Gurion University of the Negev (BGU) have developed a new test that quickly and accurately distinguishes between bacterial and viral infections in as little as five hours.

Treating viral infections with antibiotics is ineffective and contributes to the development of , allergic reactions, toxicity and greater healthcare costs. Currently tests take 24-48 hours and aren't always accurate enough for a clear-cut diagnosis. Doctors often prescribe antibiotics to provide patient relief before the test comes back, without waiting for the results.

According to a study published in the Journal of Analytical Chemistry, the BGU group has shown it is possible to distinguish a patient's infection as either viral or bacterial by adding luminol to a blood sample and measuring the glow. Luminol is a luminescent used in to locate traces of blood.

BGU's study clearly indicated that that protect the body (phagocytes) react differently to viral and bacterial infections and that the glow or "chemiluminescence" (CL) can detect those distinct reactions.

According to the study, "The method is timesaving, easy to perform and can be commercially available, thus, having predictive diagnostic value and could be implemented in various medical institutions."

In the study, 69 patients admitted to Soroka University Medical Center in Beer-Sheva with various types of infections. Rather than looking at the infection, they looked at the immune system's response to the infection.

A multi-disciplinary team, headed by Prof. Robert Marks, of the Department of Biotechnology Engineering and the National Institute for Biotechnology in the Negev (NIBN) made the discovery. Team member and doctoral student Daria Prilutsky undertook the project as part of her Interdisciplinary Technologies Fellowship from the Planning and Budgeting Committee of the Council for Higher Education.

"This is a terrific example of the multi-disciplinary approach at BGU that results in innovative research and yields results that can have a worldwide impact," explains Doron Krakow, executive vice president of American Associates, Ben Gurion University of the Negev. "A test of this type has significant implications for cutting healthcare costs, and providing more accurate treatment."

Provided by American Associates, Ben-Gurion University of the Negev (news : web)

Controlling movements with light

German researchers at the Ruhr-Universitaet have succeeded in controlling the activity of certain nerve cells using light, thus influencing the movements of mice. By changing special receptors in nerve cells of the cerebellum such that they can be activated and deactivated by light, the researchers have shown that the signaling pathways, which are activated by the receptors play a crucial role in controlling movement.

Unlike conventional methods, with the so-called optogenetics, the researchers are able to target one cell type. "We are now going to use this method to find out exactly what goes wrong in the nerve cells in movement disorders such as ataxias", said Prof. Dr. Stefan Herlitze (RUB Department for Biology and Biotechnology). The results are reported in the .

The Bochum team examined a specific signalling pathway that is controlled by a so-called G-protein-coupled receptor. This is important for the modulation of activity in complex . Disturbances of the function can, for example, have an effect on emotional and motor behaviours. "We know that the activity pattern of the Purkinje cells in the cerebellum is crucial for the coordination of movements", Herlitze explained. "It is unclear, however, what contribution is made by the individual ." In conventional studies, researchers use drugs that inhibit or stimulate specific proteins in to investigate the contribution of these proteins to the activity of the cells. However, Herlitze's team was interested in a (G-protein-coupled receptor) which occurs in various cell types. Had the researchers administered a drug, they would not only have deactivated the receptor in the Purkinje cells, but in all cell types in which it occurs. The drug method therefore makes it impossible to observe the contribution of the receptor in the Purkinje cells in isolation.

Optogenetics: replacing drugs with light

To avoid this problem, Herlitze's team replaced the drugs with proteins that are activated by light. Using genetic methods, the researchers integrated rhodopsin, the light-sensitive protein of the eye, into the Purkinje cells of mice. They also implanted a laser probe in the , with which they illuminated the rhodopsin. The light-activated rhodopsin then activated the G-protein-coupled receptor in the Purkinje cells, while the same receptors in other cell types remained inactive. The RUB Department of General Zoology and Neurobiology has been instrumental in establishing this method worldwide.

Investigated receptor is crucial for movement control

The researchers found that activation of the G-protein-coupled receptor changed the activity pattern of the Purkinje cells. Herlitze's team had to expose the rhodopsin to light for several seconds to achieve these effects. A twenty to thirty percent reduction in cell activity was sufficient to induce visible motor deficits in the behaviour of the mice, such as impaired balance or coordination problems. "We were able to demonstrate for the first time that the modulation of a specific G-protein-coupled receptor in the Purkinje cells is of crucial importance for the control and coordination of movement", summed up Herlitze.

More information: Gutierrez, D.V., Mark, M.D., Masseck, O., Maejima, T., Kuckelsberg, D., Hyde, R.A., Krause, M., Kruse, W., Herlitze, S. Optogenetic control of motor coordination by Gi/o protein-coupled vertebrate rhodopsin in cerebellar Purkinje cells. J. Biol. Chem., doi: 10.1074/jbc.M111.25367 (2011)

Provided by Ruhr-University Bochum

Modified genetic alphabet: Chemical evolution generates bacterial strain with artificial nucleotide in its genome

 Evolution is based on heredity, changes to the genetic material (mutation), and the natural selection of those organisms that are best suited to the given environmental conditions. An international team led by Rupert Mutzel at the Freie Universit├Ąt of Berlin has now successfully emulated one particular evolutionary process in the laboratory. As the researchers report in the journal Angewandte Chemie, they were able to generate a bacterial strain whose genetic material contains an artificial building block in place of a natural one. Their success results from a special automated cultivation technique.


DNA, the carrier of the genetic information of all cells, is based on a code consisting of four “letters”, the bases adenine, cytosine, guanine, and thymine. Thanks to their new artificial evolution process, the scientists have now been able to grow bacteria in which the thymine of DNA has been replaced with an analogue, the base 5-chlorouracil. This synthetic component is poisonous to other .


The researchers started with a genetically modified strain of the bacterium Escherichia coli that is no longer capable of producing thymine. These microorganisms were cultivated over many generations in the presence of increasing amounts of chlorouracil in a specially built apparatus. Whenever the size of the population sank below a certain level, the bacteria were given a brief dose of a chlorouracil-free, thymine-containing medium to give them a chance to recover. The concentration of chlorouracil was automatically increased whenever genetic variants of the bacteria that better tolerated this substance were produced.


In this way, the cells were always exposed to a quantity of chlorouracil that was just barely tolerable. After about 1000 generations, the microorganisms had adapted to the altered , that is, the presence of chlorouracil instead of thymine. They were able to build up their DNA with chlorouracil in place of thymine. Analysis of the genome showed that the process of adaptation resulted in many changes to the of the bacteria.


“Our results demonstrate the success of our evolutionary cultivation strategy,” says Mutzel. “In this way it should be possible to develop microorganisms that can convert chemical intermediates to pharmaceuticals or break down environmental pollutants.” Microorganisms that have DNA with synthetic building blocks may also be useful in hindering the spread of purposely or accidentally released modified cells in the environment. Such microorganisms would also be incapable of exchanging genes with their natural relatives.


More information: Rupert Mutzel, Chemical Evolution of a Bacterium's Genome, Angewandte Chemie International Edition 2011, 50, No. 31, 7109–7114, Permalink to the article: http://dx.doi.org/ … ie.201100535


Provided by Wiley (news : web)