Electrophoresis may be the main technique for molecular separation in today's cell biology laboratory. Because it is such a powerful technique, and yet reasonably easy and inexpensive, it has become commonplace. In spite of the many physical arrangments for the apparatus, and regardless of the medium through which molecules are allowed to migrate, all electrophoretic separations depend upon the charge distribution of the molecules being separated. 1
Electrophoresis can be one dimensional (i.e. one plane of separation) or two dimensional. One dimensional electrophoresis is used for most routine protein and nucleic acid separations. Two dimensional separation of proteins is used for finger printing , and when properly constructed can be extremely accurate in resolving all of the proteins present within a cell (greater than 1,500).
The support medium for electrophoresis can be formed into a gel within a tube or it can be layered into flat sheets. The tubes are used for easy one dimensional separations (nearly anyone can make their own apparatus from inexpensive materials found in any lab), while the sheets have a larger surface area and are better for two- dimensional separations. Figure 4.1 shows a typical slab electrophoresis unit.
When the detergent SDS (sodium dodecyl sulfate) 2 is used with proteins, all of the proteins become negatively charged by their attachment to the SDS anions. When separated on a polyacrylamide gel, the procedure is abbreviated as SDS--PAGE (for Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis). The technique has become a standard means for molecular weight determination.
Polyacrylamide gels are formed from the polymerization of two compounds, acrylamide and N,N-methylene- bis-acrylamide (Bis, for short). Bis is a cross-linking agent for the gels. The polymerization is initiated by the addition of ammonium persulfate along with either -dimethyl amino-propionitrile (DMAP) or N,N,N,N,- tetramethylethylenediamine (TEMED). The gels are neutral, hydrophillic, three-dimensional networks of long hydrocarbons crosslinked by methylene groups.
The separation of molecules within a gel is determined by the relative size of the pores formed within the gel. The pore size of a gel is determined by two factors, the total amount of acrylamide present (designated as %T) and the amount of cross-linker (%C). As the total amount of acrylamide increases, the pore size decreases. With cross- linking, 5%C gives the smallest pore size. Any increase or decrease in %C increases the pore size. Gels are designated as percent solutions and will have two necessary parameters. The total acrylamide is given as a % (w/v) of the acrylamide plus the bis-acrylamide. Thus, a 7 1/2 %T would indicate that there is a total of 7.5 gms of acrylamide and bis per 100 ml of gel. A gel designated as 7.5%T:5%C would have a total of 7.5% (w/v) acrylamide + bis, and the bis would be 5% of the total (with pure acrylamide composing the remaining 2.5%).
Proteins with molecular weights ranging from 10,000 to 1,000,000 may be separated with 7 1/2% acrylamide gels, while proteins with higher molecular weights require lower acrylamide gel concentrations. Conversely, gels up to 30% have been used to separate small polypeptides. The higher the gel concentration, the smaller the pore size of the gel and the better it will be able to separate smaller molecules. The percent gel to use depends on the molecular weight of the protein to be separated. Use 5% gels for proteins ranging from 60,000 to 200,000 daltons, 10% gels for a range of 16,000 to 70,000 daltons and 15% gels for a range of 12,000 to 45,000 daltons. 3
Cationic vs anionic systems
In electrophoresis, proteins are separated on the basis of charge, and the charge of a protein can be either + or -- , depending upon the pH of the buffer. In normal operation, a column of gel is partitioned into three sections, known as the Separating or Running Gel, the Stacking Gel and the Sample Gel. The sample gel may be eliminated and the sample introduced via a dense non-convective medium such as sucrose. Electrodes are attached to the ends of the column and an electric current passed through the partitioned gels. If the electrodes are arranged in such a way that the upper bath is -- (cathode), while the lower bath is + (anode), and -- anions are allowed to flow toward the anode, the system is known as an anionic system. Flow in the opposite direction, with + cations flowing to the cathode is a cationic system.
Two basic approaches have been used in the design of electrophoresis protocols. One, column electrophoresis, uses tubular gels formed in glass tubes, while the other, slab gel electrophoresis, uses flat gels formed between two plates of glass. Tube gels have an advantage in that the movement of molecules through the gels is less prone to lateral movement and thus there is a slightly improved resolution of the bands, particularly for proteins. It is also more economical, since it is relatively easy to construct homemade systems from materials on hand. However, slab gels have the advantage of allowing for two dimensional analysis, and of running multiple samples simultaneously in the same gel.
Slab gels are designed with multiple lanes set up such that samples run in parallel. The size and number of the lanes can be varied and, since the samples run in the same medium, there is less likelihood of sample variation due to minor changes in the gel structure. Slab gels are unquestionably the the technique of choice for any blot analyses and for autoradiographic analysis. Consequently, for laboratories performing routine nucleic acid analyses, and those employing antigenic controls, slab gels have become standard. The availability of reasonably priced commercial slab gel units has increased the use of slab gel systems, and the use of tube gels is becoming rare.
The theory and operation of slab gel electrophoresis is identical to tube gel electrophoresis. Which system is used depends more on the experience of the investigator than on any other factor, and the availability of equipment.
The original use of gels as separating media involved using a single gel with a uniform pH throughout. Molecules were separated on the basis of their mobility through a single gel matrix. This system has only occasional use in today's laboratory. It has been replaced with discontinous, 4 multiple gel systems. In multiple gel systems, a separating gel is augmented with a stacking gel and an optional sample gel. These gels can have different concentrations of the same support media, or may be completely different agents. The key difference is how the molecules separate when they enter the separating gel. The proteins in the sample gel will concentrate into a small zone in the stacking gel before entering the separating gel. The zone within the stacking gel can range in thickness from a few microns to a full millimeter. As the proteins are stacked in concentrated bands, they continue to migrate into the separating gel in concentrated narrow bands. The bands then are separated from each other on a discontinuous (i.e. disc ) pH gel.
Once the protein bands enter the separating gel, separation of the bands is enhanced by ions passing through the gel column in pairs. Each ioin in the pair has the same charge polarity as the protein (usually negative), but differ in charge magnitude. One ion will have a much greater charge magnitude than the proteins, while the other has a lesser charge magnitude than the proteins. The ion having a greater charge will move faster and is thus the leading ion, while the ion with the lesser charge will be the trailing ion. When an anionic system is employed, the Cl¯ and glycinate (glycine as its acid derivative) ions are derived from the reservoir buffer (Tris-Glycine). The leading ion is usually Cl¯ glycinate is the trailing ion. A schematic of this anionic system is shown in Figure 4.3. Chloride ions enter the separating gel first and rapidly move down the gel, followed by the proteins and then the glycinate ions. The glycinate ions overtake the proteins and ultimately establish a uniform linear voltage gradient within the gel. The proteins then sort themselves within this gradient according to their charge and size.
While acrylamide gels have become the standard for protein analysis, they are less suitable for extremely high molecular weight nucleic acids (above 200,000 daltons). In order to properly separate these large molecules, the acrylamide concentration needs to be reduced to a level where it remains liquid.
The gels can be formed, however, by the addition of agarose, a naturally linear polysaccharide, to the low concentration of acrylamide. With the addition of agarose, acrylamide concentrations of 0.5% can be used and molecular weights of up to 3.5 x 10 daltons can be separated. This is particularly useful for the separation of large sequences of DNA. Consequently, agarose-acrylamide gels are used extensively in today's genetic laboratories for the determination of gene maps. This chapter will concentrate on the separation of proteins, but Figure 4.4 demonstrates the separation of DNA fragments on an agarose gel.
http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html
Thursday, July 29, 2010
Reconstructing the Lung:William R. Wagner1 and Bartley P. Griffith2
From an engineering and materials science perspective, the lung is a paradigm of design efficiency. A gas transfer surface area of approximately 70 m2 is packed into an elastic, dynamic structure to accomplish efficient oxygen and carbon dioxide transfer. There has been modest success in organizing cells into small-scale structures that mimic pulmonary tissue, but the question of how to scale up and effectively connect such structures has loomed large. Two studies by Petersen et al. on page 538 of this issue (1) and by Ott et al. (2) describe an alternative approach by which the structural efficiencies of native lung tissue can be captured while potentially avoiding the immunogenicity barriers associated with nonautologous tissue transplantation.
Temporary reproduction of the gas transfer function of the lungs does not depend on an organized and viable pulmonary tissue, despite early clinical attempts to do this, such as the perfusion of rhesus monkey lungs with patient's blood during open-heart surgery (3). Rather, mechanical blood oxygenator designs have advanced to make cardiopulmonary support a routine aspect of such procedures. However, biocompatible artificial lung technology to provide chronic aid for patients in end-stage pulmonary failure remains elusive. Support by synthetic membrane–based lung devices remains limited to a period of days or weeks, requires aggressive therapy to prevent blood clotting (anticoagulation), greatly restricts patient mobility and quality of life, and has high mortality.
The limits of current technology are found at the interface where oxygen and carbon dioxide pass between the blood and gas sides of synthetic hollow-fiber membranes. Blood proteins adsorb to the polymeric membrane surfaces, which can trigger the activation immune cells and deposition of clots onto the fibers. This can result in immune responses and a tendency toward bleeding, respectively, when the blood reenters the patient. Further, to achieve adequate gas transfer, membrane surface areas on the order of 1 m2 are required. The size of current membrane oxygenators is incompatible with placement within the body. This is in stark contrast to the inherently anticoagulant surfaces of endothelial cell–lined capillaries that provide the 70 m2 of gas transfer area in the native human lung (see the figure).
Recent approaches to create a more biocompatible artificial lung for extended support have sought to address the surface area per volume limitations of current devices as well as the blood interface bioincompatibility. By reducing fluid boundary layers and optimizing blood flow patterns, blood-contacting synthetic surface areas can be minimized and inhibition of cellular deposition can be improved (4). Smaller oxygenators can also be designed to accommodate patients who do not require complete respiratory support (5). Microfabrication techniques have generated both branched and densely packed blood-carrying polymeric channels with high gas diffusivity that can be stacked in layers near gas channels (6, 7). The blood pathways of these devices can be lined with autologous endothelial cells to provide a surface that mimics that of blood vessels and minimizes the need for anticoagulants. In a cell-seed approach, microporous hollow-fiber membranes are chemically modified to support endothelial cell adhesion. These endothelial cell–covered fibers are bundled and rotated in an oxygenator to create mixing and reduce boundary layer limits on gas transfer efficiency (8). Such devices create a "bio-hybrid" lung that is primarily synthetic but incorporates patient endothelial cells. If they can simulate native thromboresistant endothelial surfaces, a major goal will be achieved
Artificial and limited. Membrane oxygenators currently in clinical use (left) commonly use packed hollow-fiber membranes across which blood flows. Protein and cellular deposits on the synthetic surfaces lead to patient complications and the need for anticoagulation therapy. The native lung (right) provides approximately 70 m2 of gas transfer area in alveoli with actively anticoagulant endothelial surfaces lining the blood
Petersen et al. and Ott et al. apply whole-organ decellularization (removal of all cellular constituents) to the lung, an approach recently explored for heart, liver, and kidney (9–11). This method has conceptual roots in clinically successful bone marrow transplantation. In vivo tissue devitalization, followed by introduction of a regenerating cell population to reconstruct function, was pioneered by E. Donnall Thomas and colleagues in the 1950s and was the basis of the 1990 Nobel Prize in Physiology or Medicine. Petersen et al. and Ott et al. removed the cells from isolated adult rat lungs in a manner that preserved the structural characteristics of the organ, notably the vasculature and the airways (including alveoli, the tiny air sacs in which gas transfer occurs). The remaining lung "scaffolds" were maintained in a bioreactor designed to mimic the developmental environment of lung tissue, and repopulated with epithelial and endothelial cells. Lung tissue was successfully regenerated that mimicked native tissue in appearance and could facilitate gas exchange in vitro and when grafted into rodents.
The promise of this tissue-engineering approach lies in its potential to effectively expand the pool of donor organs, particularly if lungs unsuitable for transplant can be processed for seeding with autologous cells for a given patient while the patient is supported with artificial lung technology. More important, if a tissue-engineered lung had the functional profile of an allograft lung, the removal of pharmacologic therapy to combat organ rejection would have a profound effect on the prognosis and quality of life for lung recipients. Other possible applications of this technique include processing of a patient's diseased tissue to remove problematic cellular components followed by regeneration to a healthy state.
Although the reports of Petersen et al. and Ott et al. open possibilities for treating pulmonary failure and studying mechanisms underlying cell–extracellular matrix interactions, many issues remain to be addressed. Identifying cell sources from the patient that are most effective in repopulating the decellularized lung, achieving a sound blood-gas barrier and a completely endothelialized blood pathway, and providing long-term evaluation of cellularization and differentiation in situ are considerations. The use of extracellular matrix for connective tissue repair is effective in many applications, but there can be failures associated with tissue remodeling that result in mechanically inadequate structures (12). How will the lung extracellular matrix ultimately be remodeled? If replacement lung tissue is inappropriately fibrous or weak, long-term outcomes will be insufficient. Also, the level of phenotypic organ recapitulation that can be achieved and is functionally adequate remains open. Like the native organ, will the regenerated lung recruit vascular beds that will permit increasing blood flows with low resistance? The organ decellularization paradigm has opened a breach where multidisciplinary teams of biologists, clinicians, and engineers can explore new ways to engineer complex tissues. The next generation of artificial or bio-hybrid organs may provide temporary support for patients while patient-specific regenerative solutions are prepared and implanted.
Deadly Viruses Have Been Part of Us for Millions of Years:by Jennifer Couzin-Frankel on July 29, 2010 4:18 PM
Over the past few months, researchers have found that the viruses responsible for Ebola, Marburg hemorrhagic fever, and other deadly diseases have been hanging out in the genomes of certain mammals for tens of millions of years. It turns out that this was just the tip of the iceberg. Scientists have now discovered that these viruses have integrated themselves into the DNA of a wide range of animals, including humans, zebrafish, and other vertebrates. Although the researchers don't know whether the embedded viral sequences have a function, they suspect they are helpful to the animals—otherwise, they wouldn't have endured through millions of years of evolution.
The viruses in question belong to two families: Filoviruses, which include Ebola and Marburg, and Bornaviruses, which causes neurological diseases in certain animals, such as horses. All are RNA viruses, which means they can't easily convert their genetic material into DNA, a necessary step for integrating into an animal's genome. Yet they've done just that, and the new study suggests that they've been even more successful than scientists imagined.
Anna Skalka, a virologist at Fox Chase Cancer Center in Philadelphia, Pennsylvania, was on sabbatical at Princeton University when she heard about work showing that RNA viruses had integrated into insects and plants. Along with two colleagues, Vladimir Belyi and Arnold Levine, both at the Institute for Advanced Study in Princeton, New Jersey, she decided to see whether the same was true in vertebrates. Unlike the previous studies that focused on certain species or a particular RNA virus, Skalka went broad: She and her colleagues surveyed every vertebrate genome available, 48 in all, and looked for hints of 5666 RNA viral sequences from 38 known families and nine genera that were unclassified. It was "everything available that could be looked at," Skalka says.
The three "were floored" by the results, she says, as much for what they showed as what they didn't show. Nineteen of the species, including a squirrel, a small bat, a zebrafish, and a human had RNA viral sequences embedded in their DNA. But equally intriguing was how few different families of RNA viruses turned up: just Bornaviruses and Filoviruses. "It's a mystery as to why this should be," Skalka says. The integrations happened as long as 40 million years ago, the team reports today in PLOS Pathogens.
"It would be interesting to know what's special about these two [families] of viruses," says Jonathan Stoye, a virologist at the National Institute for Medical Research in London. He wonders exactly how the RNA viruses are infecting cells without harming them, allowing them to become a part of an animal's DNA. That said, it's still too early to know whether Bornaviruses and Filoviruses are really overrepresented, says Derek Taylor, an evolutionary biologist at the University at Buffalo in New York. That's because studies like this can't help but miss genetic sequences from viruses that have changed significantly over time, and the viruses may now look very different from how they did when they inserted themselves into a genome. "There may be some ancient ghosts in there," Skalka agrees, "but the surviving viruses have evolved so far that we can't recognize them anymore."
Skalka speculates that the sequences her team and others have found might protect the host from infection. Interestingly, horses are especially vulnerable to Bornaviruses, and no Bornavirus sequences showed up in these animals. In addition, bats had Ebola-like sequences, which the scientists speculate could help them transmit the disease without succumbing to it.
The viruses in question belong to two families: Filoviruses, which include Ebola and Marburg, and Bornaviruses, which causes neurological diseases in certain animals, such as horses. All are RNA viruses, which means they can't easily convert their genetic material into DNA, a necessary step for integrating into an animal's genome. Yet they've done just that, and the new study suggests that they've been even more successful than scientists imagined.
Anna Skalka, a virologist at Fox Chase Cancer Center in Philadelphia, Pennsylvania, was on sabbatical at Princeton University when she heard about work showing that RNA viruses had integrated into insects and plants. Along with two colleagues, Vladimir Belyi and Arnold Levine, both at the Institute for Advanced Study in Princeton, New Jersey, she decided to see whether the same was true in vertebrates. Unlike the previous studies that focused on certain species or a particular RNA virus, Skalka went broad: She and her colleagues surveyed every vertebrate genome available, 48 in all, and looked for hints of 5666 RNA viral sequences from 38 known families and nine genera that were unclassified. It was "everything available that could be looked at," Skalka says.
The three "were floored" by the results, she says, as much for what they showed as what they didn't show. Nineteen of the species, including a squirrel, a small bat, a zebrafish, and a human had RNA viral sequences embedded in their DNA. But equally intriguing was how few different families of RNA viruses turned up: just Bornaviruses and Filoviruses. "It's a mystery as to why this should be," Skalka says. The integrations happened as long as 40 million years ago, the team reports today in PLOS Pathogens.
"It would be interesting to know what's special about these two [families] of viruses," says Jonathan Stoye, a virologist at the National Institute for Medical Research in London. He wonders exactly how the RNA viruses are infecting cells without harming them, allowing them to become a part of an animal's DNA. That said, it's still too early to know whether Bornaviruses and Filoviruses are really overrepresented, says Derek Taylor, an evolutionary biologist at the University at Buffalo in New York. That's because studies like this can't help but miss genetic sequences from viruses that have changed significantly over time, and the viruses may now look very different from how they did when they inserted themselves into a genome. "There may be some ancient ghosts in there," Skalka agrees, "but the surviving viruses have evolved so far that we can't recognize them anymore."
Skalka speculates that the sequences her team and others have found might protect the host from infection. Interestingly, horses are especially vulnerable to Bornaviruses, and no Bornavirus sequences showed up in these animals. In addition, bats had Ebola-like sequences, which the scientists speculate could help them transmit the disease without succumbing to it.
Island Monkeys Give Clues to Origins of HIV's Ancestor
VIENNA—Thousands of years ago, a piece of West Africa separated from the mainland and formed the island of Bioko. The monkeys that inhabit the island may be crucial to unraveling the puzzling origins of the AIDS epidemic in humans, according to a study presented here last week at the 18th International AIDS Conference.
Scientists have argued about the origin of the AIDS epidemic since it surfaced in 1981, but this much is widely accepted today: Sometime around 1931, HIV-1, the main virus driving the epidemic, likely entered humans from chimpanzees, which are infected with a related virus called SIVcpz. The chimp virus, in turn, is a blend of SIVs from two different monkey species.
Less clear is when the monkey viruses moved into chimpanzees. Last year, one prominent investigator in the origin field, evolutionary biologist Michael Worobey of the University of Arizona in Tucson, found evidence that the monkey-to-chimp jump occurred sometime between 1266 and 1685. Worobey and his team used changes in the RNA of these SIVs to calculate their age. These so-called molecular clocks depend on how they're calibrated, however, and not everyone was convinced.
Skeptics, including virologist Preston Marx of the Tulane National Primate Research Center in Covington, Louisiana, suspected the leap from monkeys to chimps occurred tens of thousands of years earlier. SIVs and SIVcpz are found everywhere from East to West Africa, and Marx reasoned that it "was just not possible" for the viruses to have spread so widely in 500 years. So he came up with a new way to calibrate the molecular clock that relied on Bioko's known separation date from the mainland, and he recruited Worobey to help him analyze the data.
Marx and his team collected samples of SIV in dead monkeys on Bioko, which were killed for bushmeat. The researchers isolated SIVs from four different species on the island. One species, the Bioko drill (Mandrillus leucophaeus poensis), has a mainland counterpart that also harbors SIV. The fact that the virus was in both drills and that the island separated from the mainland 12,000 years ago provided a precise way to calibrate the molecular clock, and comparisons of the SIVs confirmed Marx's suspicion that the jump into chimpanzees must have occurred much earlier than Worobey's previous estimates.
As it turned out, the SIV from the drills closely matches SIV from red-capped mangabeys, one of the two contributors to SIVcpz. So an ancestor of this drill could have infected chimpanzees. According to Marx's analysis, a virus related to the Bioko drill's SIV infected chimpanzees at least 22,000 years ago.
Worobey's earlier studies did not date the origin of SIV itself but suggested that it was "relatively young." Others have argued that the SIVs emerged millions of years ago. The new analysis of all four monkey species suggests that, at a minimum, the SIVs are 76,000 years old—although Marx suspects that they evolved far earlier. This longer history of primates harboring the viruses may explain why SIVs cause no harm in the African monkeys they infect, Marx noted: The hosts have had more time to evolve appropriate immune responses or cellular changes that make them less vulnerable to the viruses. (Recent reports strongly indicate that SIVcpz can cause AIDS in chimpanzees.)
"The data are excellent," says Simon Wain-Hobson, a virologist at the Pasteur Institute in Paris who has been involved in origin studies since the start of the AIDS epidemic. But he cautions that the SIV on the island may have been introduced recently, upending Marx and Worobey's clock calibration. "The jury is out," says Wain-Hobson, noting that he is not ready to discard the substantial evidence from other molecular-clock analyses that SIVcpz is younger.
Paul Sharp, an evolutionary biologist at the University of Edinburgh in the United Kingdom who first described the SIVs that led to SIVcpz, has more confidence that the new findings will hold up to scrutiny. "Molecular-clock analyses have suggested that the SIVs arose within the last few thousand years," he says. "These Bioko viruses are clear evidence that the SIVs must be much older than that."
Scientists have argued about the origin of the AIDS epidemic since it surfaced in 1981, but this much is widely accepted today: Sometime around 1931, HIV-1, the main virus driving the epidemic, likely entered humans from chimpanzees, which are infected with a related virus called SIVcpz. The chimp virus, in turn, is a blend of SIVs from two different monkey species.
Less clear is when the monkey viruses moved into chimpanzees. Last year, one prominent investigator in the origin field, evolutionary biologist Michael Worobey of the University of Arizona in Tucson, found evidence that the monkey-to-chimp jump occurred sometime between 1266 and 1685. Worobey and his team used changes in the RNA of these SIVs to calculate their age. These so-called molecular clocks depend on how they're calibrated, however, and not everyone was convinced.
Skeptics, including virologist Preston Marx of the Tulane National Primate Research Center in Covington, Louisiana, suspected the leap from monkeys to chimps occurred tens of thousands of years earlier. SIVs and SIVcpz are found everywhere from East to West Africa, and Marx reasoned that it "was just not possible" for the viruses to have spread so widely in 500 years. So he came up with a new way to calibrate the molecular clock that relied on Bioko's known separation date from the mainland, and he recruited Worobey to help him analyze the data.
Marx and his team collected samples of SIV in dead monkeys on Bioko, which were killed for bushmeat. The researchers isolated SIVs from four different species on the island. One species, the Bioko drill (Mandrillus leucophaeus poensis), has a mainland counterpart that also harbors SIV. The fact that the virus was in both drills and that the island separated from the mainland 12,000 years ago provided a precise way to calibrate the molecular clock, and comparisons of the SIVs confirmed Marx's suspicion that the jump into chimpanzees must have occurred much earlier than Worobey's previous estimates.
As it turned out, the SIV from the drills closely matches SIV from red-capped mangabeys, one of the two contributors to SIVcpz. So an ancestor of this drill could have infected chimpanzees. According to Marx's analysis, a virus related to the Bioko drill's SIV infected chimpanzees at least 22,000 years ago.
Worobey's earlier studies did not date the origin of SIV itself but suggested that it was "relatively young." Others have argued that the SIVs emerged millions of years ago. The new analysis of all four monkey species suggests that, at a minimum, the SIVs are 76,000 years old—although Marx suspects that they evolved far earlier. This longer history of primates harboring the viruses may explain why SIVs cause no harm in the African monkeys they infect, Marx noted: The hosts have had more time to evolve appropriate immune responses or cellular changes that make them less vulnerable to the viruses. (Recent reports strongly indicate that SIVcpz can cause AIDS in chimpanzees.)
"The data are excellent," says Simon Wain-Hobson, a virologist at the Pasteur Institute in Paris who has been involved in origin studies since the start of the AIDS epidemic. But he cautions that the SIV on the island may have been introduced recently, upending Marx and Worobey's clock calibration. "The jury is out," says Wain-Hobson, noting that he is not ready to discard the substantial evidence from other molecular-clock analyses that SIVcpz is younger.
Paul Sharp, an evolutionary biologist at the University of Edinburgh in the United Kingdom who first described the SIVs that led to SIVcpz, has more confidence that the new findings will hold up to scrutiny. "Molecular-clock analyses have suggested that the SIVs arose within the last few thousand years," he says. "These Bioko viruses are clear evidence that the SIVs must be much older than that."
Tuesday, July 20, 2010
Should Smuggled Madagascar Frogs Be Returned Home?
by John Bohannon on July 19, 2010 5:19 PM
Conservation biologists are celebrating last week's bust of Madagascar animal smugglers at the airport in Kuala Lumpur, Malaysia. But in an ironic twist, they're now scrambling to ensure that the animals aren't shipped back home. The 40 extremely rare tomato frogs that were found hidden in a piece of luggage "should not be returned under any circumstances," says Joseph Mendelson, a herpetologist at Zoo Atlanta. They could bring home the amphibian-killing Chytrid fungus, found in Malaysia that the entire island of Madagascar has so far avoided.
E-mails have been flying fast and furious between herpetologists today with proposals for what to do with the stranded frogs. Euthanasia seems to have been ruled out. "The best thing is to ship them to a zoo with a captive breeding program," says Mendelson. "There's a good one in Hong Kong." But Mendelson and others say that they fear that the frogs could pick up the fungus during their several-day stay in the airport. The only way the fungus moves across the ocean is on the bodies of amphibians; scientists have called for a complete ban on amphibians crossing Madagascar's borders.
The smuggling of the animals off the island is the latest sign that Madagascar's new military rulers have not brought the illegal trade of the country's unique species under control.
Scientists have been cautiously optimistic since 2 April when the regime caved to pressure to reinstate the ban on the logging of Rosewood trees. Since then, allegations of government complicity with species trafficking have not abated. But the orphaned tomato frogs pose a far greater threat to the island's biota, says Mendelson. "Madagascar is one of the few places left that has escaped the fungus," and just a single contaminated amphibian could spark "an extremely rapid loss of species."
Conservation biologists are celebrating last week's bust of Madagascar animal smugglers at the airport in Kuala Lumpur, Malaysia. But in an ironic twist, they're now scrambling to ensure that the animals aren't shipped back home. The 40 extremely rare tomato frogs that were found hidden in a piece of luggage "should not be returned under any circumstances," says Joseph Mendelson, a herpetologist at Zoo Atlanta. They could bring home the amphibian-killing Chytrid fungus, found in Malaysia that the entire island of Madagascar has so far avoided.
E-mails have been flying fast and furious between herpetologists today with proposals for what to do with the stranded frogs. Euthanasia seems to have been ruled out. "The best thing is to ship them to a zoo with a captive breeding program," says Mendelson. "There's a good one in Hong Kong." But Mendelson and others say that they fear that the frogs could pick up the fungus during their several-day stay in the airport. The only way the fungus moves across the ocean is on the bodies of amphibians; scientists have called for a complete ban on amphibians crossing Madagascar's borders.
The smuggling of the animals off the island is the latest sign that Madagascar's new military rulers have not brought the illegal trade of the country's unique species under control.
Scientists have been cautiously optimistic since 2 April when the regime caved to pressure to reinstate the ban on the logging of Rosewood trees. Since then, allegations of government complicity with species trafficking have not abated. But the orphaned tomato frogs pose a far greater threat to the island's biota, says Mendelson. "Madagascar is one of the few places left that has escaped the fungus," and just a single contaminated amphibian could spark "an extremely rapid loss of species."
Artificial Cells Communicate and Cooperate Like Biological Cells, Ants
ScienceDaily (July 19, 2010) — Inspired by the social interactions of ants and slime molds, University of Pittsburgh engineers have designed artificial cells capable of self-organizing into independent groups that can communicate and cooperate.
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Recently reported in the Proceedings of the National Academy of Sciences (PNAS), the research is a significant step toward producing synthetic cells that behave like natural organisms and could perform important, microscale functions in fields ranging from the chemical industry to medicine.
The team presents in the PNAS paper computational models that provide a blueprint for developing artificial cells -- or microcapsules -- that can communicate, move independently, and transport "cargo" such as chemicals needed for reactions. Most importantly, the "biologically inspired" devices function entirely through simple physical and chemical processes, behaving like complex natural organisms but without the complicated internal biochemistry, said corresponding author Anna Balazs, Distinguished Professor of Chemical Engineering in Pitt's Swanson School of Engineering.
The Pitt group's microcapsules interact by secreting nanoparticles in a way similar to that used by biological cells signal to communicate and assemble into groups. And with a nod to ants, the cells leave chemical trails as they travel, prompting fellow microcapsules to follow. Balazs worked with lead author German Kolmakov and Victor Yashin, both postdoctoral researchers in Pitt's Department of Chemical and Petroleum Engineering, who produced the cell models; and with Pitt professor of electrical and computer engineering Steven Levitan, who devised the ant-like trailing ability.
The researchers write that communication hinges on the interaction between microcapsules exchanging two different types of nanoparticles. The "signaling" cell secretes nanoparticles known as agonists that prompt the second "target" microcapsule to emit nanoparticles known as antagonists.
In one video of the interaction, as the signaling cell emits the agonist nanoparticles, the target cell responds with antagonists that stop the first cell from secreting. Once the signaling cell goes dormant, the target cell likewise stops releasing antagonists -- which makes the signaling cell start up again. The microcapsules get locked into a cycle that equates to an intercellular conversation, a dialogue humans could control by adjusting the capsules' permeability and the quantity of nanoparticles they contain.
Locomotion results as the released nanoparticles alter the surface underneath the microcapsules. The cell's polymer-based walls begin to push on the fluid surrounding the capsule and the fluid pushes back even harder, moving the capsule. At the same time, the nanoparticles from the signaling cell pull it toward the target cells. Groups of capsules begin to form as the signaling cell rolls along, picking up target cells. In practical use, Balazs said, the signaling cell could transport target cells loaded with cargo; the team's next step is to control the order in which target cells are collected and dropped off.
The researchers adjusted the particle output of the signaling cell to create various cell formations. One video clip shows the trailing "ants," wherein the particle secretions of one microcapsule group are delayed until another group passes by and activates it. The newly awakened cluster then follows the chemical residue left behind by the lead group.
A second film depicts a "dragon" formation comprising two cooperating signaling cells (shown as red) leading a large group of targets. Similar to these are "snakes" made up of competing signaling capsules pulling respective lines of target cells.
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Recently reported in the Proceedings of the National Academy of Sciences (PNAS), the research is a significant step toward producing synthetic cells that behave like natural organisms and could perform important, microscale functions in fields ranging from the chemical industry to medicine.
The team presents in the PNAS paper computational models that provide a blueprint for developing artificial cells -- or microcapsules -- that can communicate, move independently, and transport "cargo" such as chemicals needed for reactions. Most importantly, the "biologically inspired" devices function entirely through simple physical and chemical processes, behaving like complex natural organisms but without the complicated internal biochemistry, said corresponding author Anna Balazs, Distinguished Professor of Chemical Engineering in Pitt's Swanson School of Engineering.
The Pitt group's microcapsules interact by secreting nanoparticles in a way similar to that used by biological cells signal to communicate and assemble into groups. And with a nod to ants, the cells leave chemical trails as they travel, prompting fellow microcapsules to follow. Balazs worked with lead author German Kolmakov and Victor Yashin, both postdoctoral researchers in Pitt's Department of Chemical and Petroleum Engineering, who produced the cell models; and with Pitt professor of electrical and computer engineering Steven Levitan, who devised the ant-like trailing ability.
The researchers write that communication hinges on the interaction between microcapsules exchanging two different types of nanoparticles. The "signaling" cell secretes nanoparticles known as agonists that prompt the second "target" microcapsule to emit nanoparticles known as antagonists.
In one video of the interaction, as the signaling cell emits the agonist nanoparticles, the target cell responds with antagonists that stop the first cell from secreting. Once the signaling cell goes dormant, the target cell likewise stops releasing antagonists -- which makes the signaling cell start up again. The microcapsules get locked into a cycle that equates to an intercellular conversation, a dialogue humans could control by adjusting the capsules' permeability and the quantity of nanoparticles they contain.
Locomotion results as the released nanoparticles alter the surface underneath the microcapsules. The cell's polymer-based walls begin to push on the fluid surrounding the capsule and the fluid pushes back even harder, moving the capsule. At the same time, the nanoparticles from the signaling cell pull it toward the target cells. Groups of capsules begin to form as the signaling cell rolls along, picking up target cells. In practical use, Balazs said, the signaling cell could transport target cells loaded with cargo; the team's next step is to control the order in which target cells are collected and dropped off.
The researchers adjusted the particle output of the signaling cell to create various cell formations. One video clip shows the trailing "ants," wherein the particle secretions of one microcapsule group are delayed until another group passes by and activates it. The newly awakened cluster then follows the chemical residue left behind by the lead group.
A second film depicts a "dragon" formation comprising two cooperating signaling cells (shown as red) leading a large group of targets. Similar to these are "snakes" made up of competing signaling capsules pulling respective lines of target cells.
Scientists Proclaim Breakthrough in HIV Prevention
Cape Town — Scientists have proclaimed a breakthrough in research into the use of an antiretroviral microbicide which they say could prevent more than 500,000 new HIV infections in South Africa alone over the next decade.
The scientists, from the University of KwaZulu-Natal (UKZN), Durban, and Columbia University, New York, say that an experiment with a trial group of South African women shows that those who used a vaginal gel containing tenofovir, an antiretroviral drug, were 39 percent less likely to become infected with HIV during sex than those who did not use it.
They say the gel was also 51 percent effective in preventing genital herpes infections in the women participating in the trial. The scientists noted that women with genital herpes run a high risk of HIV infection.
Making their announcement jointly in Durban and at the International AIDS Conference in Vienna, they said it was "an important scientific breakthrough in the fight against HIV and genital herpes."
If the research results are confirmed in further studies, the gel could revolutionize the lives of women whose partners fail to practise safe sex.
Kristy Siegfried/IRIN
A grandmother cares for four children after their mothers died of Aids. In one of the biggest advances in decades, a vaginal gel containing an antiretroviral drug has been proven to protect almost four out of 10 women from HIV.
“Tenofovir gel could fill an important HIV prevention gap by empowering women who are unable to successfully negotiate mutual faithfulness or condom use with their male partners,” said Dr. Quarraisha Abdool Karim, Associate Professor of Epidemiology at Columbia University.
The trial with the gel was carried out by the Centre for the AIDS Programme of Research in South Africa (CAPRISA), a joint UKZN-Columbia University research institute based at the UKZN's medical school.
The 889 women from KwaZulu-Natal who took part were told to use the gel up to 12 hours before, and soon after, having sex. They used it over a period of between 12 and 30 months.
Of the 889, 98 became HIV positive over the course of the experiment. Of those who used the gel, 38 became HIV positive, while of those who used a placebo, 60 became HIV positive.
"Women who used the gel in more than 80 percent of their sex acts had a 54 percent reduction in HIV infections," the researchers said, "whereas those who used the gel in less than half of their sex acts had a 28 percent reduction in HIV infections."
Of 434 women who tested negative for herpes, 29 who used the gel became infected, as against 58 who used a placebo.
"The reduced rates of HIV and herpes infections among the women who used the tenofovir gel are statistically significant," the researchers added.
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South Africa
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U.S., Canada and Africa
The scientists, from the University of KwaZulu-Natal (UKZN), Durban, and Columbia University, New York, say that an experiment with a trial group of South African women shows that those who used a vaginal gel containing tenofovir, an antiretroviral drug, were 39 percent less likely to become infected with HIV during sex than those who did not use it.
They say the gel was also 51 percent effective in preventing genital herpes infections in the women participating in the trial. The scientists noted that women with genital herpes run a high risk of HIV infection.
Making their announcement jointly in Durban and at the International AIDS Conference in Vienna, they said it was "an important scientific breakthrough in the fight against HIV and genital herpes."
If the research results are confirmed in further studies, the gel could revolutionize the lives of women whose partners fail to practise safe sex.
Kristy Siegfried/IRIN
A grandmother cares for four children after their mothers died of Aids. In one of the biggest advances in decades, a vaginal gel containing an antiretroviral drug has been proven to protect almost four out of 10 women from HIV.
“Tenofovir gel could fill an important HIV prevention gap by empowering women who are unable to successfully negotiate mutual faithfulness or condom use with their male partners,” said Dr. Quarraisha Abdool Karim, Associate Professor of Epidemiology at Columbia University.
The trial with the gel was carried out by the Centre for the AIDS Programme of Research in South Africa (CAPRISA), a joint UKZN-Columbia University research institute based at the UKZN's medical school.
The 889 women from KwaZulu-Natal who took part were told to use the gel up to 12 hours before, and soon after, having sex. They used it over a period of between 12 and 30 months.
Of the 889, 98 became HIV positive over the course of the experiment. Of those who used the gel, 38 became HIV positive, while of those who used a placebo, 60 became HIV positive.
"Women who used the gel in more than 80 percent of their sex acts had a 54 percent reduction in HIV infections," the researchers said, "whereas those who used the gel in less than half of their sex acts had a 28 percent reduction in HIV infections."
Of 434 women who tested negative for herpes, 29 who used the gel became infected, as against 58 who used a placebo.
"The reduced rates of HIV and herpes infections among the women who used the tenofovir gel are statistically significant," the researchers added.
Relevant Links
Southern Africa
South Africa
AIDS
Science
U.S., Canada and Africa
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