Wanted: Dead or Alive

It would be very useful for the immune system to be able to discriminate between live and dead bacteria.

Well, that's pretty obvious.

Immunologists have long known that live bacteria induce different, and better, adaptive immune responses than dead bacteria. Again, it may seem obvious, but is hard to reconcile with current thinking about how adaptive, or antigen-specific, immune responses develop. Robust adaptive immune responses need two things: the antigen that makes antigen-specific T and B cells expand, and inflammatory signals, which tell the immune system that something wrong or dangerous is around and that its okay to try and eliminate the sources of the antigens they recognize.

(This is not the whole story, but its a good starting point, and the whole point of this blog is to try and understand the point of immunology and not to get hung up on details.)

Anyway, seminal and beautiful work by many people, primarily Charles Janeway and Ruslan Medzhitov (you'll definitely hear more about him here, I really enjoy reading what he writes), found that those inflammatory signals could be triggered by specific components of microbes, called "patterns". In other words, when the immune system ( the innate immune system, have I mentioned how strange I find these categorizations? I digress) sees a molecule X, which is made by almost all bacteria of a certain kind, it responds because X is a pattern, characteristic of "bad" bacteria. So if you combine foreign antigens and pattern X, you should get nice robust responses to the antigens because you have both antigens and inflammation.

And you do get beautiful, strong responses to antigens this way. The only problem is that dead bacteria usually retain their patterns, like X. So there must be something else that sets live bacteria apart from their dead counterparts, something other than pattern X.

In a simple, elegant study published in Nature, Julie Blander's lab at Mount Sinai found a possible candidate-a pattern that is characteristic of bacterial life, a vita-PAMP they call it ( not my favorite term, but...). They show that ribonucleic acid, or RNA is rapidly lost from bacteria when they are killed, and that if you just add RNA back to dead bacteria, they become as good at inducing strong immune responses as live bacteria. Specifically, they found that messenger RNA (mRNA), so called because they carry the information coded by a gene in a message that is "translated" into a protein, is a vita-PAMP.

What is so strange, and so cool, about their findings is that they found that all bacteria had vita-PAMPs and the ability to induce certain immune responses. Not just pathogenic, or harmful bateria, but al bacteria, including the billions that live in our digestive tracts and at our mucosal surfaces, our commensal families. Why then do we not explode in a mass of inflammation and immune responses-after all we share body space with billions of bacteria? Is it because of strict specialization and compartmentalization: are the cells that recognize vita-PAMPs far away from our natural bacterial buddies, so that their detecting vita-PAMPs is a sign that bacteria are where they have no business being? Or do our immune systems keep responding to vita-PAMPs at a low level, and so get de-sensitized? Or, to get a little fanciful, is it good for us to keep responding to some levels of vita-PAMPs all the time-keep the immune muscles warm and in tone?

Much food for thought. And the nice thing? All this comes from such a simple question and an old observation-someone just asked "But, why?"

(Kind of like-why do ultra-tired babies cry their heads off instead of going to sleep? It's so obvious, you're tired, go to sleep. Don't sit up in bed, throw all your cuddly sleep toys out of your crib and howl to the high heavens. Apparently it is not that obvious. Sigh.)

It's been a while

It's been a long time since I wrote here. I started blogging as a new postdoc, to vent about the frustrations, and to join the community of scientists on the web who describe the vagaries of the scientific life.

I haven't enjoyed being a postdoc very much, and I'm distinctly ambivalent about still having the same job. There are many ups to being a postdoc and many downs, but I' not going to talk about them anymore, there are so many people who say it better. Besides, all that talking blinded me to why I do it in the first place: I think the immune system is pretty damn awesome, and I find something interesting to read about it nearly every day.

A lot has changed since I last wrote here: I have an energetic toddler, I'm far more downbeat about my ambitions to cure disease, I'm tired, I feel guilty most of time, and I'm afraid I'm losing my spark, the zip that makes my mind interesting and unique. All of which said, I find that I'm still jazzed about immunology, and I'm going to just try and focus on that. The ideas, the innovations and the incredible pace with which our understanding of the immune system progresses.

So the next post will be about my new favorite idea. And will be posted in a time period less than three years.

Ending a Statement With a Question Mark?

Have you ever conciliated your way through a scientific discussions by ending negative statements or disagreements with an implied question mark? I do it a lot, and I'm not sure I like the reason why.

As a woman (and maybe as a man too, though I have not had direct personal experience with this, any insight would be welcome), I tend to walk the fine line between directness and conciliation. I am naturally emphatic, and used to state my opinions strongly-maybe too strongly-carried along by the force of my convictions and the absolute determination not to give in to anybody. Some of this crazy certainty faded with age and some wisdom, but I still am pretty definite in what I believe and inclined to be forceful in what I say.

One big reason why I stopped being loud and emphatic and thumping my metaphorical fist on the table is because I realized I hated being at the receiving end of such treatment. I hate being steamrollered, its uncomfortable and puts me off the discussion. I also found that not thumping table meant I could hear other people, a nice change. So politeness and a genuine curiosity to hear other people's thoughts started me off on my path of less declaiming and more questioning. As an added bonus, people warm to gentle conciliation more than they do to vocal steamrollers.

That there is the rub: within the reasonable demands of courtesy, how much should one conciliate? It's a reflex now, I always take the diplomatic path rather than just say what I think outright. I like to think that I stand by my convictions-I'm just more mellow about them-but is that really true? Have I gone too far down the road of conciliation? In lab meeting particularly, or during seminars, I ask questions and challenge people almost apologetically. In some cases, it makes the questioned feel more comfortable, and in some cases it makes them more dismissive of your question. Is a reputation for being thoughtful and a disinclination to put people on the spot worth being dismissed?

Why so I have this need to instinctively subdue my challenges? The saddest thing is that I think I do it because I am a woman. There are men in my group who are distinctly less indirect, often outright rude and in-your-face with their challenges and it doesn't affect the esteem in which they are held. And in the most bitter of stereotypes, when other women do the same they are called aggressive, bitchy and unpleasant. I used to be against overt feminism because I thought it was loud, exclusionary and exhaustingly unproductive, not to mention bound to get you laughed at. It galls me though that the natural instinct of intelligent ambitious women of my generation is to tone it down, to try and not become one of those women who became PIs in the 1970s and -80s. While I am certainly not a fan of rudeness or putting someone else down out of a sense of your own superiority, I am so tired and fed up of ending every sentence with an implied question mark.


This post is my 50th on this blog(I'm slow), and its also for my mother, the kind of feminist I would like to be.

A Link Between Environemental Toxins and Autoimmunity?

The title of a paper in the advanced online publication section of Nature refers to a possible link between environmental toxins and autoimmune disease: an irresistible hook, and of course I could not resist. So followed some excited perusal of Veldhoen et al (doi:10.1038/nature06881) and another paper on the same subject, Quintana et al (doi:10.1038/nature06880), also in the AOP section.

So is there a link between environmental toxins and autoimmunity? Well, maybe, though I am not entirely convinced of that aspect of things, and here is why. Both papers discuss the effect of triggering the aryl hydrocarbon receptor (AHR) on T cells (description in the sidebar, for those of you are interested). The AHR, to my chemistry-ignorant mind, is a protein that binds hydrocarbon molecules with aromatic rings. The AHR is a type of protein, called a transcription factor, that decides when genes are turned on. In tune with this known ability of the AHR, both studies find that triggering the AHR on a subset of T cells called helper T cells turns on genes that influence the function of these T cells.

Helper T cells can direct the immune response along different paths, towards a more allergic response, one that is better suited to the fighting of infection, or towards a more inflammatory response which can result in the immune system attacking oneself—“auto”immunity. Helper T cells work by producing cytokines, small proteins that zip around the body carrying messages from one cell to another. Helper T cells that produce the cytokine IL-17, called Th17 cells, play a large role in autoimmune diseases: for instance, blocking IL-17 in a mouse model of multiple sclerosis, experimental autoimmune encephalitis (EAE), protects mice from getting the disease. Other helper T cells—regulatory T cells or Tregs—combat autoimmunity by battening down active T cells, and giving mice Tregs can protect them from a variety of autoimmune diseases. So helper T cells can go either way, either promoting or suppressing autoimmune disease.

Veldhoen et al find that triggering the AHR on helper T cells through its ligand FICZ* enhances the generation of both mouse and human Th17 cells. Treating mice with FICZ also accelerates the progress of EAE, suggesting that triggering the AHR enhances autoimmunity. FICZ is generated by UV treatment of tryptophan, an essential amino acid, and the authors suggest that it may be generated by exposure of the skin to UV light, thereby connecting environmental factors to an autoimmune disease. A connection I find a bit tenuous, but the idea is interesting and novel, and I am very curious to know what they find next.

Quintana et al present a more detailed study, with results that suggest something different. They find that triggering the AHR through a more famous ligand, TCDD**, leads to the enhanced development of Tregs, the tranquilizers of the immune response. Now, TCDD is thought to be very toxic, and is found in the environment (most infamously, in Agent Orange), but it seems to actually counteract autoimmunity, because treating mice with TCDD considerably slows down their development of EAE. Qunitana et al also agree that FICZ treatment enhances the generation of Th17 cells, and simultaneously retards Treg formation.

Both compounds bind to the AHR with different affinities, and possibly in different ways, which might explain their opposing actions. In any case, aromatic hydrocarbons are capable of modulating the immune response and shaping its eventual outcome, an exciting and novel set of findings. That they have such strong effects (reversing or accelerating EAE is pretty strong) certainly suggests that they bear further study, and back up the idea that things in the environment can have an effect on our immune systems. After all, having too many Tregs because you were exposed to TCDD may save you form some autoimmunity, but too-tranquil T cells are not very good at fighting off infections. Similarly, too much sun can turn tryptophan—which we absolutely need—into FICZ, which can then diffuse off into our bodies and do something to exacerbate autoimmunity somewhere. Food for thought.

*6-Formylindolo[3,2-b]carbazole (FICZ)
**2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin)

The aryl hydrocarbon receptor links TH17-cell-mediated autoimmunity to environmental toxins
Marc Veldhoen, Keiji Hirota, Astrid M. Westendorf, Jan Buer, Laure Dumoutier, Jean-Christophe Renauld & Brigitta Stockinger
doi:10.1038/nature06881

Control of Treg and TH17 cell differentiation by the aryl hydrocarbon receptor
Francisco J. Quintana, Alexandre S. Basso, Antonio H. Iglesias, Thomas Korn, Mauricio F. Farez, Estelle Bettelli, Mario Caccamo, Mohamed Oukka & Howard L. Weiner
doi:10.1038/nature06880

Multi-tasking Madness

"I hate running PCRs to type mice, because I could have been doing X experiment instead."

No I could not have done X experiment instead. I have two hands and one head and unless I can master the usage of my toes, its one thing at a time. The typing of mice (or testing of serum for tissue culture, restocking of boxes, running of FACS samples) is as essential as anything else, despite being boring as hell. This feeling of "I could have been doing something else, obviously more useful or constructive" is a fallacy, and a stressful one at that.

I feel such a strong impulse to multi-task all the time, but often its the multi-tasking that slows me down. I am naturally a quick mover and I tend to dart around doing things, keeping myself busy. This tendency goes to the extreme in lab, where I don't feel occupied unless I have three threads running. And guess what? I make mistakes and have to repeat things, which adds to my not-inconsiderable work load (I have a particular PCR jinx).

There is this drive today to get things done, faster and better, now instead of soon. Lab work is easier and research is more competitive, so it's easy to see how this has come about. Is it constructive though? If something takes a certain amount of time to do, what is the point of rushing it or wishing it along faster? Or, inserting other tasks into the gaps? One does get more done eventually, but at more cost to oneself. And is that really efficient in the end?

I have discovered that I do not like operating at full stretch all the time. Sometimes I really enjoy it, I am in the zone and buzzing. But the rest of the time, I think my research would be better served by my working with more discipline. Unfortunately, when one can work in the zone, one starts to expect it of oneself always, and that is just not realistic. So I am going to run PCRs and DNA gels, and only those, this Saturday and look out of my window at the sunny trees.

(I might reshuffle my papers, read a few, trackback a few references, no matter.)
(And yes, its Saturday, I know)

Postdoc as Domestic Goddess (or God)

Skill 1: Combines ingredients with precision, follows instructions to the letter, but adds the occasional touch of improvisational flair with interesting outcomes. Occasionally works with the reagents at hand instead of what may be ideal, with perfectly acceptable results. Not afraid to use alcohol as necessary.

Skill 2: At the end of a process, puts everything away in its allotted space, wipes down all work surfaces, neatly collects trash. Surveys area, spots missed spots with the eagle eye trained by years of experience. Has no patience with others' sloppy work areas, is disgusted by ill-maintained and unclean surfaces. Is zealous about the weekly wipedown. Again alcohol accompanies as needed.

Skill 3: Monitors the health, diet and breeding habits of several small dependent creatures. Always has a mental map of which little creature is where.

Skill 4: Plans menus and processes days ahead. Has activities allotted to each day, to be carried out according to a strict schedule. Adheres to said schedule with admirable focus.

Skill 5: Does the shopping for supplies as needed. Prefers to do so on a regular schedule (schedule is important), but is willing to pop down to the shop when running short. Combines trips to shop with transport of laundry.

An alternative career maps itself out...

Blog List

Oh I am so stoked by this new feature on blogger (Draft blogger for now), it imports blogs from my google reader and posts them in my blog list! That is so good because I could not figure out blogroll and have been feeling bad about being a lazy one who hasn't manually entered everyone yet.
Love it. Because you are all awesome bloggers, and I am so glad to have found you.

Paranoia and the Destruction of the Soul

I wanted to write a happy post today since I'm in a really good life-mood (research mood is low-level fatalistic tending towards zen acceptance), but I've been thinking about paranoia a lot lately. A good friend and colleague is having a really hard time in lab because they feel like slices of their project are being given away without their consent. And they are right, the project is hard to partition in the first place, and there are three bright, invested individuals working on it at the same time. However my friend has made particular innovations, and is uniquely qualified to do some things, that they had suggested in the first place. This slicing and dicing has been going on for nearly two years now.

Enter full blown, pull out all the stops paranoia. Behaviour-altering, mind-bending paranoia. I won't share reagents, I'll never discuss a good idea in lab again paranoia. It's getting to the point where they are saying and doing things I don't think are characteristic or believable, and they are morphing into human jelly. And I just want to stand up and scream at them to stop it. I have tried to bring up the subject more gently and constructively than that, but an unavoidable side-effect of paranoia is that one perceives judgment and betrayal in what everyone says. So what do I do?

I think paranoia is the single most destructive emotion one can give in to. I know this regarding emotion from deep personal experiences; and regarding work from having been scooped three times in grad school. I have worked with many brilliant and paranoid people, and the one thing that always leaps out at me is the amount of energy they waste in spinning their paranoid wheels. It is such a waste of all that brilliance, all those (rapidly diminishing) neurons firing salvos of negative emotion. It drains you, makes you bitter, changes you in fundamental ways, alters your equation with everyone you work with, and is utterly pointless in the end.

It is both insulting and patronizing to tell someone they are being paranoid when they feel, legitimately or otherwise, that they are being deprived of what is rightfully theirs. However protecting one's territory can go too far, and when that line is crossed, it really messes things up. Academic research as it is today relies on the goodwill and respect of one's peers, and paranoia and its close companion suspicion, are the surest way to erode all goodwill and respect that anyone has for you. And in the end, it destroys your own self-respect, and no paper is worth that.

The Endgame

I've been thinking about "the endgame" a lot these days.
(Style note: I swear not to begin anymore posts with "I've been thinking about..." anymore, it might be time for "It was a dark and stormy night...")

What is my endgame? With this postdoc and all that I am doing now, involving low-level politicking, writing etc. Let's say its an academic position in India, doing cutting edge research in infectious diseases. What then am I doing to go there? How specifically do I go about getting there, as opposed to just trying to do good research and sending up prayers to the gods of funding and publishing?

This is a novel way of thinking for me, and one that I think should have started a long time ago. I drifted into science because I was bright and academically-inclined. The drift wasn't aimless, it was influenced by the fact that I love talking to intelligent people about intelligent things, my mother is a scientist, and I thought science and scientists were so cool. I came to the US because it was kind of the done thing at my (competitive) college: my friends all wrote the GRE and sent out applications. Most of us did well, and nearly all of us went to good schools. I floated into biology because I liked both biology and chemistry, and biology incorporated elements of chemistry. I went where I did for grad school because they offered me a place, and it seemed really cool.

I don't mean to say that I just faffed through life and things happened to me. I think it was more that I hadn't found all the the dislikes and loves that I have now. I liked most things, I was interested in studying most things, and I looked on travelling as an adventure. I had no conception of the mind-bending cultural changes I was about to face, and I did not think for one second about how hard it would be to consider living and working in India after nearly ten years as an American scientist. I am pretty fortunate that things have turned out well, and I am very fortunate to have discovered both ambition and immunology. Fulfilling that ambition would be much easier if I had actually started thinking about my life and career choices more actively a long time ago. I didn't, though, and whether that was a function of culture, personality, upbringing, I can't say with certainty. But it is what it is, and I'm going to find out, soon enough.

Now I know better, its all about the endgame. Which includes immunology, India, a family, my large and extended family, writing, travelling and a faculty position.

Too ambitious?

One Week at a Time

So my last post was about how charged up I am about an academic career.
With that in mind, let me describe my week:

Saturday: Transfected cells, let's say A cells, to use for experiments. Fed them, put them away, felt very good about the adventurousness of the experiment being undertaken.

Sunday: Realized while 40 miles away that I had completely forgotten to get another set of cells, call them B cells, ready for the experiment. B cells are needed for the final step of the experiment. Crap. Bummer. Well will just have to get them ready on Monday.

Monday: Got B cells, and third and final type of cells, called C, ready. A cells, of course are nice and confluent and ready to use, only I am not going to use them. F*cking A. Efficiently set up more of A cells to start the whole things again, but nested so I don't waste any time.

Tuesday: Set it all up, A cells, B cells and C cells. They are happy in the incubator, ready for me to read them tomorrow. Whew, that didn't work out too badly did it?
Transfected second batch of A cells.

Wednesday: Started off readings, all negative. Completely, and it doesn't even look like A cells make the protein I transfected into them (which is the whole entire point of transfection!). Double sh*t. No f*ck it, triple sh*t. Then the blinding realization dawns that I threw away my leftover B cells after using them yesterday, and I don't have any more going. There are no words, only anti-endorphins.

Thursday: Started round 2, with some alternative B cells. Also, smartly decided to check whether transfecting served any purpose this time, since I screwed up the previous time and let A cells go much longer than they should have. Turns out that transfecting A cells doesn't work when I do it (though it is routine procedure in my lab). Nothing, nothing at all. There's no point in doing the next step, with alternative or otherwise B cells. Now what? Blog, I suppose. Maybe some beer.

Friday: Strategize? Re-evaluate? Retire?

I am a shining example, I tell you.

I Like Being a Scientist?

Something interesting happened to me recently. I've had a busy few months: I went to India, for a wedding (mine), came back to the US and to work. Pounded out a bunch of experiments before I left and few since I returned. Struggled badly with jetlag (it only gets worse!), cultural disaffection, being tired and ill, loads of pressure from the boss and truly pernicious lethargy.

And now I want to work. I want to do experiments, read immunology, gossip about science. I even looked up job openings in India, because I think I want to start my own lab there. I want to keep being a scientist.

It's difficult, rarely rewarding, massively underpaid and a niche profession if I ever heard of one. My job prospects in my home country are limited, to say the least, aside from the fact that I have never actually worked in India. I have done all my research in the States and am, for all practical purposes, an American scientist. I have a new husband and our busy life together. I need some sexy papers, and some powerful, original ideas. I need to push and slog and labour till I can't stand it anymore and my family can't stand it anymore.

Why would I do this? I guess its because I really like being a scientist.
Who knew.

Managing Time

Long blog absences generally mean that someone isn't doing a very good job of managing their time. Sighs. It's been a busy-postdoc phase, which is a good thing because it means that I actually have work to do on multiple projects. It also means, however, that I have really had to re-evaluate how I manage my time.

As an experimental scientist one's single greatest asset is the ability to manage time. I've always been a fan of schedules, I used make little timetables for my day even as a little kid. I don't make schedules because I like to, I do it because I am chronically lazy and unless I set myself concrete targets to meet (preferably in writing), I will waffle and procrastinate. As a young grad student I was high on "doing science", and thrilled with the maverick aspects of research and liked to "go with the flow", "see where my data lead me" etc etc. It didn't work out so well as you can imagine.

So, I started making monthly schedules with the invaluable help of iCal. First big roadblock: my work computer and home computer did not have the same calendar entries or alerts. I tried synchronizing them, then decided that the best possible way was to have a paper copy. And that system has served me extraordinarily well ever since. I print out a monthly calendar with standing meetings on it, and then add all my experiments and other things on it. In pencil, because I do chop and change my plan a lot. I post that schedule on the corkboard above my desk and it stares balefully at me all day.

That worked really well in grad school, where I usually had to plan my experiments up to three or four weeks in advance, coordinate cell sorting schedules, GM-CSF addition, FACS time (the bane of all immunologists) etc. Now I find that I need to plan three months ahead because of the nature of the experiments. Three months! It's crazy and more anal than I'd like, but the whole house of cards is distinctly precarious when I don't plan that far ahead.

It really irks me to have to map out so much so far ahead. I detest feeling circumscribed by my schedule. Seriously, I am now one of those people who has to check their calendar all the time. There aren't FACS time calendars printed for the time I need to use them. The plain truth is, however, that my productivity has shot up since I started planning so far ahead. And I can say now, with relative confidence, that we can go out of town at X time since I won't have a pressing cell commitment then. Along with the calendar, I make a list of objectives for the next two months. What are the questions that need to be addressed now, how can I prioritize them, and what should I do when to optimize the use of my time. Together with the calendar, I felt really on top of things, on top of my game and in charge of my science.

Then I find myself mentoring two rotation students (first year grad students checking out the lab) and all my carefully made plans crumble. I am here all the time, rushing rushing rushing, trying to perform mad feats of time-juggling and trying to keep four projects and three people on track. And I have to say, it's not going to happen. This has exposed the crucial flaw in my scheduling system: inflexibility. If you're just one person, you can organize your time perfectly, plan all you need to do and execute with all the precision your heart can desire. You can't do that when you're mentoring other people. So I suppose the choice is whether you mentor people or not, and I feel very strongly that one should mentor, having been the recipient of some kickass mentoring myself. I am forced to conclude therefore that while planning and time management and the key to being productive in lab, I must schedule some wiggle room.

Responsibility for Scientific Fraud

Who is responsible when a paper is declared to be the product of fraudulent research? This is a tough question to answer, and is becoming more relevant every day. Is it the first author? The PI? If you're the third author, or even the second on a paper that's found to be fraud, how responsible are you? Should it be allowed to negatively impact your career (it probably will)? Can you claim that you do not bear responsibility if you are not the "direct perpetrator" of the fraud?

Nature has an interesting proposal.

I'm not sure making one author sign such a declaration is necessarily the solution, but at least it has the advantage of holding the senior author truly accountable for the work that bears their name. I don't think one can force responsibility. I think that a really responsible PI will have checked the work in a paper, and one that is inclined to be less responsible will not be made more so easily. Perhaps enforcing accountability with a binding (although I don't know how binding this will be) signed document may lead to greater responsibility.

Thoughts?

D'yer Mak'r

The inner dialogue: confessions of a Friday evening.

This postdoc's inner dialogue tends to be frenetic. "Was that 12 or 14 microliters? 12? no 14? How much did I add? ???? Sh*t forget it, its X.03mM instead of XmM" "Okay finish finish, Next Postdoc is signed up to start in the hood in ten minutes. Do all those cells really need to be split yet? Can all those cells be split in ten minutes? F*ck no way. So f*ck it, these cells will survive overgrowing better than those, so be it." "Ugh only halfway through the injections, ten more to go..." "F*cking A, the meeting with the Boss begins in three minutes, can I process three flowjo layouts in three minutes??" "F*ck f*ck F*CK"

And so on. There are calendars and to-do lists to remember, mice to take care of, experiments to design, papers to read, meetings to be gone to, socializing to be done and the special people to see and talk to. The average postdoc treadmill, and I love every minute of it-surfing deadlines, the pace, the multi-tasking, the nearly constant motion.

That said, the best inner dialogue is the silent one. It's 5.30 on Friday evening. The postdoc picks up all her detritus from the hood, puts it away. Mops up her bench, puts the media away. Sits down at her desk, collects all the little yellow stickies with cell counts, concentration calculations, dates of births and general experimental miscellanea and pastes them into the current lab notebook page. She looks at the calendar on the cork board in front of her, and everything is crossed out. The to-do list is similarly complete. All the mice are happily asleep or running around in their cages. The fluorescent lights hum, the lab is nearly empty, the radio plays on, for once not the driving rhythm of work but just music in the background. And the postdoc squirrels further into her chair and just listens to the sound of silence, the inner voice quiet.

The Battle of Evermore

Antigen Presentation: The first in a series of basic concepts in Immunology

Recognition of a pathogen as a pathogen is one of the most fundamental functions of the immune system. The immune system can be divided into two basic classes, partly based on the ways in which pathogens are recognized: the innate and the adaptive, or acquired, immune system. The innate immune system is the early arm of the immune system, acting rapidly, within minutes of encountering a pathogen. Innate immunity is inherently broad in its specificity—cells of the innate immune system broadly recognize pathogens as pathogens, not specifically as X virus of Y bacteria. This is not to say that innate immunity is indiscriminate, far from it. The way in which innate immune cells recognize microbes tends to identify whole classes of pathogens, labeling a microbe generally as “gram-negative bacterium” instead of specifically “E. coli”. That is not the whole story, and will be the subject of another exposition another day.

What I want to discuss today are some of the ways in which the adaptive immune system sees pathogens. There two principal types of cells that constitute the adaptive immune system are antigen presenting cells and effector cells. (Though these distinctions are not hard and fast, some APCs are effectors and vice versa.) Antigen presenting cells, affectionately called APCs, “present” antigens to effector cells. What does this mean?

Starting with the effector cells, in this case T cells (see sidebar). T cells express an antigen receptor called the T cell receptor (TCR) that recognizes protein antigens presented to them by APCs. Once T cells recognize antigens, they react in a variety of ways: cytotoxic (literally: toxic to cells) T cells (CTLs) kill the cells that present the antigen to them, while helper T cells (TH cells) produce cytokines (see earlier) that communicate with other cells. Given that antigen recognition by a T cell can have powerful and far-reaching consequences, it is evident that it should be a tightly controlled process. And tightly controlled it is—by two elegant little biological caveats.

The first is that TCRs only recognize small fragments of said protein antigens called peptides, usually between 8 and 15 amino acids in length. T cells are very picky about the length and nature of these peptides, and APCs therefore have to “process” whole proteins down into the precise fragments the TCR can see. This process of degradation usually happens only inside a cell and requires the cell to have a fairly extensive and specialized “antigen processing” machinery. So not just any cell can present antigens to T cells, and the ones that can, cannot present just any antigen.

The second is that TCRs can only see peptides when they are carried on the surface of the APC on specific carrier proteins called MHC molecules. MHC expands into major histocompatibility complex (and I am violating every canon of science writing that says you have to define your term before you use an abbreviation, but the expansion only clouds the issue here), and refers to a set of genes that encode proteins that present antigens, called antigen presenting molecules. Something I won’t go into now is that these MHC genes are what determine whether organs are compatible (histo means tissue) for transplants, they are called HLA molecules in human, HLA typing anyone? Anyway, the function of MHC molecules is to carry peptides and present them to T cells. This is particularly nifty because T cells are restricted to recognizing only the antigens presented to them by self-MHC, which basically says that the T cells in my body only recognize antigens presented on the MHC molecules my body has.

So a T cell is constrained to recognize antigens only in a certain form, and then only when self-MHC molecules present those antigens. MHC molecules fall into two classes (of course, its not that simple, but its broadly true), MHC class I and class II. MHC class I molecules present peptide antigens that are made inside the cell and MHC class II those that are swallowed from outside the cell. Cells only make proteins from a microbe when they are infected by that microbe, and MHC class I molecules signal that a cell is infected by presenting the microbial protein as an antigen. The best way to deal with an infected cell in to destroy it, and cytotoxic T cells see antigens presented by MHC class I and kill the cells that present them the antigen. MHC class II molecules on the other hand present antigens from proteins they find floating around outside the cell, to helper T cells. Microbial proteins floating around indicate that there in an infection somewhere around, not necessarily in the cell that is presenting the antigen. So when helper T cells see their antigens on MHC class II, they don’t kill the messenger, but send out cytokine summons to all other immune cells to come a-hunting for the infection in the area.

Thus does antigen presentation form a critical part of immune recognition, battling on evermore in the standoff between bugs that don’t want to be recognized and bodies that want to recognize and eliminate them. The evolution and function of antigen presentation is an absorbing and continuous field of study, and has lead to some fascinating insight into the ways in which cells organize their insides. Some other areas that continue to interest are the sources of various antigens, and the ways in which T cells are educated to recognize foreign antigens but not the body itself. While antigen presentation is the most basic platform of immune recognition, it is also the jumping off point for some truly awesome research. Coming up in future posts…

Good Times Bad Times

Inspired by one of Sunil's posts, at balancing life.

The key question is whether one can put a value on all the research that isn't published. Anyone who does research knows that there are good times and bad times, and that one has very little control over when those times occur. Postdocs need hot papers to get academic jobs-assuming, for the moment that we are talking about postdocs who want academic positions. Grad students would also like to publish high, but its less critical at that stage in one's career. It's usually the quality of one's postdoctoral work that is evaluated for jobs.

So what happens if one's well designed, innovative and technically superb project has no results? No publishable, sexy, revolutionary results? That the null hypothesis is true? It is extraordinarily difficult to publish negative results, especially since one can always encounter the ultimately dismissive critique that one hadn't tried everything yet. Does that mean that the two years spent on the project are toast? One's thought, analysis and expertise are worthless because they cannot be proved in print? Five more years as a postdoc?

So how then can we quantify effort and ability if not by publications? If its an especially technically difficult field, years of experience should count for a lot. If the idea behind the project is not mainstream (as Sunil discusses) and doesn't have any of the fashionably fund-able keywords, should one get points for risk-taking? The willingness to take on challenges without guarantees, at least the guarantees implied in "current-hot-topics" research, is uncommon and to be prized. So should CVs include a paragraph briefly describing one's project and the ideas behind it? Or will that just be seen as an attempt to flesh out the CV in the face of the conspicuous absences in the publications section? Probably.

After all new fields are created by people who can think out of the box. And sometimes luck only shows up late, and it takes three failed attempts to come up with truly revolutionary ideas. Or, the three failed attempts could reflect the complete absence of any BS-detector. Which is it? Does one always need to have a publishable side project, which will generate small reliable papers, thus demonstrating that one can actually do publishable research as well as study risky and unusual subjects? That one's out-there ideas are the product of intelligent thought, hopefully as demonstrated by the stuff that did get published.

Seriously though, is this a workable solution? Most postdocs I know do have two projects, just in case and to keep oneself occupied, particularly in immunology, where some experiments just take so long. Isn't it somewhat ridiculous to require people to have two projects? Or more?

Or, should one just ascribe it to the nature of the game, and let it go if things don't work out? After all, there are just way too few academic positions, and luck may just be another way to filter people out. Just because some intelligent and qualified people get thrown out with the bathwater, that doesn't mean that other equally intelligent and qualified people don't get lucky, publish and get academic positions. This way of thinking goes against everything I personally believe, because it just is not fair.

But who said life would be fair?

What is and What Should Never Be

Will asking postdoc mentors to document their mentoring help the current mentoring-or-lack-thereof situation?

I don't know, seems like yet another opportunity to write things in a grant proposal that you don't necessarily mean to do. More verbal padding, more watchwords. On the other hand, writing the watchwords may start you think about them. Will it change what is, and what should never be, or have been for that matter?

Hm.

Stairway to Heaven

I may be late, but you can still jump on the comment stairway! De-lurk please, I would love to meet you.

From Schmutzie

No Quarter

(Persevering with the Led Zeppelin theme)

Can you succeed in academia only if you are a shark?

This is something I started to think about when I came to the USA. In India, in my experience, academic scientists are idealistic, workaholic, fatalistic and gossipy. Money is always tight and you rarely get to publish in the good journals (and I'm not talking only about the big three or five) because of where you're from. The salaries for PI-level scientists are nowhere as high as they are here, and respect from the public and one's peers in other countries can be in short supply. The keen-edged aggression that one sees among scientists (specifically, biologists) here is not at all common. But, and this is crucial, once you enter the system of government science labs, you will have a career. Every X years you will be promoted, every Y years you will get a salary. You have tons of holidays, your kids have opportunities. Many post-postdoctoral scientists I know in India have jobs and some measure of security.

Many things are common between the scientific world in India and the United States, the most glaring absence in the latter is the absence of any prospects of security in academia until you have tenure. So the situation is then that you have really bright people who work and work and work, with limited pay and even more limited prospects. To make things more interesting, these people are often enormously motivated and justly ambitious: So where does all the energy go?

We all know there aren't enough PI positions. There aren't very many non-PI permanent researcher positions either. So the only thing to do then is to fight for the positions there are, right? To give no quarter, to your peers, to the possibility of failure, to your life, or to yourself. To be aggressive and up-to-date, to work harder, better and more successfully than everyone else. To know things and have connections that others don't have. Not that there is anything wrong with any of these things, I get a buzz out of the hunt just as much as anyone. My point is that it is not really sustainable.

Or not sustainable for the majority anyway. what happens to the people who cannot, do not want to or will not be sharks? The laws of luck and averages dictate that some such will succeed in academia, but in the balance I think the sharks don't succeed. Then you have a situation in which the system "selects" for the most aggressive people, and often does not encourage other more nurturing or considerate professional behaviour. The lack of consideration and sensitivity, coupled with a reluctance to show "mommy qualities" because that would invite professional ridicule, leads to bosses who demand and do not teach, who hector not mentor, and whose personal advancement is their primary goal.

Shark eat shark then. Which doesn't seem like much fun to me, and maybe to more people. Why is it that the system is ok with people who are excellent scientists but dreadful people? Why is that acceptable? I don't know. However I do see some incremental changes (not where I work at, but), and I think the key to any improvements in the system can only occur with recognition of these issues. I hope so, because the joy of research is being subsumed by the nastiness of its execution.

When the Levee Breaks

Plug it as best you can and keep going.

This is probably the best, and only advice I would have to give to an emerging scientist. This is also the most valuable lesson I have learned over the years from my various mentors. I don't think it was explicitly stated as such, and certainly not in the words of Led Zeppelin, but it was a strong message I got.

I did my Ph.D. in a fairly unusual situation. I was one of very very few students where I was, in a very postdoc-heavy environment. Some of the labs in Ph.D. place didn't had only postdocs and technicians. It was an intimidating environment in the beginning, especially when I was a mousy first-year. But what happened over they years is that I ended up having a plethora of mentors.

I learned how to do FACS from one postdoc, dissect mice from another, inject mice from yet another. I developed a deep respect for "bullshit detectors" and a strong seminar habit (which I really don't get enough of here), learned to ask "Why?" instead of "How?". I learned to think ahead, to plan for figures, to never run out of mice. I learned how to deflect tantrums, how to stand up for myself, how to speak at conferences, all from my Ph.D. advisor and various postdoc mentors. I didn't really have any interaction with student peers, didn't have a student milieu, but it turned out to be a phenomenal experience overall.

Not that it was all happy days and everybody being helpful. But when times were hard and feelings were hurt, the people I really admire kept going. They put their heads down, ignored their feelings of being neglected by the boss, ignored ridicule from other lab members (yes, ridicule) and kept working, and working smart. That's the best thing to do- let the work speak for itself. Don't let yourself drown when the levees break. Swim.

Some of the nicest people were the most useless as mentors and some of the most seemingly curmudgeonly the best mentors. The hard lessons are not learned easily. The mentors I have the most respect for now, in retrospect, are those who told it like it was. Directness can be unpalatable, but it is the only way to clarity, scientific and otherwise.

Another thing I feel strongly about is that you have to pass mentoring on. If you have been treated well, you have to treat people well. If you have not, you have to be extra vigilant not to take it out on people who you will be mentoring. Everyone starts somewhere and impatience is absolutely incompatible with mentoring. This may be seem obvious, yet it is surprisingly easy to forget.

Its easy to resent time taken away from experiments, its easy to be annoyed by constant interruptions, I certainly am. That doesn't mean that one should indulge that annoyance.

So as a mentor, I want to be direct, firm, hopefully gentle, accessible. But most of all, I would like to be constructively critical, and pass on the importance of a bullshit detector. Let's see.

scientiae-carnival