A few of us had lunch with the seminar speaker today. Very nice guy, interesting work, but somewhere in the middle we got into a discussion about grants, funding, and alternative careers. He filled us in on the bleak atmosphere out there: too little money, too many grants.
All and all, it was a little depressing.
When I told my friend about what we talked about, all she could say was, "Well of course you don't tell grad students about how hard funding is. That's like a mean adult telling a child there is no Santa Claus"
Tuesday, October 13, 2009
Sunday, May 24, 2009
NK Memory, the descriptive phase (Part II)
NK Memory in Contact Hypersensitivity
First described (to my knowledge) in Nature on May 2006, Ulrich von Andrian's group was experimenting in hapten-induced contact hypersensitivity (CHS) when they noticed a recall response in the absence of B and T cells (via a RAG2 knock-out mouse). They pinpointed NK cells as the source and performed follow-up experiments to support NK cell memory
Introduction
Contact hypersensitivity (such as responses to poison ivy) occurs when the immune system is primed against foreign antigen, inducing specific T and B cell responses (T cells are more important). Upon secondary encounter, memory T cells are activated and begin secreting cytokines such as interferon gamma. These cytokines in turn activate macrophages and other cells, which ultimately induce inflammation: pain, heat, swelling, redness (and the godawful itch).
Contact hypersensitivity can be measured in a variety of ways, but in the von Andrian's paper, it was mostly measured by ear-swelling and by identifying the type and number of cellular infiltrates in the ear.
The authors' used three different chemicals in order to induced CHS. In most of their experiments, they used 2,4-dinitrofluorobenzene (DNFB), but oxazolone (OXA) and picryl chloride was also used. These are small molecule haptens which react with cellular proteins to create new, foreign epitopes for which the immune system can respond.
Secondary-response to DNFB in RAG2-/- mice
The authors' original intent was to examine various tissues in skin-induced CHS. They were characterizing the role of T cells in hypersensitivity of the bladder when they discovered that RAG2-/- mice have a recall response to antigen: furthermore, the magnitude of the response was similar to WT mice. They wanted to know if hypersensitivity in the bladder was T cell dependent, but what they found is that other cell types might have memory-like properties.
Their work in the bladder gave an indication of what cell type might be involved. In WT sensitized mice, T cells predominate in the infiltrate. However, NK cells are a close second. It should also be mentioned that PMNs [mostly neutrophils] are also high; but as they live for only a few days, these can be ruled out. But before they could begin to work on NK cells, they had to prove that antigen specificity actually occurs.
Antigen specificity of recall response in CHS using RAG2-/- mice
The "recall response" could have been nothing more than innate inflammation that was stronger the second time around (given the environment produced a month previously). In order to show antigen specificity, mice were sensitized to one hapten, like DNFB, and then the recall response to an unrelated antigen (OXA) was assessed. In both WT and RAG2-/- mice, increased ear swelling was shown only when the mouse was primed, and then boosted with, the same antigen. This satisfies one requirement of memory-antigen specificity, and suggested the involvement of a memory-like cell in the absence of a B or T cell response.
Increase in NK cell numbers after challenge
As mentioned previously, NK cells were shown to be present in large numbers in CHS, second only to T cells. The numbers of NK cells were assessed both during sensitization and after challenge. As expected, in RAG2-/- mice, there was an increase in NK cells after challenge with hapten, as measure by flow cytometry using the markers NK1.1 and CD45. Immunofluoresence also shown increased numbers of NK cells. But again, it wasn't clear if the increase in NK cells are a byproduct of the model or the mediators.
Hypersensitivity in the absence of NK cells?
If NK cells are the population mediating this recall response, RAG2-/- mice that have a defect in NK cells shouldn't have any secondary response. Thus, the authors chose two different mouse models in which to perform their studies, only one of which I'll talk about here. RAG2-/- mice were crossed with mice lacking the common gamma chain, resulting in a double knock out mouse (RAG2-/-IL2Rgamma-/-). The common gamma chain is shared by a family of cytokine receptors and results in defective responses to multiple cytokines, some of which are also important for NK cell development. Thus, this double knockout has no B, T, and NK cells. As expected, without NK cells the challenge response to haptens was greatly reduced (when compared to a RAG2-/-). This experiment increases the argument for NK cell memory, but there are a few caveats: other cell types might also use these cytokines and there is probably reduced activation in these cells. Therefore, in addition to using another model, SCID x beige mice, further experiments had to be performed.
NK cells were also depleted using specific antibody(such as NK1.1) Again, when compared to WT secondary responses to haptens, inflammation in RAG-/- mice was much lower.
Adoptive transfer of naive and sensitized NK cells
Because other cell types might have been affected by lacking the common gamma chain, adoptive transfer assays were used to confirm NK cells as the mediator of this memory-like phenotype. Compared to naive NK cells or sensitized cells depleted of NK cells, previously sensitized NK cells adopted into naive mice had greater hapten-specific ear swelling.
Other
The authors also perform other experiments in order to narrow-down the exact subset of NK cells mediating this effect. However, this isn't as convincing. There's nothing wrong with the data, which narrows down the NK cell population to that in the liver, but not enough was done to confirm that the subset actually comes from the liver. For example, activation of NK cells might have increased localization to the liver. Or, because they were only comparing NK cells between lymph node, liver, and spleen, they might have missed an increase in the skin population.
Likewise, the attempt to characterize the NK population using expression of activating receptors (via flow cytometry) has the same problem. Without knowing the exact receptor the NK cell uses to respond to the hapten, it's like looking for a needle in a haystack.
Conclusion
Based on these experiments, it was clear NK cells mediate a memory-cell like function. The authors demonstrate increased recall responses after 4 weeks in RAG2-/- mice, antigen specificity(in the broad sense), and adoptive transfer of antigen-experienced NK cells mediating this function.
That being said, there were still many things left to be proved. The lack of a specific receptor-ligand pair hindered quantification of these memory-like NK cells, without which the scientific community was apt to be skeptical. In addition, without a better model (ear swelling isn't the greatest assay in a non-CHS setting) future experiments having to do with pathogen models and vaccine development couldn't be accomplished.
The next paper deals with a specific receptor-ligand pair that was able to extend the knowledge on these memory-like NK cells. In the meantime, checkout the 2006 paper and see for yourself:
O'Leary JG, Goodarzi M, Drayton DL, and von Andrian UH. (2006) T cell-and B cell-independent adaptive immunity mediated by natural killer cells. Nature 7(5): 507-14
Pubmed
http://www.ncbi.nlm.nih.gov/pubmed/16617337
First described (to my knowledge) in Nature on May 2006, Ulrich von Andrian's group was experimenting in hapten-induced contact hypersensitivity (CHS) when they noticed a recall response in the absence of B and T cells (via a RAG2 knock-out mouse). They pinpointed NK cells as the source and performed follow-up experiments to support NK cell memory
Introduction
Contact hypersensitivity (such as responses to poison ivy) occurs when the immune system is primed against foreign antigen, inducing specific T and B cell responses (T cells are more important). Upon secondary encounter, memory T cells are activated and begin secreting cytokines such as interferon gamma. These cytokines in turn activate macrophages and other cells, which ultimately induce inflammation: pain, heat, swelling, redness (and the godawful itch).
Contact hypersensitivity can be measured in a variety of ways, but in the von Andrian's paper, it was mostly measured by ear-swelling and by identifying the type and number of cellular infiltrates in the ear.
The authors' used three different chemicals in order to induced CHS. In most of their experiments, they used 2,4-dinitrofluorobenzene (DNFB), but oxazolone (OXA) and picryl chloride was also used. These are small molecule haptens which react with cellular proteins to create new, foreign epitopes for which the immune system can respond.
Secondary-response to DNFB in RAG2-/- mice
The authors' original intent was to examine various tissues in skin-induced CHS. They were characterizing the role of T cells in hypersensitivity of the bladder when they discovered that RAG2-/- mice have a recall response to antigen: furthermore, the magnitude of the response was similar to WT mice. They wanted to know if hypersensitivity in the bladder was T cell dependent, but what they found is that other cell types might have memory-like properties.
Their work in the bladder gave an indication of what cell type might be involved. In WT sensitized mice, T cells predominate in the infiltrate. However, NK cells are a close second. It should also be mentioned that PMNs [mostly neutrophils] are also high; but as they live for only a few days, these can be ruled out. But before they could begin to work on NK cells, they had to prove that antigen specificity actually occurs.
Antigen specificity of recall response in CHS using RAG2-/- mice
The "recall response" could have been nothing more than innate inflammation that was stronger the second time around (given the environment produced a month previously). In order to show antigen specificity, mice were sensitized to one hapten, like DNFB, and then the recall response to an unrelated antigen (OXA) was assessed. In both WT and RAG2-/- mice, increased ear swelling was shown only when the mouse was primed, and then boosted with, the same antigen. This satisfies one requirement of memory-antigen specificity, and suggested the involvement of a memory-like cell in the absence of a B or T cell response.
Increase in NK cell numbers after challenge
As mentioned previously, NK cells were shown to be present in large numbers in CHS, second only to T cells. The numbers of NK cells were assessed both during sensitization and after challenge. As expected, in RAG2-/- mice, there was an increase in NK cells after challenge with hapten, as measure by flow cytometry using the markers NK1.1 and CD45. Immunofluoresence also shown increased numbers of NK cells. But again, it wasn't clear if the increase in NK cells are a byproduct of the model or the mediators.
Hypersensitivity in the absence of NK cells?
If NK cells are the population mediating this recall response, RAG2-/- mice that have a defect in NK cells shouldn't have any secondary response. Thus, the authors chose two different mouse models in which to perform their studies, only one of which I'll talk about here. RAG2-/- mice were crossed with mice lacking the common gamma chain, resulting in a double knock out mouse (RAG2-/-IL2Rgamma-/-). The common gamma chain is shared by a family of cytokine receptors and results in defective responses to multiple cytokines, some of which are also important for NK cell development. Thus, this double knockout has no B, T, and NK cells. As expected, without NK cells the challenge response to haptens was greatly reduced (when compared to a RAG2-/-). This experiment increases the argument for NK cell memory, but there are a few caveats: other cell types might also use these cytokines and there is probably reduced activation in these cells. Therefore, in addition to using another model, SCID x beige mice, further experiments had to be performed.
NK cells were also depleted using specific antibody(such as NK1.1) Again, when compared to WT secondary responses to haptens, inflammation in RAG-/- mice was much lower.
Adoptive transfer of naive and sensitized NK cells
Because other cell types might have been affected by lacking the common gamma chain, adoptive transfer assays were used to confirm NK cells as the mediator of this memory-like phenotype. Compared to naive NK cells or sensitized cells depleted of NK cells, previously sensitized NK cells adopted into naive mice had greater hapten-specific ear swelling.
Other
The authors also perform other experiments in order to narrow-down the exact subset of NK cells mediating this effect. However, this isn't as convincing. There's nothing wrong with the data, which narrows down the NK cell population to that in the liver, but not enough was done to confirm that the subset actually comes from the liver. For example, activation of NK cells might have increased localization to the liver. Or, because they were only comparing NK cells between lymph node, liver, and spleen, they might have missed an increase in the skin population.
Likewise, the attempt to characterize the NK population using expression of activating receptors (via flow cytometry) has the same problem. Without knowing the exact receptor the NK cell uses to respond to the hapten, it's like looking for a needle in a haystack.
Conclusion
Based on these experiments, it was clear NK cells mediate a memory-cell like function. The authors demonstrate increased recall responses after 4 weeks in RAG2-/- mice, antigen specificity(in the broad sense), and adoptive transfer of antigen-experienced NK cells mediating this function.
That being said, there were still many things left to be proved. The lack of a specific receptor-ligand pair hindered quantification of these memory-like NK cells, without which the scientific community was apt to be skeptical. In addition, without a better model (ear swelling isn't the greatest assay in a non-CHS setting) future experiments having to do with pathogen models and vaccine development couldn't be accomplished.
The next paper deals with a specific receptor-ligand pair that was able to extend the knowledge on these memory-like NK cells. In the meantime, checkout the 2006 paper and see for yourself:
O'Leary JG, Goodarzi M, Drayton DL, and von Andrian UH. (2006) T cell-and B cell-independent adaptive immunity mediated by natural killer cells. Nature 7(5): 507-14
Pubmed
http://www.ncbi.nlm.nih.gov/pubmed/16617337
NK Cells: Do these cells have Memory? (Part 1)
Introduction: A Primer
Memory
The ability of the immune system to recognize previously encountered pathogens and initiate a better immune response is the hallmark of memory. Responses to previously encountered organisms are characterized by, among other things, a large clonal population of cells that are uniquely specific for the offending pathogen. As an oversimplification, this large population then out-competes the rate of pathogen spread and brings the infection under control.
These memory populations have been thought to come from the adaptive immune system, consisting of both B and T cells. Upon primary infection, naive B and T cells specific for the pathogen exist at low precursor frequency. In order to be activated and clonally expand into a large effector population, these cells must be activated by the innate immune system, which provides the necessary co-stimulation in order to fully induce an adaptive response. Immune system responses can be thought of as a game of escalation: the innate arm recognizes pathogen-associated molecular patterns common to most pathogens and responds early in the infection process. If, however, the innate system is unable to control pathogen spread, it activates the adaptive immune system. Once activated, B and T cells expand into large number of cells directed against the specific pathogen, proceeding to contract into a small population of memory cells after clearance of antigen. These memory cells protect against future infection (or damage to the host) through a variety of ways
Inducing memory (and thus a B and T cell response) is the mechanism by which vaccines work.
NK cells
Natural killer cells are part of the innate immune system. Like all innate cells, NK cells are characterized by their ability to recognize patterns and respond early in infection. However, unlike macrophages and dendritic cells, NK cells do not interact directly with pathogens.* Instead, they recognize patterns on host cells associated with cellular abnormality, which can either be induced by viral infection or if the cell has become cancerous.
NK cells have both activating and inhibitory receptors on their surface which provide the ability to surveil the state of the host. These receptors interact with ligands on other cells and the combined signal from both types of receptors ultimately determine the response from the NK cell. If there are more inhibitory signals than activating ones, the NK cell does not respond. If, however, there are more activating stimuli, any inhibitory signal is overrode and the NK cell carriers out its effector function (death of the target cell, cytokine production). Examples of activating ligands include cellular stress molecules expressed during virus infection. In another example, the absence of an important inhibitory molecule called MHC is an indication that there is something wrong with the cell.
Because NK cells possess a limited set of receptors that respond to patterns of positive and negative signals instead of specific pathogens, it makes sense that these cells should not have memory. An expanded subset of relatively nonspecific NK cells (as compared to B and T cells) might actually be detrimental to the host in certain circumstances. When control can be handled by the innate arm of the immune system, inducing a large memory population of powerful NK cells might cause more damage to the host than the offending pathogen.
The Current Picture
However, within the past few years, the idea of what cell types constitute memory has been challenged. While this area is still largely the domain of B and T cells, recent evidence points to memory-like properties of NK cells such as: increased immune response to secondary encounter with antigen, adoptive transfer of NK cells providing pathogen-specific responses, and long- lived** subsets of NK cells induced by primary infection with pathogens.
In the following posts, my goal is to give a very brief introduction to this new finding.
*There are some virus-specific receptors, and we will talk about one of them in the near future.
**long-lived in this case meaning up to 90 days
Memory
The ability of the immune system to recognize previously encountered pathogens and initiate a better immune response is the hallmark of memory. Responses to previously encountered organisms are characterized by, among other things, a large clonal population of cells that are uniquely specific for the offending pathogen. As an oversimplification, this large population then out-competes the rate of pathogen spread and brings the infection under control.
These memory populations have been thought to come from the adaptive immune system, consisting of both B and T cells. Upon primary infection, naive B and T cells specific for the pathogen exist at low precursor frequency. In order to be activated and clonally expand into a large effector population, these cells must be activated by the innate immune system, which provides the necessary co-stimulation in order to fully induce an adaptive response. Immune system responses can be thought of as a game of escalation: the innate arm recognizes pathogen-associated molecular patterns common to most pathogens and responds early in the infection process. If, however, the innate system is unable to control pathogen spread, it activates the adaptive immune system. Once activated, B and T cells expand into large number of cells directed against the specific pathogen, proceeding to contract into a small population of memory cells after clearance of antigen. These memory cells protect against future infection (or damage to the host) through a variety of ways
Inducing memory (and thus a B and T cell response) is the mechanism by which vaccines work.
NK cells
Natural killer cells are part of the innate immune system. Like all innate cells, NK cells are characterized by their ability to recognize patterns and respond early in infection. However, unlike macrophages and dendritic cells, NK cells do not interact directly with pathogens.* Instead, they recognize patterns on host cells associated with cellular abnormality, which can either be induced by viral infection or if the cell has become cancerous.
NK cells have both activating and inhibitory receptors on their surface which provide the ability to surveil the state of the host. These receptors interact with ligands on other cells and the combined signal from both types of receptors ultimately determine the response from the NK cell. If there are more inhibitory signals than activating ones, the NK cell does not respond. If, however, there are more activating stimuli, any inhibitory signal is overrode and the NK cell carriers out its effector function (death of the target cell, cytokine production). Examples of activating ligands include cellular stress molecules expressed during virus infection. In another example, the absence of an important inhibitory molecule called MHC is an indication that there is something wrong with the cell.
Because NK cells possess a limited set of receptors that respond to patterns of positive and negative signals instead of specific pathogens, it makes sense that these cells should not have memory. An expanded subset of relatively nonspecific NK cells (as compared to B and T cells) might actually be detrimental to the host in certain circumstances. When control can be handled by the innate arm of the immune system, inducing a large memory population of powerful NK cells might cause more damage to the host than the offending pathogen.
The Current Picture
However, within the past few years, the idea of what cell types constitute memory has been challenged. While this area is still largely the domain of B and T cells, recent evidence points to memory-like properties of NK cells such as: increased immune response to secondary encounter with antigen, adoptive transfer of NK cells providing pathogen-specific responses, and long- lived** subsets of NK cells induced by primary infection with pathogens.
In the following posts, my goal is to give a very brief introduction to this new finding.
*There are some virus-specific receptors, and we will talk about one of them in the near future.
**long-lived in this case meaning up to 90 days
Tuesday, May 19, 2009
Personal: Passed Quals!
So I passed my qualifying exam and am now an official Ph.D. candidate! That means I can get back to work on research. It also means that I can start posting more science-related articles. I've got a few that I think are really interesting and pave the way for breakthroughs in immunology.
The exam itself was a humbling experience. I mean, I knew I know 0.0001% of the total amount of immunology there is to know, but I found out I could work on some areas in addition to that.
Overall, I'll remember to work harder from the experience, but also to be confident in what I do know
The exam itself was a humbling experience. I mean, I knew I know 0.0001% of the total amount of immunology there is to know, but I found out I could work on some areas in addition to that.
Overall, I'll remember to work harder from the experience, but also to be confident in what I do know
Sunday, May 3, 2009
Passing on a link: Michael Palm Basic Science, Vaccines, and Prevention Project Blog
No, I haven't gotten hit by the swine flu, I've just have been busy studying for my qualifying examine in immunology. Hopefully, I'll be a more regular poster around June.
In the meantime, I wanted to pass along this link to Michael Palm's blog for the Treatment Action Group (http://www.treatmentactiongroup.org/). It's a great resource for everything HIV related, and is current on HIV research findings.
If you really want to understand the immune system, just look at a virus which directly disrupts it.
His blog can be found at http://tagbasicscienceproject.typepad.com/
In the meantime, I wanted to pass along this link to Michael Palm's blog for the Treatment Action Group (http://www.treatmentactiongroup.org/). It's a great resource for everything HIV related, and is current on HIV research findings.
If you really want to understand the immune system, just look at a virus which directly disrupts it.
His blog can be found at http://tagbasicscienceproject.typepad.com/
Friday, April 17, 2009
Passing on a link: Mystery Rays From Outer Space
Mystery Rays
If you like anything I put up on this blog, you'll definitely like Mystery Rays. Dr. Ian York does a great job of presenting new and interesting aspects of immunology/virology. Topics range from virus evolution to cancer and tend to center around immune escape. In addition to good reading, I have also used this site to find background information and paper suggestions. Mostly, this is because Dr. York covers some of the stuff they don't mention much in introductory immunology courses (for example, immunodominance). And, he does it in a clear and informative manner.
He has a new post up concerning the possibility of HIV actually increasing its virulence.
So, if that sparked your interest, go check it out!
Mystery Rays from Outer Space
http://www.iayork.com/MysteryRays/2009/04/16/is-hiv-becoming-more-virulent/
If you like anything I put up on this blog, you'll definitely like Mystery Rays. Dr. Ian York does a great job of presenting new and interesting aspects of immunology/virology. Topics range from virus evolution to cancer and tend to center around immune escape. In addition to good reading, I have also used this site to find background information and paper suggestions. Mostly, this is because Dr. York covers some of the stuff they don't mention much in introductory immunology courses (for example, immunodominance). And, he does it in a clear and informative manner.
He has a new post up concerning the possibility of HIV actually increasing its virulence.
So, if that sparked your interest, go check it out!
Mystery Rays from Outer Space
http://www.iayork.com/MysteryRays/2009/04/16/is-hiv-becoming-more-virulent/
Saturday, April 4, 2009
System.out.println("The answer is 42");
The Dawning Age of Robot Scientists
Introduction
Friday has become my favorite day. Not because it's the weekend, oh no. No, Fridays are my favorite because I'm such a geek that I'm excited when The Journals come out. And even if you don't regularly scan their table of contents, you'd be hard pressed to miss the headlines in the science sections from the general news media: The first steps to a robot scientist are here people!
(All on the same story, but from different sources)
http://www.nytimes.com/2009/04/07/science/07robot.html
http://news.bbc.co.uk/2/hi/science/nature/7979113.stm
http://sciencenow.sciencemag.org/cgi/content/full/2009/402/1
http://blog.wired.com/wiredscience/2009/04/robotscientist.html
The actual scientific paper in Science is called The Automation of Science by King et al. at Aberystwyth University. There is also a comment on it and a related paper in the same issue (though the news outlets do a good job as well.)
Essentially, this robot, termed Adam, is able to complete the scientific process. Whereas computers and automation today are capable of collecting vast amounts of data, they have so far been unable to process it. Human scientists usually need to look at the data and determine what's useful. However, King et al. designed a system that can not only interpret the data and form a hypothesis about it, but can also perform followup experiments to test this hypothesis. To do this, they designed and implemented several different programs, ranging from hardware interaction to data collection and data analysis.
Just a note on the hardware: Adam is pretty beautiful, consising of multiple fridges, incubators, and robot arms. And, it never gets tired. The experiments involved growth curves of various yeast strains, which Adam can do hundreds of times a day. Just thinking about a human doing that amount of pipetting gives me thumb arthritis.
What Did Adam Actually Do?
Being a machine, Adam is great at hundreds of simple tasks and measurements. After taking specific yeast strains from the freezer, Adam can inoculate them in media and measure the ODs at various time points to establish a growth curve.
In order to test Adam, the authors decided to look at orphan enzymes, or enzymes for which the corresponding gene(s) haven't been identified. These enzymes have been well characterized in terms of their biochemical actions, so Adam is able to screen for enzyme activity by recording growth curves in specific media. Adam was also able to take advantage of yeast knockout strains for which the gene was known. Thus, Adam is able to take yeast that lack a gene and see if it is still able to grow when tested in a variety of limiting medias supplemented with certain metabolites. From this data, Adam is able hypothesize about the unknown gene encoding the orphan enzyme and is able to run further tests to confirm this hypothesis.
But Wait, There's More! (Bioinformatics)
One of the software packages I didn't talk about was their relational database of proteins and genes known to be present in yeast. Although the authors aren't descriptive, this is probably something similar to the DAVID Bioinformatics database (http://david.abcc.ncifcrf.gov/), Gene Ontology(http://www.geneontology.org/), or Pathways Analysis(http://www.ingenuity.com/products/pathways_analysis.html%20to)-- to name a few. I think this is the most important part of Adam, its "memory". Without such databases, it would be hard to understand gene and protein interaction and formulate an educated hypothesis.
Conclusion
Mass data collection, expanding bioinformatics tools, increasingly sophisticated programming, and efficient hardware are converging at the biological sciences, extending capability and knowledge at an exponential pace. Adam is only the beginning.
(If you don't believe me, read Ray Kurzweils The Singularity is Near, which, though on a slightly different topic, is on the same general trend. Or read about Eve at the group’s website, below.)
The paper is:
King et al. The Automation of Science. Science, vol. 324, April 3 2009.
http://www.ncbi.nlm.nih.gov/pubmed/19342587
The group's website can be found at:
http://www.aber.ac.uk/compsci/Research/bio/robotsci/
There are videos of Adam in action and other interesting things on the site
Introduction
Friday has become my favorite day. Not because it's the weekend, oh no. No, Fridays are my favorite because I'm such a geek that I'm excited when The Journals come out. And even if you don't regularly scan their table of contents, you'd be hard pressed to miss the headlines in the science sections from the general news media: The first steps to a robot scientist are here people!
(All on the same story, but from different sources)
http://www.nytimes.com/2009/04/07/science/07robot.html
http://news.bbc.co.uk/2/hi/science/nature/7979113.stm
http://sciencenow.sciencemag.org/cgi/content/full/2009/402/1
http://blog.wired.com/wiredscience/2009/04/robotscientist.html
The actual scientific paper in Science is called The Automation of Science by King et al. at Aberystwyth University. There is also a comment on it and a related paper in the same issue (though the news outlets do a good job as well.)
Essentially, this robot, termed Adam, is able to complete the scientific process. Whereas computers and automation today are capable of collecting vast amounts of data, they have so far been unable to process it. Human scientists usually need to look at the data and determine what's useful. However, King et al. designed a system that can not only interpret the data and form a hypothesis about it, but can also perform followup experiments to test this hypothesis. To do this, they designed and implemented several different programs, ranging from hardware interaction to data collection and data analysis.
Just a note on the hardware: Adam is pretty beautiful, consising of multiple fridges, incubators, and robot arms. And, it never gets tired. The experiments involved growth curves of various yeast strains, which Adam can do hundreds of times a day. Just thinking about a human doing that amount of pipetting gives me thumb arthritis.
What Did Adam Actually Do?
Being a machine, Adam is great at hundreds of simple tasks and measurements. After taking specific yeast strains from the freezer, Adam can inoculate them in media and measure the ODs at various time points to establish a growth curve.
In order to test Adam, the authors decided to look at orphan enzymes, or enzymes for which the corresponding gene(s) haven't been identified. These enzymes have been well characterized in terms of their biochemical actions, so Adam is able to screen for enzyme activity by recording growth curves in specific media. Adam was also able to take advantage of yeast knockout strains for which the gene was known. Thus, Adam is able to take yeast that lack a gene and see if it is still able to grow when tested in a variety of limiting medias supplemented with certain metabolites. From this data, Adam is able hypothesize about the unknown gene encoding the orphan enzyme and is able to run further tests to confirm this hypothesis.
But Wait, There's More! (Bioinformatics)
One of the software packages I didn't talk about was their relational database of proteins and genes known to be present in yeast. Although the authors aren't descriptive, this is probably something similar to the DAVID Bioinformatics database (http://david.abcc.ncifcrf.gov/), Gene Ontology(http://www.geneontology.org/), or Pathways Analysis(http://www.ingenuity.com/products/pathways_analysis.html%20to)-- to name a few. I think this is the most important part of Adam, its "memory". Without such databases, it would be hard to understand gene and protein interaction and formulate an educated hypothesis.
Conclusion
Mass data collection, expanding bioinformatics tools, increasingly sophisticated programming, and efficient hardware are converging at the biological sciences, extending capability and knowledge at an exponential pace. Adam is only the beginning.
(If you don't believe me, read Ray Kurzweils The Singularity is Near, which, though on a slightly different topic, is on the same general trend. Or read about Eve at the group’s website, below.)
The paper is:
King et al. The Automation of Science. Science, vol. 324, April 3 2009.
http://www.ncbi.nlm.nih.gov/pubmed/19342587
The group's website can be found at:
http://www.aber.ac.uk/compsci/Research/bio/robotsci/
There are videos of Adam in action and other interesting things on the site
Sunday, March 29, 2009
Passing on a link: The Virological Synapse
Virogical Synapses and HIV Transmission
A new article at MIT's Technology Review highlights a recent paper about the way HIV is transmitted between CD4 T cells. Using a GFP-expressing virus, the authors ( show direct cell-to-cell transmission of HIV via formation of what's known as the virological synapse. Check it out at http://www.technologyreview.com/blog/editors/23244/
Mini Introduction to the topic:
Viruses (as well as bacteria) can infect cells in a variety of ways. Normally, we think of the route of entry as being mediated by cell surface receptors. The virus attaches to the cell through this method and is taken into the cell, where it begins to replicate itself. Later, the cell bursts, spilling thousands of viral progeny into the host; alternatively, the virus can continually bud off from the plasma membrane. (The virus can either do this right away or form a latent infection, where viral DNA is present but little viral protein is made. In this method, the virus rides along with the cell until conditions are right to start producing viruses again.)
The key point here is that at some point in their lifetime, viruses are thought to go extracellular; viral particles have to spread out and infect cells. And when they're out in the open like that, the host immune system can see them better; more arms are available to fight the virus. For example, a virus can be neutralized by antibody or a lipid envelope can be degraded by complement (to name only a few things). If a virus wasn't exposed to this arm at all, the immune system is at a disadvantage.
Relatively recent work has proposed the existence of a virological synapse which effectively transmits virus between cells without progeny having to ever enter the extracellular space. Essentially, the virological synapse is an immune synapse (in this instance, without the priming stage). MHC II, as well as adhesion molecules, are upregulated upon T cell activation. The adhesion molecules attach to other T cells and MHC II interacts with TCR, collectively forming a tight seal between T cells. Usually, this is good for T cell priming, in addition to probably playing a pertinent role in cytotoxic T cell ability.
However, CD4 T cells are a main target for human immunodefiency virus (HIV). Instead of a protective immunological synapse forming, infected CD4 T cells interacting tightly with uninfected CD4 T cells can pass on the virus through the immunological/virological synapse. This might happen a variety of ways (i.e. trogocytosis, active infection, etc) but the thing to keep in mind is, again, that this exchange is thought to keep out antibody and other effector molecules. Incidentally, if the immunological/virological synapse is presenting cognate antigen, it will help to activate (or keep activated) the uninfected CD4 T cell, which provides an excellent environment for the virus to replicate in.
Not having read the paper (sorry to so uninformed, more pressing papers to read), I don't know whether the authors prove this beyond a doubt. (For example, notice how long it takes for the uninfected CD4 T cell to become infected. Usually an infection is quicker- maybe there was virus in the supernatant that was the cause of the late GFP explosion in that CD4 T cell.) But regardless, it certainly strengthens the argument for a virological synpase and is another avenue scientists have to explore if we are ever going to effectively combat this virus.
It’d also be interesting if they took a look at FAS/FASL on these cells. If they are going to interact that strongly, what happens when activation-induced cell death (AICD) starts to really get going? (…Or does HIV inhibit this, I just don’t know.)
The actual paper can be found at:
Sunday, March 22, 2009
Prions are What?
Normally, I think of prions as a bad thing. Anyone hear of bovine spongiform encephalopathy(BSE)? BSE, or Mad Cow, was featured prominently in the news a few years ago and probably made a vegetarian out of a few people. That's because BSE is caused by prions, which are hard to detect, hard to prevent (prions are highly resistant to heat and other sterilizing devices), and ultimately result in death. So what are prions?
A prion consists of a single infective protein. Unlike other pathogens, nucleic acid isn't necessary to make future generations. Instead, a prion is capable of making more of itself by converting a particular host protein (called prion protein- PrP) into a prion. In essence, the prion protein functions to change the conformation of PrP such that it now takes on the function of a prion; these converted prions in turn recruit others. Most of the pathogenicity occurs because these prions also form aggregates in cells of the brain, ultimately resulting in death.
Prions, as we know them, are dangerous, but because PrP can be so easily converted into a prion, many groups have begun asking, "What function does normal PrP have inside a cell?” Or more generally, what are these potential prions doing there?
Eleven prions have been identified in yeast so far and are a good place to start for finding out what these things do. Interestingly, a new paper published in Nature Cell Biology (http://www.nature.com/ncb/) by Dr. Susan W. Liebman's group at the University of Illinois Chicago has discovered another method of non-Mendelian inheritance via prion function (note: this is my take, the paper is more scientifically conservative and accurate in that its title is The yeast global transcriptional co-repressor protein Cyc8 can propagate as a prion).
It's a good paper and there's a insightful comment on it in the issue, so I won't go into much detail here, but I did want to list a few key points.
Cyc8 (called OCT in its prion form) is a global transcriptional co-repressor protein that, among other things, regulates yeast growth on lactate. This protein suppresses other proteins important for growth on lactate plates. However, by transiently overexpressing the putative prion domain of Cyc8 (and thus increasing the chance of misfolding for the naturally occurring Cyc8) , the authors were able to show increased colonies growing on lactate plates versus controls. The authors were then able to do further experiments using this Lac+ phenotype
One of the most important things they did was an experiment to show that these Lac+ colonies were not spontaneous mutations resulting in revertants. (i.e. mutated nonfunctional Cyc8). Overexpression of functional Cyc8 did not restore the Lac+ phenotype.
However, as an aside, there was always the possibility of a gain-of-function mutation. Perhaps the gene required for lactate growth lost its ability to be repressed by Cyc8. I feel that simple sequencing of the most important genes involved in the Lac+ pathway could have been useful, but the authors’ main concern was elsewhere and they focused their remaining experiments in proving that OCT has all the characteristics of a prion.
This was the most fundamental, and convincing, aspect of the paper. The OCT+ mutation was dominant in an OCT- cross (not characteristic of Cyc8 loss mutations), cytoplasm of OCT+ cells could transfer the Lac+ growth phenotype, and fluorescence of OCT fused to YFP showed aggregates within the cells.
This paper continues to extend the knowledge of prion function. They ain't just fer disease anymore! Instead, proteins with prion-like characteristics might serve to regulate cell function in a rapid and inherited manner, without the need to continuously monitor transcriptional and translational levels of mRNA. (In fact, this paper cites other work with the prion Swi, a protein involved in chromatin remodeling.)
While this paper involves propagation of OCT and didn't look at any possible regulation of Cyc8 to OCT conversion, the fact that there are 11 putative prions in yeast is suggestive. Instead of relying on mutation and unchangeable consequences, perhaps some prion-like proteins can be converted between the two states. (The paper might have a citation on this; I'll need to check it out further)
Again, if you're really interested, check out the paper:
Patel B.K., Gavin-Smyth J., and S.W. Liebman. The yeast global transcriptional co-repressor protein Cyc8 can propagate as a prion. Nature Cell Biology. March 11, 2009. DOI: 10.1038/ncb1843
http://www.ncbi.nlm.nih.gov/pubmed/19219034
A prion consists of a single infective protein. Unlike other pathogens, nucleic acid isn't necessary to make future generations. Instead, a prion is capable of making more of itself by converting a particular host protein (called prion protein- PrP) into a prion. In essence, the prion protein functions to change the conformation of PrP such that it now takes on the function of a prion; these converted prions in turn recruit others. Most of the pathogenicity occurs because these prions also form aggregates in cells of the brain, ultimately resulting in death.
Prions, as we know them, are dangerous, but because PrP can be so easily converted into a prion, many groups have begun asking, "What function does normal PrP have inside a cell?” Or more generally, what are these potential prions doing there?
Eleven prions have been identified in yeast so far and are a good place to start for finding out what these things do. Interestingly, a new paper published in Nature Cell Biology (http://www.nature.com/ncb/) by Dr. Susan W. Liebman's group at the University of Illinois Chicago has discovered another method of non-Mendelian inheritance via prion function (note: this is my take, the paper is more scientifically conservative and accurate in that its title is The yeast global transcriptional co-repressor protein Cyc8 can propagate as a prion).
It's a good paper and there's a insightful comment on it in the issue, so I won't go into much detail here, but I did want to list a few key points.
Cyc8 (called OCT in its prion form) is a global transcriptional co-repressor protein that, among other things, regulates yeast growth on lactate. This protein suppresses other proteins important for growth on lactate plates. However, by transiently overexpressing the putative prion domain of Cyc8 (and thus increasing the chance of misfolding for the naturally occurring Cyc8) , the authors were able to show increased colonies growing on lactate plates versus controls. The authors were then able to do further experiments using this Lac+ phenotype
One of the most important things they did was an experiment to show that these Lac+ colonies were not spontaneous mutations resulting in revertants. (i.e. mutated nonfunctional Cyc8). Overexpression of functional Cyc8 did not restore the Lac+ phenotype.
However, as an aside, there was always the possibility of a gain-of-function mutation. Perhaps the gene required for lactate growth lost its ability to be repressed by Cyc8. I feel that simple sequencing of the most important genes involved in the Lac+ pathway could have been useful, but the authors’ main concern was elsewhere and they focused their remaining experiments in proving that OCT has all the characteristics of a prion.
This was the most fundamental, and convincing, aspect of the paper. The OCT+ mutation was dominant in an OCT- cross (not characteristic of Cyc8 loss mutations), cytoplasm of OCT+ cells could transfer the Lac+ growth phenotype, and fluorescence of OCT fused to YFP showed aggregates within the cells.
This paper continues to extend the knowledge of prion function. They ain't just fer disease anymore! Instead, proteins with prion-like characteristics might serve to regulate cell function in a rapid and inherited manner, without the need to continuously monitor transcriptional and translational levels of mRNA. (In fact, this paper cites other work with the prion Swi, a protein involved in chromatin remodeling.)
While this paper involves propagation of OCT and didn't look at any possible regulation of Cyc8 to OCT conversion, the fact that there are 11 putative prions in yeast is suggestive. Instead of relying on mutation and unchangeable consequences, perhaps some prion-like proteins can be converted between the two states. (The paper might have a citation on this; I'll need to check it out further)
Again, if you're really interested, check out the paper:
Patel B.K., Gavin-Smyth J., and S.W. Liebman. The yeast global transcriptional co-repressor protein Cyc8 can propagate as a prion. Nature Cell Biology. March 11, 2009. DOI: 10.1038/ncb1843
http://www.ncbi.nlm.nih.gov/pubmed/19219034
Sunday, March 15, 2009
Host Adaptation of Influenza, Part II
Other Influenza Proteins
Neuraminidasen (NA)
Neuraminidase is the other protein major glycoprotein on the surface of the virion. Unlike hemagglutinin, it's not involved in binding to the surface of the cell. Instead, this protein will actually cleave sialic acid from other carboyhydrate linkages. This feature is especially important in virus release; as new virons are made, hemagglutinin binds to sialic acid on the infected cells. The virus wouldn't be able to escape and spread without neuraminidase cutting sialic acid and freeing the virus.
The same function helps with virus entry. Think of hemagglutinin sticking too strongly; it wouldn't be able to change its conformation and fuse with the plasma membrane. It’s true, but I haven't come across too many papers on this subject.
In terms of host range, I'm not aware of any drastic changes that need to be made in NA in order for an avian strain to infect humans or vice versa. But keep in mind that the neuraminidase activity of any virus will evolve to be directly proportional to HA's ability to bind sialic acid (i.e. a weaker binding hemagglutinin has a weak neuraminidase)
Matrix (MA) and Nonstructural (NS) Proteins
Ok, these proteins are important. The matrix proteins do two things: M2 provides an ion channel. When the virus invades the cell, the low pH of the endosome is essentially transported into the virus (H+) resulting in release of ribonucleotide particles (the genome is released). M1 is definitely matrix and forms the outer capsid-like structure of the virus.
At least one of the nonstructural proteins slows down the host immune response by interfering with the interferon pathway (It stops RIG-I recognition of nucleic acid) The other frameshift, which results in NS1, does something else, like assisting in viral replication, but it's pretty vague for me.
So, while these proteins play major roles in virus biology, I'm not aware of any specific changes that need to occur in order for influenza to adapt from human to avian and vice versa. However, these changes do occur, they are just not studied extensively.
Next: Polymerase proteins and nucleoprotein.
Neuraminidasen (NA)
Neuraminidase is the other protein major glycoprotein on the surface of the virion. Unlike hemagglutinin, it's not involved in binding to the surface of the cell. Instead, this protein will actually cleave sialic acid from other carboyhydrate linkages. This feature is especially important in virus release; as new virons are made, hemagglutinin binds to sialic acid on the infected cells. The virus wouldn't be able to escape and spread without neuraminidase cutting sialic acid and freeing the virus.
The same function helps with virus entry. Think of hemagglutinin sticking too strongly; it wouldn't be able to change its conformation and fuse with the plasma membrane. It’s true, but I haven't come across too many papers on this subject.
In terms of host range, I'm not aware of any drastic changes that need to be made in NA in order for an avian strain to infect humans or vice versa. But keep in mind that the neuraminidase activity of any virus will evolve to be directly proportional to HA's ability to bind sialic acid (i.e. a weaker binding hemagglutinin has a weak neuraminidase)
Matrix (MA) and Nonstructural (NS) Proteins
Ok, these proteins are important. The matrix proteins do two things: M2 provides an ion channel. When the virus invades the cell, the low pH of the endosome is essentially transported into the virus (H+) resulting in release of ribonucleotide particles (the genome is released). M1 is definitely matrix and forms the outer capsid-like structure of the virus.
At least one of the nonstructural proteins slows down the host immune response by interfering with the interferon pathway (It stops RIG-I recognition of nucleic acid) The other frameshift, which results in NS1, does something else, like assisting in viral replication, but it's pretty vague for me.
So, while these proteins play major roles in virus biology, I'm not aware of any specific changes that need to occur in order for influenza to adapt from human to avian and vice versa. However, these changes do occur, they are just not studied extensively.
Next: Polymerase proteins and nucleoprotein.
Monday, March 9, 2009
Host range adaptation of Influenza, Part I
Introduction
Influenza is a major problem worldwide. Even without a pandemic year (the 1918 "Spanish" flu killed between 20-50million people), influenza kills 36,000 people a year in the US and results in 200,000 hospitalizations. Why, when people get vaccinated every year, is influenza such a problem?
One reason is that the virus is not the same from year to year. This virus can evade host immunity in two main ways. One mechanism, called antigenic drift, is when the virus gradually changes its proteins such that it is no longer recognized as effectively by the immune system. Although this results in some viral escape, there is usually enough cross-reactivity in the population so that there are no large-scale pandemics. Think of it as panting a car. It's now harder to find in a parking lot, but it still has the basic shape and look of the original enough so that you can tell the difference.
Antigenic drift can cause many problems for the host, but antigenic shift is much scarier. Instead of a gradual change in the virus, a completely new one is created. But to explain what antigenic shift is, it's necessary to delve a little deeper into the biology of the actual virus
Influenza virus biology.
Influenza is an enveloped, single-stranded negative sense RNA virus that mainly infects epithelial cells of the respiratory tract. This virus has ~11 proteins (new ones continue to be discovered) contained in 8 separate genomic pieces. What are those proteins, and what are they doing? What role do they play in host adaptation?
Hemagglutinin (HA)
The hemagglutinin (HA) glycoprotein on the virus surface binds sialic acid, ubiquitous on many cell types besides epithelial cells. (Though for the most part, influenza does not go systemic and remains a respiratory virus)
Hemagglutinin's ability to bind sialic acid is a major factor of host range. For example, the H5N1 avian virus binds alpha 2'3 linked sialic acid. Although it has been known to infect humans, one of the major reasons it hasn't been spread by human-to-human contact (except in rare, close contact familial cases) is that our lungs mostly contain alpha 2'6 linked sialic acid in the upper respiratory tract. Thus, most human-adapted influenza viruses are 2'6 linked while those of avian origin are 2'3 linked and these viruses rarely cross species barriers.
Worry about a pandemic H5N1 outbreak is centered on the virus mutating its binding preference to alpha 2'6 so that it can readily infect humans. Although this switch is the major concern, binding preference is not the only determinant of host range. In fact, much of the genome is involved.
Detour, so what actually is antigenic drift?
What happens if a virus that is human-adapted except for its HA protein suddenly acquires this H5? This isn't just science fiction. The nature of influenza's segmented genome means that these separate pieces can be mixed and matched and in the Darwinian struggle for evolution, those that have the advantage will survive (This mixing and matching between different viruses is what's known as antigenic shift). So, even though the H5 is still avian-adapted, are the remaining proteins enough to let the virus infect humans? Will this spur the adaptation of a human (alpha 2'6 sialic acid binding) H5 subtype HA?
This is the concern that keeps scientists up at night, and why it's important to continue to study influenza as much as possible. Of course, it's unlikely that a fully human adapted virus will acquire the H5 HA, or that an avian H5 that becomes human adapted will be as pathogenic as the original. Why? Well this goes back to the role the other viral proteins play in infection. So what are those proteins?
Next topic: Getting back to the rest of influenza's proteins
Monday, February 23, 2009
Statement of Purpose
What this blog is about:
Reviews in the journals are great, but sometimes a short synthesis of the basics is just as good. As scientists, we all know our research inside and out, but a larger perspective on biology as a whole is needed. Diving into unchartered waters helps draws connections about our research that we might not have otherwise seen; this is essential for progress.
Here, I am attempting to blog weekly about a specific area of immunology, briefly summarizing background and then the current state of the research. The point is not to list everything that happens with pathogen A, then B and C, but to generalize what is known and provide some thoughts for future directions. Sometimes, I will talk about only one paper. Ideally, although my main focus is immunology, this blog will also be somewhat interdisciplinary, encompassing such areas as bioinformatics, synthetic biology, nanotechnology, and genetics.
As a graduate student pursuing a doctorate in immunology, I’ve started this blog for four reasons revolving around science education and discussion.
1) To provide links and resources from people much smarter than I to people much smarter than I.
The web is full of information, so much so that now computer scientists have to develop data mining tools and strategies in order to make some sense of all this new data. Bioinformatics and computational biology are emerging fields encompassing Web2.0, but have more traditional biologists been able to keep up? I will be posting links to tools and webpages that make research easier
2) As a resource for the dreaded upcoming qualifying exam.
I’m a tactile learner, what can I say? But beyond that, in order to write about a subject clearly, I have to know it inside and out. And, this is more interesting than reading a textbook. I also hope other students will find the information posted here useful.
3) As an educational tool.
Ever read a thick primary paper? How many papers did you have to backtrack before you found out what the authors were actually taking about when they used transgenic XYZ system (validated in paper 1) to extend research (first described in paper 2) on a new T helper subtype ( reviewed in paper 3)? Besides reviews in the top journals, another perspective might be useful.
4) Incite discussion
Science is always a discussion of ideas. That’s where progress is made.
Reviews in the journals are great, but sometimes a short synthesis of the basics is just as good. As scientists, we all know our research inside and out, but a larger perspective on biology as a whole is needed. Diving into unchartered waters helps draws connections about our research that we might not have otherwise seen; this is essential for progress.
Here, I am attempting to blog weekly about a specific area of immunology, briefly summarizing background and then the current state of the research. The point is not to list everything that happens with pathogen A, then B and C, but to generalize what is known and provide some thoughts for future directions. Sometimes, I will talk about only one paper. Ideally, although my main focus is immunology, this blog will also be somewhat interdisciplinary, encompassing such areas as bioinformatics, synthetic biology, nanotechnology, and genetics.
As a graduate student pursuing a doctorate in immunology, I’ve started this blog for four reasons revolving around science education and discussion.
1) To provide links and resources from people much smarter than I to people much smarter than I.
The web is full of information, so much so that now computer scientists have to develop data mining tools and strategies in order to make some sense of all this new data. Bioinformatics and computational biology are emerging fields encompassing Web2.0, but have more traditional biologists been able to keep up? I will be posting links to tools and webpages that make research easier
2) As a resource for the dreaded upcoming qualifying exam.
I’m a tactile learner, what can I say? But beyond that, in order to write about a subject clearly, I have to know it inside and out. And, this is more interesting than reading a textbook. I also hope other students will find the information posted here useful.
3) As an educational tool.
Ever read a thick primary paper? How many papers did you have to backtrack before you found out what the authors were actually taking about when they used transgenic XYZ system (validated in paper 1) to extend research (first described in paper 2) on a new T helper subtype ( reviewed in paper 3)? Besides reviews in the top journals, another perspective might be useful.
4) Incite discussion
Science is always a discussion of ideas. That’s where progress is made.
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