Saturday, June 18, 2011
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Tuesday, June 14, 2011
Single GFP-Expressing Cell Is Basis of Living Laser Device
It sounds like something out of a comic book or a science fiction movie -- a living laser -- but that is exactly what two investigators at the Wellman Center for Photomedicine at Massachusetts General Hospital have developed. In a report that will appear in the journal Nature Photonics and is receiving advance online release, Wellman researchers Malte Gather, PhD, and Seok Hyun Yun, PhD, describe how a single cell genetically engineered to express green fluorescent protein (GFP) can be used to amplify the light particles called photons into nanosecond-long pulses of laser light.
"Since they were first developed some 50 years ago, lasers have used synthetic materials such as crystals, dyes and purified gases as optical gain media, within which photon pulses are amplified as they bounces back and forth between two mirrors," says Yun, corresponding author of the report. "Ours is the first report of a successful biological laser based on a single, living cell."
Adds Gather, a research fellow and the paper's lead author, "Part of the motivation of this project was basic scientific curiosity. In addition to realizing that biological substances had not played a major role in lasers, we wondered whether there was a fundamental reason why laser light, as far as we know, does not occur in nature or if we could find a way to achieve lasing in biological substances or living organisms."
The investigators chose GFP for their exploration of those questions because the protein -- originally found in a species of jellyfish -- can be induced to emit light without the application of additional enzymes. Its properties are well understood, and there are established techniques to genetically program many organisms to express GFP. To determine the protein's potential for generating laser light, the researcher first assembled a device consisting of an inch-long cylinder, with mirrors at each end, filled with a solution of GFP in water. After first confirming that the GFP solution could amplify input energy into brief pulses of laser light, the researchers estimated the concentration of GFP required to produce the laser effect.
Using that information, their next step was to develop a line of mammalian cells expressing GFP at the required levels. The cellular laser was assembled by placing a single GFP-expressing cell -- with a diameter of from 15 to 20 millionths of a meter -- in a microcavity consisting of two highly reflective mirrors spaced 20 millionths of a meter apart. Not only did the cell-based device produce pulses of laser light as in the GFP solution experiment, the researchers also found that the spherical shape of the cell itself acted as a lens, refocusing the light and inducing emission of laser light at lower energy levels than required for the solution-based device. The cells used in the device survived the lasing process and were able to continue producing hundreds of pulses of laser light.
"While the individual laser pulses last for only a few nanoseconds, they are bright enough to be readily detected and appear to carry very useful information that may give us new ways to analyze the properties of large numbers of cells almost instantaneously," says Yun, who is an associate professor of Dermatology at Harvard Medical School. "And the ability to generate laser light from a biocompatible source placed inside a patient could be useful for photodynamic therapies, in which drugs are activated by the application of light, or novel forms of imaging."
Gather adds, "One of our long-term goals will be finding ways to bring optical communications and computing, currently done with inanimate electronic devices, into the realm of biotechnology. That could be particularly useful in projects requiring the interfacing of electronics with biological organisms. We also hope to be able to implant a structure equivalent to the mirrored chamber right into a cell, which would the next milestone in this research." The study was supported by grants from the National Science Foundation and the Korea National Research Foundation.
Journal Reference:
Malte C. Gather, Seok Hyun Yun. Single-cell biological lasers. Nature Photonics, 2011; DOI: 10.1038/nphoton.2011.99
"Since they were first developed some 50 years ago, lasers have used synthetic materials such as crystals, dyes and purified gases as optical gain media, within which photon pulses are amplified as they bounces back and forth between two mirrors," says Yun, corresponding author of the report. "Ours is the first report of a successful biological laser based on a single, living cell."
Adds Gather, a research fellow and the paper's lead author, "Part of the motivation of this project was basic scientific curiosity. In addition to realizing that biological substances had not played a major role in lasers, we wondered whether there was a fundamental reason why laser light, as far as we know, does not occur in nature or if we could find a way to achieve lasing in biological substances or living organisms."
The investigators chose GFP for their exploration of those questions because the protein -- originally found in a species of jellyfish -- can be induced to emit light without the application of additional enzymes. Its properties are well understood, and there are established techniques to genetically program many organisms to express GFP. To determine the protein's potential for generating laser light, the researcher first assembled a device consisting of an inch-long cylinder, with mirrors at each end, filled with a solution of GFP in water. After first confirming that the GFP solution could amplify input energy into brief pulses of laser light, the researchers estimated the concentration of GFP required to produce the laser effect.
Using that information, their next step was to develop a line of mammalian cells expressing GFP at the required levels. The cellular laser was assembled by placing a single GFP-expressing cell -- with a diameter of from 15 to 20 millionths of a meter -- in a microcavity consisting of two highly reflective mirrors spaced 20 millionths of a meter apart. Not only did the cell-based device produce pulses of laser light as in the GFP solution experiment, the researchers also found that the spherical shape of the cell itself acted as a lens, refocusing the light and inducing emission of laser light at lower energy levels than required for the solution-based device. The cells used in the device survived the lasing process and were able to continue producing hundreds of pulses of laser light.
"While the individual laser pulses last for only a few nanoseconds, they are bright enough to be readily detected and appear to carry very useful information that may give us new ways to analyze the properties of large numbers of cells almost instantaneously," says Yun, who is an associate professor of Dermatology at Harvard Medical School. "And the ability to generate laser light from a biocompatible source placed inside a patient could be useful for photodynamic therapies, in which drugs are activated by the application of light, or novel forms of imaging."
Gather adds, "One of our long-term goals will be finding ways to bring optical communications and computing, currently done with inanimate electronic devices, into the realm of biotechnology. That could be particularly useful in projects requiring the interfacing of electronics with biological organisms. We also hope to be able to implant a structure equivalent to the mirrored chamber right into a cell, which would the next milestone in this research." The study was supported by grants from the National Science Foundation and the Korea National Research Foundation.
Journal Reference:
Malte C. Gather, Seok Hyun Yun. Single-cell biological lasers. Nature Photonics, 2011; DOI: 10.1038/nphoton.2011.99
Life-History Traits of Extinct Species May Be Discoverable, Large-Scale DNA Sequencing Data Suggest
For the first time, scientists have used large-scale DNA sequencing data to investigate a long-standing evolutionary assumption: DNA mutation rates are influenced by a set of species-specific life-history traits. These traits include metabolic rate and the interval of time between an individual's birth and the birth of its offspring, known as generation time.
The team of researchers led by Kateryna Makova, a Penn State University associate professor of biology, and first author Melissa Wilson Sayres, a graduate student, used whole-genome sequence data to test life-history hypotheses for 32 mammalian species, including humans. For each species, they studied the mutation rate, estimated by the rate of substitutions in neutrally evolving DNA segments -- chunks of genetic material that are not subject to natural selection. They then correlated their estimations with several indicators of life history.
The results of the research will be published in the journal Evolution on June 13, 2011.
One of the many implications of this research is that life-history traits of extinct species now could be discoverable. "Correlations between life-history traits and mutation rates for existing species make it possible to develop a hypothesis in reverse for an ancient species for which we have genomic data, but no living individuals to observe as test subjects," Makova explained. "So, if we have information about how extant species' life history affects mutation rates, it becomes possible to make inferences about the life history of a species that has been extinct for even tens of thousands of years, simply by looking at the genomic data."
To find correlations between life history and mutation rates, the scientists first focused on generation time. "The expected relationship between generation time and mutation rate is quite simple and intuitive," Makova said. "The more generations a species has per unit of time, the more chances there are for something to go wrong; that is, for mutations or changes in the DNA sequence to occur." Makova explained that the difference between mice and humans could be used to illustrate how vastly generation time can vary from species to species. On the one hand, mice in the wild usually have their first litter at just six months of age, and thus their generation time is very short. Humans, on the other hand, have offspring when they are at least in their mid-teens or even in their twenties, and thus have a longer generation time. "If we do the math we see that, for mice, every 100 years equates to about 200 generations, whereas for humans, we end up with only five generations every 100 years," Makova said. After comparing 32 mammalian species, her team found that the strongest, most significant life-history indicator of mutation rate was, in fact, the average time between a species member's birth and the birth of its first offspring, accounting for a healthy 40% of mutation-rate variation among species.
Makova's team also found that generation time affects male mutation bias -- a higher rate of DNA mutation in the male sperm versus the female egg. "Females of a species are born with their entire lifetime supply of oocytes, or egg cells. These cells have to divide only once to become fertilizable," Makova explained. "However, males of a species produce sperm throughout their reproductive life, and, compared with egg cells, sperm cells undergo many more DNA replications -- many more chances for mutations to occur." Previous researchers had demonstrated a higher DNA mutation rate in mammalian males than in mammalian females, a phenomenon called male mutation bias. However, until now, no one had shown that generation time was the main determinant of this phenomenon.
The second life-history trait that Makova's team examined was metabolic rate -- the amount of energy expended by an animal daily -- and how it correlates with genetic mutations. Wilson Sayres explained that some of the team's 32 test species, such as shrews and rodents, fell into the high-metabolism category, while others, such as dolphins and elephants, fell into the low-metabolism category. Previous researchers had hypothesized that the higher the metabolic rate, the greater the number of mutations. "According to this idea, sperm cells should be more affected than egg cells by a higher metabolic rate," Wilson Sayres said. "A sperm cell is very active and constantly moving, and, in addition, its cell membrane is not very dense. But an egg cell basically sits there and does nothing, while being protected by a thicker membrane, much like a coat of armor." Wilson Sayres explained that the combination of high energy and meager protection leaves sperm cells more susceptible to bombardment by free radicals -- atoms or molecules with unpaired electrons -- and that these free radicals can increase mutations. "The hypothesis is that a high metabolism greatly increases this already volatile situation, especially for sperm; so, in our study, we expected stronger male mutation bias in organisms with high metabolic rate," Wilson Sayres said.
Makova's team found that, unlike generation time, metabolic rate appeared to be only a moderate predictor of mutation rates and of male mutation bias. "While this finding was not as significant as the generation-time result, I suspect that further studies may provide stronger evidence that metabolic rate exerts an important influence on mutation rates and male mutation bias," Makova said. She explained that the challenge is to disentangle metabolic rate as a separate factor from generation time. "The two factors strongly correlate with one another, so it's hard to get a clear fix on how metabolism might be acting independently of generation-time intervals."
Third, Makova and her team explored another life-history trait that other researchers had hypothesized might affect mutation rates -- sperm competition. "Sperm competition is just that -- the struggle between the sperm of different males to fertilize egg cells," Wilson Sayres said. "In a species such as the chimpanzee, where females mate with many different males during a given cycle, intense sperm competition results in large testicle size, and thus, high sperm production. But in a harem species such as the gorilla, where each female is basically exclusive to one male, sperm competition is much less relevant, and the result is small testicle size and low sperm production." Makova explained that sperm competition should, in theory, correlate positively with sperm mutation and thus a higher male mutation bias. "The more sperm that are produced, the more cell divisions are needed and the greater the chances are of mistakes during DNA copying, or replication," Makova said.
However, in the case of sperm competition, the results were surprising. "We did not find as strong an association between male mutation bias and sperm competition as other researchers had hypothesized, although we speculate that future studies might yield different results if the data on sperm competition are collected in different ways," Wilson Sayres explained.
In addition to Makova and Wilson Sayres, the other authors of the soon-to-be-published scientific paper include Chris Venditti of the University of Hull in the United Kingdom, and Mark Pagel of the University of Reading in the United Kingdom and the Santa Fe Institute in New Mexico. The research was funded by the National Institutes of Health and the National Science Foundation.
The team of researchers led by Kateryna Makova, a Penn State University associate professor of biology, and first author Melissa Wilson Sayres, a graduate student, used whole-genome sequence data to test life-history hypotheses for 32 mammalian species, including humans. For each species, they studied the mutation rate, estimated by the rate of substitutions in neutrally evolving DNA segments -- chunks of genetic material that are not subject to natural selection. They then correlated their estimations with several indicators of life history.
The results of the research will be published in the journal Evolution on June 13, 2011.
One of the many implications of this research is that life-history traits of extinct species now could be discoverable. "Correlations between life-history traits and mutation rates for existing species make it possible to develop a hypothesis in reverse for an ancient species for which we have genomic data, but no living individuals to observe as test subjects," Makova explained. "So, if we have information about how extant species' life history affects mutation rates, it becomes possible to make inferences about the life history of a species that has been extinct for even tens of thousands of years, simply by looking at the genomic data."
To find correlations between life history and mutation rates, the scientists first focused on generation time. "The expected relationship between generation time and mutation rate is quite simple and intuitive," Makova said. "The more generations a species has per unit of time, the more chances there are for something to go wrong; that is, for mutations or changes in the DNA sequence to occur." Makova explained that the difference between mice and humans could be used to illustrate how vastly generation time can vary from species to species. On the one hand, mice in the wild usually have their first litter at just six months of age, and thus their generation time is very short. Humans, on the other hand, have offspring when they are at least in their mid-teens or even in their twenties, and thus have a longer generation time. "If we do the math we see that, for mice, every 100 years equates to about 200 generations, whereas for humans, we end up with only five generations every 100 years," Makova said. After comparing 32 mammalian species, her team found that the strongest, most significant life-history indicator of mutation rate was, in fact, the average time between a species member's birth and the birth of its first offspring, accounting for a healthy 40% of mutation-rate variation among species.
Makova's team also found that generation time affects male mutation bias -- a higher rate of DNA mutation in the male sperm versus the female egg. "Females of a species are born with their entire lifetime supply of oocytes, or egg cells. These cells have to divide only once to become fertilizable," Makova explained. "However, males of a species produce sperm throughout their reproductive life, and, compared with egg cells, sperm cells undergo many more DNA replications -- many more chances for mutations to occur." Previous researchers had demonstrated a higher DNA mutation rate in mammalian males than in mammalian females, a phenomenon called male mutation bias. However, until now, no one had shown that generation time was the main determinant of this phenomenon.
The second life-history trait that Makova's team examined was metabolic rate -- the amount of energy expended by an animal daily -- and how it correlates with genetic mutations. Wilson Sayres explained that some of the team's 32 test species, such as shrews and rodents, fell into the high-metabolism category, while others, such as dolphins and elephants, fell into the low-metabolism category. Previous researchers had hypothesized that the higher the metabolic rate, the greater the number of mutations. "According to this idea, sperm cells should be more affected than egg cells by a higher metabolic rate," Wilson Sayres said. "A sperm cell is very active and constantly moving, and, in addition, its cell membrane is not very dense. But an egg cell basically sits there and does nothing, while being protected by a thicker membrane, much like a coat of armor." Wilson Sayres explained that the combination of high energy and meager protection leaves sperm cells more susceptible to bombardment by free radicals -- atoms or molecules with unpaired electrons -- and that these free radicals can increase mutations. "The hypothesis is that a high metabolism greatly increases this already volatile situation, especially for sperm; so, in our study, we expected stronger male mutation bias in organisms with high metabolic rate," Wilson Sayres said.
Makova's team found that, unlike generation time, metabolic rate appeared to be only a moderate predictor of mutation rates and of male mutation bias. "While this finding was not as significant as the generation-time result, I suspect that further studies may provide stronger evidence that metabolic rate exerts an important influence on mutation rates and male mutation bias," Makova said. She explained that the challenge is to disentangle metabolic rate as a separate factor from generation time. "The two factors strongly correlate with one another, so it's hard to get a clear fix on how metabolism might be acting independently of generation-time intervals."
Third, Makova and her team explored another life-history trait that other researchers had hypothesized might affect mutation rates -- sperm competition. "Sperm competition is just that -- the struggle between the sperm of different males to fertilize egg cells," Wilson Sayres said. "In a species such as the chimpanzee, where females mate with many different males during a given cycle, intense sperm competition results in large testicle size, and thus, high sperm production. But in a harem species such as the gorilla, where each female is basically exclusive to one male, sperm competition is much less relevant, and the result is small testicle size and low sperm production." Makova explained that sperm competition should, in theory, correlate positively with sperm mutation and thus a higher male mutation bias. "The more sperm that are produced, the more cell divisions are needed and the greater the chances are of mistakes during DNA copying, or replication," Makova said.
However, in the case of sperm competition, the results were surprising. "We did not find as strong an association between male mutation bias and sperm competition as other researchers had hypothesized, although we speculate that future studies might yield different results if the data on sperm competition are collected in different ways," Wilson Sayres explained.
In addition to Makova and Wilson Sayres, the other authors of the soon-to-be-published scientific paper include Chris Venditti of the University of Hull in the United Kingdom, and Mark Pagel of the University of Reading in the United Kingdom and the Santa Fe Institute in New Mexico. The research was funded by the National Institutes of Health and the National Science Foundation.
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