On Recognizance of Competence

Recently I had some business cards with my personal contact information printed up to help me network more effectively and remain in touch with the awesome people involved in science and technology that I've been meeting. As these aren't for work, per se, they have a picture of a Velociraptor skeleton, a quote from Mark Twain ("The more you explain it the more I don't understand it." A good reminder to keep things simple and short, I think), and "Awesome Scientist #17" as my job title, even though it doesn't signify exactly where I work. I'm not sure exactly who the other 16 are. About this time, I realized something: people think I'm smart. This is strange.

I don't know that I'm smart. I don't feel any differently and I cannot imagine being any other way. Frankly, most of the time I feel stupid because there is so much that I do not know and that which I have captured in my mind is so insignificant compared to how much awesome there is to learn. I only know that I am smart by interacting with other people and seeing the reflection thereof. I used to be more reclusive and introverted, counting bacteria by day (doing science) and making music alone at night, but now I have been forcing myself out of that bubble and taking on challenges and actually talking to real people*, doing SCIENCE in the new lab job, and dropping awesomeness and scientific knowledge through the informal molecular biology classes I've been teaching in the Makerspace (Youtube soon). Through all of this, I have come to realize that people actually value my knowledge, experience, perspective, and opinion. I have more opinions than anything else, but it's still a bit strange because I still feel as though I haven't done anything truly awesome that deserves any respect yet.

I probably never will. I realize this, because I know that my ambitions and analytical nature will never let me rest because there will always be more science and/or awesome always going up or something else shiny will captivate my interest. The positive side of this, I think (self-delude?) is that it'll keep me busy, and that's a very good thing as a bored Toaster digs out his backpack of tools and starts taking things apart. The other positive of this is that by taking on more, I hold myself to doing more and am forced to learn how to synthesize all the enzymes to help masticate all that I've bitten off. I used to read scientific papers for fun, but now my boss sends me papers faster than I can read them. I used to make music just for my own enjoyment, but after doing in impromptu live set I have been volunteered to make live music at an art show in 10 days (and all I have prepped are 2 remixes of the Leekspin song). I used to tinker using just my own limited tools and experience, but now that I've helped create a cooperative makerspace I have access to a much wider range of tinkerables and tools (I even coined a new term last week; making a silly, simple mistake now has the swear word: "BLOOTLELOOT!" attached to it after I mistook a diode for a resistor and soldered it to an LED). I used to play with data for weeks just out of curiosity, long after the main aim of the experiment had been answered, just to see what I could draw out of it, but now I have so many data sets to mine that pure play has transmuted into teasing the data for the prettiest results.

These are good changes. It was silly of me to ever think I should first know what I was doing before trying. This is much more informative.

*Not that bloggers aren't real people, but Internet-based communication is easier because it has a backspace key.

AHAA Addiction

Every day we labor in the lab, hoping to build knowledge into a coherent and publishable narrative, pursuing understanding. Often I have heard scientists speak of the "A-HAAAA!!!!!" moment where months' worth of laborious experiments snap into place and reveal a new bit of truth, and how addicting that moment is, how it is akin to pure joy or The Perfect Cookie. This is a wonderful goal, and somehow it keeps us working long hours for little pay and even less respect. But, admittedly, it's hard work.

I have found that every once in a while, as I'm reading along through some papers, I come across a well-supported point or elegant experiment that is just SO DAMN AWESOME that my mind has to stretch a little farther to accommodate the new view of the Entire Frickin' Universe that that paper has just jammed into my eye sockets. It's like licking lightning*, but less painful. And these papers don't have to be recognized across the scientific sea as revolutionary to make that difference. I still know approximately nothing, inasmuch as my scientific knowledge is but a 5uL drop in a 3L pond of ignorance. And as such, papers that fasten together what were previously disparate informations (for me, although that may have already been old hat to the greybeards) can be just as mind-boggling.

Apparently I have a knack for finding these papers. The other day in the lab after his committee meeting, the grad student turns to me and sez, "You failed me there! You're my Master Downloader, and you didn't catch the paper that completely changes 1/5th of my thesis!". He was kidding, I think. I have yet to read the paper in question. From the abstract, it's been recently found that IL-12Rp35 doesn't just participate in IL-12 signaling, but also in IL-35 signaling, which has an antagonistic effector function against IL-12 (IL-12 is pro-T_H1 T-cell response, IL-35 is pro-Treg T-cell response). This is on top of previous knowledge that the IL-12Rp40 subunit is also part of the IL-23R complex (IL-23 is involved in T_H17 T-cell differentiation and function).

These frequent revolutions of our understanding are unequivocally Good Things. Sure, it makes our lives difficult and increases the range of possibilities in which we make asses of ourselves in front of our colleagues, but at the same time it's the very stuff Science is made of. If we cling to out-dated theories and disproven information, we will not produce accurate new information and we will fail as scientists. This necessary nimblemindedness is sometimes frustrating, but at the same time it allows us to have those A-HAAA!!!!! moments all the more often without doing all the benchwork ourselves.

To date, there are 3 papers that stick out in my mind as having been particularly eye opening:

1) Käfer J, Hogeweg P, Marée AFM (2006) Moving Forward Moving Backward: Directional Sorting of Chemotactic Cells due to Size and Adhesion Differences. PLoS Comput Biol 2(6): e56. doi:10.1371/journal.pcbi.0020056

I didn't see this when it first came out, but when I finally read it and worked through the differential equations it really made a huge difference in how I view organogenesis. I wasn't much interested in the functional dynamics of Dictyostellium morphology, but the work on cell sorting and migration based entirely on size and net kinetic energy was really really really cool. Blogged here.

2) Venegas J, Winkler T, Musch G, Vidal Melo M, Layfield D, Tgavalekos N, Fischman A, Callahan R, Bellani G, Harris R (2005) Self-organized patchiness in asthma as a prelude to catastrophic shifts. Letters to Nature. doi: 10.1038/nature03490

This was some major computational sweetness wrapped up in massive physiological awesomeness. It integrated all the separate physical forces at work on individual alveoli to integrate them into a model of whole-lung pathology. This was tight work, very well-done, and immediately relevant to clinical practice as their model revealed that inhaled anti-asthma drugs would likely affect only the non-spasming portions of the lung airways, although it also didn't exactly make an alternative recommendation. Anyway, still relevant. Blogged here.

3) Artis D. Epithelial-cell recognition of commensal bacteria and maintenance of immune homeostasis in the gut. Nat Rev Immunol. 2008 Jun;8(6):411-20. doi: 10.1038/nri2316

This paper assembled a major heap of evidence and thoroughly blasted the simple view of intestinal epithelial cells as humble nutrient transporters that I had been taught to ragged shreds. Intestinal epithelial cells aren't just lining for the gut, they actively interact with the massive volume of microorganisms inhabiting our guts and modulate both the ecology thereof as well as the responsiveness of the innate and adaptive immune systems. Intestinal epithelial cells even express MHCII! As far as I can discern, this paper has helped to provoke a flurry of gastroenterology papers that discuss the rapidly evolving view of the microbiome, even going so far as to label our intestinal microbiome another metabolic organ**. Blogged here.

So, dear reader, what papers have changed your world-view and why?

*Don't try that at home. It tastes terrible.
**If you want to look smart to a gastroenterologist, drop "TSLP" casually into conversation. TLSP = thymic stromal lymphopoeitin, it's secreted by intestinal epithelial cells to attenuate inflammatory T_H1-type immune responses to the bacterial antigenic signal from the gut microbiota. It kinda skews the response towards T_H2 effectors instead.

Reading

Aside from scientific papers and comic books*, I don't do much reading any more. I can't remember the last new thing I read for fun. Maybe it was World War Z by Max Brooks, and that was well over a year ago. I mean, I have read Neuromancer by William Gibson since then, but I tend to do that annually, so it doesn't really count.

The problem is that whenever I sit down to read anything, even a comic book, which is quick and awesome, I feel guilty that I'm not off reading scientific literature instead. I've got a stack of unread papers rapidly approaching the thickness of my head, and I have a large noggin. I see something that looks really interesting, read the abstract, and print it off to read later. Problem is that all too often it doesn't actually get read later, it just gets buried under everything else I've been meaning to analyze and forgotten.

But at the same time I really miss curling up with a good book and reading away.

So, dear reader, what do you recommend? Any great books you've recently parsed?

*Although I am also reading "Makers" by Cory Doctorow as it comes out, serializedy, on Tor's website. It's what made me realize I miss reading stories.

Most Nefarious Competition

This will not do.

NOT AT ALL!

Some of you may have noticed a recent poll over at the cave of mein übel-(ekel)-archenemy Mein Hermitage. The poll asked: "Who's more nefarious? Toaster, Hermitage, Ninjas, or Pirates?"

This results are in, and Hermie and I have tied.

TIED!!!

How is this even possible when I am clearly far more nefarious than her!?

So to settle this, we had a cease-fire dinner of cookies and Bento and discussed methods by which we might resolve this. Although we each generated several good ideas, from a race to discover a new metazoan species and then find a way to cook it to a giant robot battle tournament to an air guitar competition, we also found that each of these proposals lacked one critical element to ensure a fair outcome: that of transparency.

Without transparency we could each easily engage in heinous subterfuge and use our respective resources to set traps. As such, we eventually found, the fairest way to settle this matter is a Battle of Scientific Acumen and Wit!!!!!!!!!!!!

Therefore Hermie and I will be facing off in the commons of the Internet and battling through the cyber journal club format, one round only, winner takes title of Most Nefarious*.

But we need your help. Dear reader, we are surrendering the material and terms of this contest to you. Here's how it'll work:

1) Submit interesting and substantial papers to Hermitage and I. For the sake of fairness it should be something outside of our respective purviews (Hermie = Peruvian Flying Fish, Toaster = Immunology).
2) Hermitage and I will publicly deliberate upon the papers and choose one to compete upon. This part may take a while.
3) We will each post a blog entry about the paper.
4) You will judge it, and by extension, us. We understand that some of you may have mixed loyalties, so to make it fair, each reader gets 100 points that they can divide between Hermie and I as they see fit. Points can simply be assigned in comments, which will remain open for 96h following posting.
5) Winner will be chosen by who has more points.

So reach into that stack of papers you keep meaning to eventually read and send them on in. This matter MUST be settled!

*Trophy to be displayed in blog side-bar.

5.4 #2: In Which Toaster Is Mostly Incoherent and Unintelligible

1) I think I've found a new favorite word: "der Unfug". Short, simple, sweet, and too the point. It's a synonym of "der Blödsinn" meaning 'nonsense' or 'mischief'. I mean, you can't invent such a great word out of the air, and even better that the opposite of Unfug exists only as an idiosyncronism "mit Fug und Recht" meaning 'with order and justice'. Sometimes I really do wish English were as silly as German, it'd make speaking a lot more fun.

2) An diesem Punkt soll ich es verkünden, dass jetzt meine lieber, aber unheilvolle und finstere Schwester Verhängnis, und ich einander mit "Zum Unfug!" grüssen soellen. Wenn ihr alle es woellt, koennen wir auch diese Begrüssung als verborgene Meldung nützen, um zu zeigen, dass wir (verrückte) Wissenschaftlern und Wissenschaftlerinnen sind. Manche von euch schriebt, dass ihr die Lust zu einem Ehrfurchtgebietenden Wissenschaftlersverein schaffen haben. Veilleicht koennen wir hier anfangen. [Für einer geheime Grund arbeitet meine "oe" Täste nicht...]

3) Presentation went OK today. I presented, for really real journal-club kung-fu style, the paper that I research bloggered here. I included about a dozen slides of immunology run-down for the people in my lab group meeting that don't know much about it, and then ran through the paper itself for another 15 slides. I was told that I'd done a very good job breaking down a very large and complex paper, and best of all neither my boss nor TechnoGrad, who've been drinking the Immunology Champagne for far longer than I, disputed my immunology run-down! This must mean I am finally getting somewhere with that whole learning thing, which is somewhat relieving. Getting into immunology is awesome, but it's so deep and complex and chaotic and, most of all, dependent upon very finely detailed contexts that at first I felt like I was drowning in a slurry of cytokines, cellular determinants, and maturation dynamics.

But it was due to my inexperience with immunology that I didn't catch the many problems with the paper that TechnoGrad and my boss did in just their cursory examination. For example, the Ohnmacht et al 2009 paper had a flow cytometry figure showing increased CD4+ T-cell infiltration into different tissues (axes were stained for CD4 and CD8) that showed massive increase (21%) CD4+ T-cells in kidneys of mice without dendritic cells and they explained this as autoreactive T-cells surviving without tolerogenic dendritic cells. But the control kidney had 5% CD4+ T-cells, which apparently is really really unusually high. Materials and methods said that these were obtained by collagenase digestion of whole tissues into single cell suspensions, but then they didn't mention whether the 21% and 5% were of total cells isolated or just total lymphocytes isolated and didn't even mention or show their side- or forward-scatter gating. I had no idea that 5% CD4+ T-cells in the kidney was unusual as the papers I've been reading about lymphocyte dynamics in peripheral tissues make it sound like we're crawling with lymphocytes all the time. So it didn't set off a flag and I'm kind of left feeling like I should have caught it.

But I guess that's what journal club stuff is for, to teach us all how to read papers more critically and extract useful information.

Not-So-Mad Science: IL-13 vs. IL-4 In The Battle For Asthma!

(Previous asthma research-blogging here)

Marsha Wills-Karp, Jackie Luyimbazi, Xueying Xu, Brian Schofield, Tamlyn Y. Neben, Christopher L. Karp, Debra D. Donaldson (1998). Interleukin-13: Central Mediator of Allergic Asthma Science, 282, 2258-2261

Abstract:
The worldwide incidence, morbidity, and mortality of allergic asthma are increasing. The pathophysiological features of allergic asthma are thought to result from the aberrant expansion of CD4(+) T cells producing the type 2 cytokines interleukin-4 (IL-4) and IL-5, although a necessary role for these cytokines in allergic asthma has not been demonstrable. The type 2 cytokine IL-13, which shares a receptor component and signaling pathways with IL-4, was found to be necessary and sufficient for the expression of allergic asthma. IL-13 induces the pathophysiological features of asthma in a manner that is independent of immunoglobulin E and eosinophils. Thus, IL-13 is critical to allergen-induced asthma but operates through mechanisms other than those that are classically implicated in allergic responses.

There are many morbidly fascinating pathological changes associated with onset and clinical asthma. To wit, these include eosinophilia, mucus overproduction, mast cell/other inflammatory cell airway infiltration, and increased smooth muscle. There may also be scarring of the airways.

From what I currently understand about it, airway hypersensitivity generally happens after immune effector cells have infiltrated the underlying airway tissues. When these effector cells, which can include allergen-specific T-cells, mast cells, eosinophils, basophils, and even macrophages, are activated by an irritant (the allergen) they more or less cut loose and let wild with the localized inflammation. The localized inflammation, in turn, leads to more immune cell infiltration over time and concurrently the tissue itself undergoes histopathologically apparent changes, including thickening of the base layer of smooth muscle.

For example of immune effector cells getting activated, let's consider the most dramatic case: the mast cell. Mast cells are a type of white blood cell that expresses Fc receptors for IgE (IgE is the immunoglobulin most associated with allergic and anti-parasite responses) on it's surface. The Fc-bound IgE act as allergen-specific receptors that, when bound to their ligand, send a signal into the cell to degranulate. Mast cells store relatively massive amounts of inflammatory cytokines and peptides in large granules (e.g., histamines, prostaglandins, and leukotrienes) and they can, effectively, disgorge them all at once. This can lead to a very rapid spike in the systemic concentration of inflammatory effector molecules and subsequently extremely rapid onset of asthmatic symptoms. The same process is at work in acute food allergies.

Figure A: The mast cell is the one with the big lumpy nucleus in the center. The black dots surrounding it are granules packed with inflammatory molecules, just waiting to be released and wreak havoc. Those other 2 cells to the right are lymphocytes (according to the original caption on this TEM).

But what inflammatory molecules are required to invoke and/or sustain a hypersensitive airway response?

This paper examined the role of IL-13 in allergic asthma. According to Janeway's Immunobiology, IL-13 is involved in the differentiation of naive CD4+ T-cells into TH2 cells, which have been shown to be more involved in allergy than TH1 cells. IL-13 is also secreted by TH2 cells, apparently, and has been shown to have a direct effect on airway epithelial cells by which their proliferation in increased and differentiation into goblet cells (goblet cell metaplasia) is increased, which in turn leads to the increased mucus production seen in allergy and asthma. And when your organs are infected with multicellular parasites, IL-13 is there to help the organs make the changes they need to get rid of those parasites. And as if that weren't enough, IL-13 also increases smooth muscle contractility.

But IL-13 doesn't really do anything without the context of a TH2 immune response. Th2 cells are characterized by secretion of IL-4, and it should be noted that IL-4 and IL-13 share a subunit in their receptors.

Figure B: The left column has a normal lung biopsy (top) and a normal airway (bottom) from a Tbet+/+ mouse. The right column has the same measurements, but showing airway inflammation with lymphocyte and eosinophil infiltration (top) and remodeled airway with increased collagen (bottom) from a Tbet-/- mouse. The picture is blurry because I took it with my phone. It is from Janeway's Immunobiology, 7th ed., page 575. Tbet is a transcription factor that is necessary for the development of TH1 cells, so its abscence will invariably result in a TH2 inflammatory response (right column). Tbet is analogous to GATA3 in TH2 cells.

Allow me to explain T-cell differentiation really briefly:

1) Naive T-cells arrive in thymus.
2) Naive T-cells have to decide whether or not to be CD4+ or CD8+, which will result in being able to recognize MHCII or MHCI, respectively.
3) CD4+ T-cells get stimulated by DCs or stuff, and the resulting cytokine mileau determines whether they become TH1, TH2, Treg (also refered to as TH3), or TH17. They can also become memory T-cells of any of those variety later on in. Respectively, these cell types are characterized by secretion of IFNg, IL-4/IL-5, IL-10, and IL-17.
4) TH1, TH2, Treg, and TH17 all more or less have distinct biological roles, although the cytokine soup that gives rise to different types is messy (e.g., IL-2 just drives T-cell proliferation irrespective of subset) and often overlaps, and they'll even compete against each other (IL-12 drives TH1 proliferation but inhibits TH2 proliferation while IL-4 does the same for TH2 cells).

So, anyway, the group behind this paper found that while IL-4 is sufficient to initiate asthmatic events, IL-13 is required for the development of the airway hypersensitivity response (AHR). They used the standard ovalbumin (OVA) induced model of AHR and found that blocking IL-13 with an neutralizing fusion protein prevents the development of AHR. Apparently blocking IL-13 in mice who already have AHR results in their measures of AHR decreasing (specifically goblet cell metaplasia and mucus production). However, with all of this, blocking IL-13 had no effect whatsoever on net circulating IgE or eosinophilia.

These findings prove that IL-13 has a significant role in asthma. But they also imply that IL-13 does not play this role through classical allergy pathways, as IL-13 is found to be elevated in patients with both allergic and non-allergic asthma. This is further supported by the group's finding that daily intratracheally administration of IL-13 is sufficient to induce asthmatic pathology even in the abscence of antigen sensitization.

What I wonder about here is: how does it make biological sense for a molecule involved heavily in the production of allergen-specific TH2 cells to also operate completely independently of that cellular phenotype?

But what is important to human health is that this paper demonstrates that adminstration of IL-13 agonists or blockers may be of great therapeutic value to human asthmatics. This paper is 11 years old, and I don't currently know if anything has come of their findings, but still, it'd be cool if this really did have therapeutic value because, as my last post on asthma discussed (link up top), inhaled acute anti-inflammatories may only be getting to the pieces of lung that need it least (because they're the pieces that can still pump air, and if reacting tissue isn't pumping tidal volume, how can inhaled medicine get to it?). If this could be used daily as a preventative, I think it could greatly improve the quality of life for asthmatics everywhere.

Mendeley

Mendeley just ate ~2.5h of work.

Mendeley is a free (for now at least) program that aims to do much what Endnote, Zotero, and Papers do, but to do so better. It's main edge seems to be that you can outsource your reference library's metadata, and even the .pdfs themselves, to their cloud and thus carry them around from computer to computer without having to continually update lagging versions of Endnote that run on different machines. This seems like a good idea so I figured I'd try it, especially since Endnote has a very annoying habit of freezing my lab computer.

Mendeley's good ideas:
1) Keep reference metadata and .pdfs in the cloud for maximum portability.

2) Automatically parse .pdf data.

3) Parse and display all references of all .pdfs added.

4) Full text-searching and user-editable tagging.

5) Social sharing of your library, notes, and metadata with other users. This seems like it'd be really useful for grad students in the same lab, post-docs in a new lab, and collaborating prinicple investigators.

Sounds good in theory, right? Yeah! However, even for a beta, Mendeley is rather clunky and awkward.

Mendeley's awkward dance moves:
1) Syncing the reference metadata I added (tags, abstracts, notes, etc.) to the cloud completely erased everything I had added. Sure, the .pdfs are now in the cloud, which is convenient, but ALL my notes about the papers are gone like dust. So is all of the cleaning up after Good Idea #2.

2) I didn't expect this to be perfect, and I was fairly satisfied that it worked perfectly in ~15% of papers and got most of it right in 75% more. But now that I've fixed it, and then lost all of that in syncing it, I'm not eager to go back and have to do all of it again.

3) Usually works. Doesn't seem to recognize most, or any in some cases, of the references in papers where the references are formatted at 50% the font size of the text. Haven't tried adding files or notes to references yet, or clicking through to their abstracts.

4) Works, but sometimes pulls in "accepted day X, month Y, year Z" or pieces of correspondence information as well. And not all papers have keywords set aside for this kind of parsing, which the program shows as a big angry exclamation point.

5) I have yet to try this. I figured I'd give it a spin after syncing by data, but now...well, not right now.

I'll most likely give Mendeley another try sometime soon, especially if the bug of deleting corrected metadata (and yes, I remembered to save) when syncing gets fixed. It looks promising and I hope that it is able to remain free (or at least, free for beta adopters?).

At the moment, I'm just glad that I didn't rashly delete my Endnote library.

UPDATE:
I contacted Mendeley about the problem and we're working on it. Hopefully their fix will work. Also, they apparently Google themselves daily, so beware (they're watching).

UPDATE 2:
Fix worked. Now I have 2 folders, one with raw extracted .pdfs, the other with my annotated .pdfs. However, I'm not sure how I'm going to maintain them both.