arsenic redux

What better way to spend a brisk december morning than by probing deeper into the landmark isolation of a bacteria that is able to grow in the absence of phosphorus?

The paper starts by noting the fundamental nutritional elemental requirements of all prokaryotic and eukaryotic cells: carbon, hydrogen, nitrogen, oxygen, sulfur, and phosphorus. (Other trace elements are necessary, often as enzyme co-factors, but are generally interchangeable) Arsenic lies below phosphorus on the periodic table giving it similar valency (3-) which contributes to its toxicity as it is mistakenly incorporated into metabolic pathways. Unfortunately, unlike chemically stable phosphorus-based interactions, arsenate-mediated bonds are much more easily hydrolyzed giving them a half-life too short to generate stable compounds. However, researchers the NASA Astrobiology Institute wondered whether, in certain nutrient-starved climates, bacteria may have adapted to substitute arsenic for phosphorus despite the inherent instability of arsenate compounds.

Their search began in Mono Lake (left), a water body with unusually high arsenic concentrations that would facilitate directed evolution of organisms that could cope with the inherent instability of arsenate-containing compounds. They inoculated media of pH 9.8 supplemented with glucose, vitamins, and trace metals but no phosphorus and increasing arsenate concentrations. They note that the inoculations underwent “many decimal dilution transfers greatly reducing any potential carryover of phosphorus.” The background phosphate in the medium was 3 micromolar, due to “trace impurities in the major salts.” They then isolated a growth which was reintroduced into phosphate-free medium, which grew out a strain, GFAJ-1, a member of the halomonadaceae family of gammaproteobacteria. The gammaproteobacteria are a large class of gram negative bacteria which include escherichia coli, psuedomonas aeruginosa, vibrio cholera, salmonella typherium, and yersinia species. The halomonadaceae family, specifically, has previously been shown to accumulate intracellular arsenic and has been suggested as a potential therapeutic agent for bioremediation of arsenic-contaminated water.

It is notable that this bacteria required either arsenic or phosphorus – it grew faster with phosphorus but was able to grow with exclusively arsenic. The bacteria grown in arsenic-exclusive media were larger in size (right, C vs. D) by scanning electron microscopy, potentially due to accumulation of large vacuoles seen on transmission EM (right, E). The authors appear to resolve the issue of phosphate impurity in the background media by showing that this bacteria could not grow in the As-/P- media, suggesting that the background phosphorus was insufficient to support growth in the absence of exogenous arsenic.

The group then went on to demonstrate arsenic incorporation into these bacteria; first by mass spectrometry of isolated bacteria and then by radiolabeled arsenic oxide, which appeared expectedly in protein, metabolite, lipid, and nucleic acid fractions (given this distribution of phosphate in other bacteria). The percentage of arsenic in nucleic acid, while small, appears to be consistent with the proportion of intracellular phosphate within nucleic acids – approximately 4% They then went on to show that arsenic was enriched in purified genomic DNA from bacteria grown in arsenic-exclusive media by ‘high-resolution secondary ion mass spectrometry’ (I don’t really know what this is). They then used “synchrotron X-ray studies” (what?) to assess how the arsenic was configured and found a structure consistent with As bound to 4 oxygen ions (similar to a phosphate group) as well as within small molecular weight metabolites (consistent with the role of phosphate in NADH, ATP, etc.) and modification of proteins on serine, threonine, and tyrosine residues (the main sites of protein phosphorylation – think of serine/threonine or tyrosine kinases).

Seems pretty solid to me.

arsenic???

OK, this is crazy. Apparently, researchers at NASA have found a new bacteria, GFAJ-1, which engineers all of the components required for life -- DNA, RNA, proteins, sugars -- with arsenic instead of phosphorus, making it a new form of life entirely different from anything else we've ever seen. They apparently isolated it from some poisonous lake in California??

Here are two links. I need to read more about this. Basically, the inherent idea that all life requires six basic elements: carbon, oxygen, hydrogen, nitrogen, phosphorus, and sulfur -- is wrong. Not requiring phosphorous might not seem like a big deal - until you realize that it is:

• the linkage unit between every base pair of DNA or RNA
• the critical on/off switch for every signal transduction pathway that drives pretty all metabolic activity in cells
• oh, right, the fundamental basis of cellular energetics (ATP)

Now, arsenic is from the same family of elements as phosphorus, lying just below it on the periodic table. In fact, its substitution for phosphorus is largely responsible for its toxicity (as shown by its competitive inhibition by exogenous phosphorus added to fungal cultures).

Crazy.

eplerenone redux

In response to my snark about the recent EMPHASIS-HF study, I wondered if what I said - that patients with EF less than 35% are already on their way to class III/IV heart failure, therefore lessening the impact of this study - was true.

I guess not, according to the American Journal of Cardiology, almost 30 years ago. In their assessment, resting ejection fraction did not correlate whatsoever with symptoms or exercise capacity. But in that case, if symptomatic heart failure is totally dissociated from resting EF, and clinicians are going to institute therapy based on symptoms, why even include an EF cutoff? Because now, physicians considering eplerenone for their mildly symptomatic patients are going to need to do an echo first. Why not have just done a trial of eplerenone for all mildly symptomatic pts with a diagnosis of HF and then look at how patients did depending on their EF?

preventing GVHD

So I'm working on a big post about lupus (and the the the first approval of a drug specifically for lupus in fifty years), but in the meantime, I've assembled some other random thoughts that won't see the light of day unless I kind of word-vomit them out first. So, here goes.

Leukemia and lymphoma, the "liquid tumors," are so named because they arise from malignant transformation of cells whose precursors originate in the bone marrow. Leukemia occurs from overproliferation of precurors to mature hematopoietic cells which lack the function of fully developed WBCs. Lymphoma occurs from acquisition of malignant properties by differentiated white blood cells which have exited the bone marrow. While chemotherapy has shown success in some limited situations (diffuse large B cell lymphoma, for example), these tumors often recur. The last available option is often to replace the patient's entire hematopoietic lineage, via the so-called "bone marrow transplant." Traditionally, this involved two steps; irradition of the host bone marrow, followed by transplantation with new bone marrow from someone whose cells were least likely to be recognized as foreign by any remaining immune cells in the host (aka, maximal HLA matching).

This procedure was first successfully attempted in mice in 1956, and subsequently attempted in humans with refractory disease in 1957 (Thomas et al. N Engl J Med 257:491-6). The same group first successfully achieved temporary remission with BMT in 1959, in a study published in the Journal of Clinical Investigation.

Unfortunately, almost as soon as successful remissions with transplants were describe, so too were significant adverse effects, in which transplanted white blood cells would see the host as foreign and attack it. This destruction, first described in 1957 was largely focused in the skin (causing a sclerosing skin rash), and in the intestine (causing inflammatory diarrhea), known collectively as graft versus host disease. It was demonstrated in 1978 that T cells were critical for the graft versus host response. In order to try and minimize this devastating, and potentially lethal, side effect, early emphasis in BMT development was placed on achieving maximal HLA matching.

Interestingly, in 1979, a paper in the New England Journal of Medicine demonstrated an unexpected finding; patients receiving a mismatched SCT were actually less likely to relapse if they developed GVHD. Based on this, the theory was put forward that donor T cells could have both positive and negative effects; that is, they would seek out and reject tumor cells, but would inflict life-threatening collateral damage on host tissue at the same time. It has since been demonstrated that selective transfer of donor lymphocytes in patients with relapsed disease following non-selective BMT can achieve complete and permanent remission in some cases. Given this, the call for optimizing allogeneic (aka, non-HLA matched) stem cell transplants for graft-mediated tumor rejection while controlling graft versus host disease continues to grow.

One method of minimizing graft versus host disease while permitting anti-tumor immunity seems to be via increasing the presence of regulatory T cells. As shown on the left, in mice, co-transfer of regulatory T cells appears to suppress GVHD while still permitting tumor rejection and significantly improving survival. A role for Tregs is further evidenced by the relatively low rate of GVHD in patients receiving transplants of umbilical cord blood, which appears to be enriched in Tregs. Therefore, the search continues for treatments that might continue to shift this balance in favor of anti-tumor immunity and away from off-target anti-host tissue effects.

A new study in the Journal of Experimental Medicine shows some promise in doing just that. This study focuses on targeting BTLA, an inhibitory, immunoglobulin-family receptor that is expressed on Th1, but not Th2 cells. BTLA interacts with both B7 family members (similar to other co-stimulatory molecules such as CD28) but also with TNF family receptors. Ligation of BTLA induces activation of SHP family phosphatases that limit T cell activation and IL-2 production. It is required for the generation of antigen-specific Tregs in response to antigen without co-stimulation. Interestingly, BTLA expression appears to be downregulated by the presence of bacterial CpG nucleotides. It is further notable that studies have shown persistent BTLA4 expression on anti-tumor, but not anti-viral cytotoxic lymphocytes, suggesting that BTLA may have a critical role in supressing anti-tumor immunity.

In this paper, the authors treated donor bone marrow with an an agonist anti-BTLA antibody prior to transfer and found that it virtually eliminated development of GVHD as measured by clinical scoring, loss of body weight, and development of intestinal mucosal inflammation and ulceration. Further investigation showed that BTLA stimulation decreased effector T cell development while maintaining Treg development, which is in line with evidence of BTLA in inducing tolerance. Of note, treatment with agonist anti-BTLA did not successfully treat GVHD when given 14 days after transfer, suggesting that BTLA's tolerance inducing effects are restricted to initial development and do not diminish effector function of differentiated Teff cells.

Finally, the authors showed in a minimal residual disease model that agonist anti-BTLA treatment did not significantly inhibit graft versus tumor responses, which still required T cell function as evidenced by the poor response of animals receiving T cell depleted transplants. The search for highly efficacious, broadly applicable cell therapy remains elusive, but providing the proper mileu to support development of an optimal balance of anti-tumor immunity and host tolerance will rely on a combination of careful cell selection and subtle skewing of cell differentiation.

I know, that's kind of a weak ending, but I'm just not ready to jump into heart failure, stroke, or asthma tonight. (Yeah and definitely not lupus either)

spring cleaning

I know, I said a big post on lupus was coming, imaginary blog readers. But I haven't been able to read the reviews yet. So let me leave you with the few other interesting pieces I've read in the past few weeks before I vanish until December.

Eplerenone in CHF (the EMPHASIS-HF study)

Yeah, there's nothing that's going to rock your world in this recent NEJM study; following the RALES study which showed an indication for spironolactone treatment in patients with class III/IV CHF, this study looked at almost 3,000 patients with class II CHF (mild symptoms only) and an EF of no greater than 35% and found a significant morbidity and mortality benefit. Yeah. If you really want to see if mineralocorticoid antagonism works as preventive therapy, look in patients who already don't have frank evidence of pathological remodeling. Aren't these patients on the doorstep of class III heart failure already? Boo.

Tonic inhibition and recovery after stroke

Ischemic strokes are a leading cause of morbidity and mortality, especially in the elderly, whose risk factors for stroke (hypertension, diabetes, hyperlipidemia) tend to be more prevalent. Aggressive risk factor control clearly reduces the risk of developing a stroke, or a future stroke after one happens. But almost nothing has been shown to reduce functional impairment caused by a stroke after it happens. Really, the only effective intervention is early rehabilitation, which has been shown in multiple randomized control studies to improve functionality by inducing cortical remodeling following a stroke. Subsequent studies have shown that the cortical remodeling that drives partial recovery of function following a stroke is not the infarcted tissue but rather the peri-infarct tissue. Excitation of peri-infarct tissue following stroke drives long-term potentiation, resulting in effective remodeling. However, long-term potentiation requires sustained synaptic neurotransmission, which is limited in peri-infarct cortex. Why exactly is this?

Count on Nature to give us the answers. A letter published a few weeks ago describes a breakthrough in understanding the physiologic brakes on peri-infarct cortical remodeling. The study focuses on GABA, an inhibitor neurotransmitter with both activity that is both synaptic (released in response to stimulus) and tonic (released at a constant rate to set a post-synaptic excitability threshold). Tonic GABA release has been shown to oppose long term potentiation and memory. The authors of the above study wondered if similar GABA-mediated tonic inhibition repressed cortical remodeling following a stroke.

Indeed, tonic GABA-mediated inhibition was found to be markedly increased in peri-infarct cortex following photothrombotic stroke in mice. This increased GABA tone increased the excitability threshold and was completely reversed by the GABA inhibitor gabazine.

Having established the role for GABA in post-infarct tonic inhibition, the authors then addressed whether GABA inhibition following stroke could restore functional capacity. Indeed, GABA blockade improved foot paw symmetry during locomotion and decreased foot faults during measured tasks.

But is there actual utility to GABA blockade in pts who undergo a stroke? Well, its complicated. The main problem is that GABA mediated inhibition immediately following a stroke limits infarct size; accordingly, GABA blockade improved murine mortality when given 3 days after a stroke but not when given immediately afterwards. Furthermore, prolonged GABA inhibition eventually led to worsening of functional performance. While this calls for careful modulation of GABA inhibition, it actually provides a potentially clincially applicable regimen: initiation of therapy a few days after a stroke, when symptoms are sure to be present, and stopping therapy after a limited amount of time (although improvements in functional status are not completely sustained after treatment is withdrawn.

Uh, CX3CR1 will have to wait for a time when I'm not actively going into a coma.

the white tiger

"In the old days there were one thousand castes and destinies in India. These days, there are just two castes: Men with big bellies, and men with small bellies. And only two destinies: eat or get eaten up."

"The darkness will not be silent. There is no water in our taps, and what do you people in Delhi give us? you give us cell phones. Can a man drink a phone when he is thirsty?"

"Go to Old Delhi,and look at the way they keep chickens there in the market. Hundred of pale hens and brightly colored roosters, stuffed tightly into wire-mesh cages. They see the organs of their brothers lying around them.They know they are next, yet they cannot rebel. They do not try to get out of the coop. The very same thing is done with humans in this country."

If you ask Aravind Adiga, he wouldn't go so far as to say that democracy is a dirty word, but its clear from his searing first novel that its quite far from a panacea. The book speaks from the point of view of Balram Halwai, a member of India's poor, who serve largely as a servant class for the few, but highly powerful elite. Through Balram's eyes we see how the rapid concentration of wealth into India's upper crust allows them to enter into a corrupt bargain with the politicians in India; the wealthy use their resources to keep politicians on power; the politicians permit the continued inequality of wealth distribution. The net effect of this is that the resources that are generated from India's economic growth (aka, cell phones, laptops, etc.) are useless in uplifting the majority of India that lacks basic irrigation or sanitation. Adiga doesn't spare his wrath or disdain for the majority of Indians in "the darkness" who accept their fate (see his comparison of the citizenry to chickens in the coop watching their brothers be slaughtered), which is reflected in the persona of Halwai, a boy driven so crazy by his station in life that he commits murder in order to obtain true freedom. It is surprising in a country in the majority of the citizenry are held captive by the concentrated power of the few and wealthy that generally nonviolent farmers have been captivated by the promise of freedom and equality from fringe groups such as the Naxalites?

I like standing next to you, Sean...it makes me feel tough.

I know I said I'd blog about asthma and chemokines and neurotransmitters and strokes, but...I'm all amped up over The Social Network (two weeks after everyone else) so, here's a "stuff I've been watching and/or listening to" post!

1. The Social Network

There are so many things that made this movie brilliant, and no one who acted in this movie is undeserving of praise, to be sure, but ultimately, this movie is about a central iconoclastic figure, and its what Jesse Eisenberg, Aaron Sorkin, and David Fincher bring to his character that defines the movie. After all, this is a movie that takes place largely in front of a computer, yet the tension (brought brilliantly to life by Trent Reznor and Atticus Ross, hoorah!) remains palpable for the entirety of the movie. And I think its because we can't make up our mind about Zuckerberg. While it's clear that he is ruthless, whatever emotions we suspect lie under the surface range from haughty to childish to spurned to vindictive to needy to regretful without so much as an eyebrow furrow. Unsurprisingly, I think the New Yorker is right when they say that that tension is upheld by the the essential conflict between Fincher and Sorkin's worldview:

The portrait of Zuckerberg, I would guess, was produced by a happy tension, even an opposition, between the two men—a tug-of-war between Fincher’s gleeful appreciation of an outsider who overturns the social order and Sorkin’s old-fashioned, humanist distaste for electronic friend-making and a world of virtual emotion.

I think different people will likely come down on different sides of the fence on the issue of Zuckerberg's ethics. On one hand, one could easily make an argument that his was the only moral code that was transparent (aka he respected only productivity) and that his all-consuming commitment to Facebook validates his desire to control the involvement of all other partners (notice how he was programming in virtually every scene in the movie and how he was content to let his associates take some marginal credit until the second that they compromised the integrity of the product). On the other hand...the job he pulled on Eduardo was cold, man. And that makes me wonder - does so-called social media like Facebook actually promote dehumanization by allowing people to only present to the world what they choose and not reform the parts of themselves that are petty, angry, vindictive, or otherwise damaging?

2. Cee-Lo

Let's switch gears from the razor-sharp, coldly perfect Social Network soundtrack (as mentioned before, by the incomparable Trent Reznor) to 70s soul-loving fun-fest that is Cee-Lo's newest album, The Lady Killer. It's almost unnecessary to review this album; just listen to and/or watch the video of his first single, "Fuck You." I mean, how can you get better than "I guess she's an X-Box, and I'm more Atari?" You can't. But for the record, almost equal to this song in quality is his transformative cover of Band of Horses' "No One's Gonna Love You." Video for "Fuck You" below.

3. The Arcade Fire

It's flown under the radar alot more than their previous albums, I really think that The Suburbs is equal in quality to Arcade Fire's past two albums. In terms of of range, they continue to feature uplifting, somewhat bombastic pieces like "Sprawl II (Mountains Beyond Mountains)" and "Rococo," uptempo NYC-rock pieces like "Ready to Start" and "Month of May" and more pensive pieces like "City With No Children" or Modern Man."

Where the band has really taken a step forward is lyrically. After an ill-advised turn for the faux-political (no doubt Obama 'Yes We Can' inspired), they turn inward and deliver an album that is about getting old, giving up the dreams of your youth, and accepting a life that feels unfulfilled. Now, as a former suburbanite, and someone who has little tolerance for people who sneer at people from the suburbs, I would ordinarily hate on this hipsteresque superiority complex, but the album finds the hipster obsession with what is transiently 'cool' to be equally stupid (see their song "Rococo"). Ultimately, the album seems to be about themselves, as artists at an intermediate point, caught between two disappointing choices. Just some snippets from some of my favorite songs from the album:

"So can you understand/why I want a daughter while I'm still young?/I want to hold her hand/and show her some beauty/before this damage is done." - The Suburbs

"They heard me singing and they told me to stop/quit these pretentious things and just punch the clock." - Sprawl II (Mountains Beyond Mountains)

"I feel like I've been living in/a city with no children in it/A garden left for ruin by/a billionaire inside of a private prison." - City with No Children

"Let's go downtown and talk to the modern kids./They will eat right out of your hand/using great big words that they don't understand" - Rococo

4. Kanye West's Beautiful Dark Twisted Fantasy

Kanye West also seems to be having a struggle with his identity, although it might just be between whether he has Narcissistic Personality Disorder or is full-on bipolar. I acutally think that Kanye is incredibly savvy, and realizes that acting out as he does is a way more effective marketing tactic than a music video is nowadays (and we all know how frustrated he is about his lack of music video recognition.) I happen to actually agree with him when it comes to his treatment on the Today show - as if people are going to listen to his explanation when they get to watch him yell at Taylor Swift again. Shut up, Matt Lauer. Meanwhile, his music continues be the absolute gold standard for hip-hop. The guy comes out with a sneering challenge to all of his critics with his first single, "Power," then issues a free remix in which he completely rewrites the song, and its even better than the original. His collabo with Jay-Z and Nicki Minaj, "Monster," is probably the strangest, coolest single since the era of Missy Elliot and Busta Rhymes. For me, personally, it will be difficult to match Graduation in quality, but i certainly agree with Rolling Stone's opinion that he is one of the few artists in music with any interest in challenging us:

There’s a famous story about Queen making "Bohemian Rhapsody": Whenever the band thought the song was finished, Freddie Mercury would say, "I’ve added a few more ‘Galileos’ here, dear." But nobody can out-Galileo Kanye. With Fantasy, he makes everybody else on the radio sound laughably meek, but he’s also throwing down a challenge to the audience. Kanye West thinks you’re a moron if you settle for artists who don’t push as hard as he does. And that means pretty much everybody.

5. The Young Money Crew: Lil' Wayne, Drake, and Nicki Minaj

My love for Lil' Wayne is well-established in this blog, so I'll just say that his assembly of the Young Money crew is yet another feather in his cap. Unlike other successful hip-hop artists who ascend to the level entrepreneurs (Jay-Z, P. Diddy, Master P, etc.) Wayne's hires reflect an eye for talented, unique young artists. Like Drake, a half-Jewish (and bar mitzvah-ed!) former teen soap actor who had released a mixtape in which he rapped over unconventional artists like Peter Bjorn & John. Or Nicki Minaj, a young, african/indonesian/trinidadian rapper who raps under multiple pseudonyms, sports a strange Barbie obsession, and a rapping style that morphs from British accents to reggaeton. Now, Drake is probably the most popular hip-hop artist on the charts, and Nicki Minaj's performance on Kanye's "Monster" is probably the best verse delivered all year. Also, they were both in the New York Times. Also, Nicki Minaj's single, "Check it Out," which samples "Video Killed the Radio Star" is one of the most clever things I've ever heard. And Wayne? Well, he just got out of prison, released a new album (which is apparently only a predecessor to the forthcoming Tha Carter IV), and will drop in on fellow Young Money albums to occasionally drop a few blistering verses (like his performance in Drake's "Miss Me" where he reminds us "I got so many styles - I am a group.")

I'll leave you with Nicki Minaj's video for "Check it Out." Haters, you can kill yourself.