Monday, July 23, 2012

A special soup of Mn-DP-Pi for better vaccine design

Bangawoyo fellows,

          If you are just gazing at first line and guessing what that could mean, its greetings in Korean. My blog is going through a darwinian evolution, i must admit. The structure of my writing has entirely changed. In a good sense  by the way. A new structure has emerged for the posts, where i talk about a couple of most interesting facts that grabbed my attention, and then move on slowly to a paper that i want to talk about. And then a few tit bits, here and there.... Oh! Yes, this is on path of improvement just like microbiology.

       Most big journals are busy updating their impact factor. Just open a journal (any), and you will find new impact factor flashing on a side. Oh, yeah. Thats news for journals. And what could be breaking news in science for us? In science, Astrophysics research (Heavy words), reveals the presence of a massive black hole at the center of milky way galaxy (Link). Why do i care, that supposedly a dump-bin of space is minding its own business. If i say this to a physicist he would feel the microbes are minding their own and why we care. (Well i better not venture into their territory).

Fig 1: A jellyfish made of silicone and rat heart cells
       A hot news in the field of biology with clinical impact and probably the game changer of future is approval of genetic therapy for a lipid disorder. China was the first country to approve a gene therapy product for commercial use, this new approval in europe has triggered a new debate (Link to the story here).  The second pathbreaking science of the week is reverse engineering a species. Kit Parker comments “Morphologically, we’ve built a jellyfish. Functionally, we’ve built a jellyfish. Genetically, this thing is a rat”. Sounds interesting!! (The quote and fig 1 taken from Nature News).

Fig 2: Motif D as an important player in viral replication
      So much for the cover story? No, no!!!! The cover story for me is finding an important function for Motif D that enhances our understanding of viral replication (Link to story here). Another, a unique potential target has been identified in bacteria to develop new antibiotics. The target here is RNase R that has a few unique properties. (Link to story here). If you can bear with me, A quick question for you. What is the most important hurdle in delivering live attenuated vaccines?? Yes you are right. Maintaining a cold chain. That problem looks like has its solution in a moth!! No kidding. Researchers at Tufts University School of Engineering, have found that the problem can be solved by using  a silk protein as stabilizer. If you ask me will this mixture of silk into vaccines effect immune titers, "I don't know". (Link to the story)

      Other than all the stories there is one important article that is the centre point of the blog today. Radiation is a very important tool in biology. be it for mutation studies or for creating a strain that is very weak. The attenuated strains can be used for vaccination. To get a high mutation, and thereby sterility a high dose radiation is employed, that often culminates in heavy DNA damage. But there is a bystander effect. The proteins that are supposedly immunogenic may also be tweaked in the process. So how do you solve the problem? The article in cell host and microbe by Gaidamakova etal is about how to evade this.

The paper is titled "Preserving Immunogenicity of Lethally Irradiated Viral and Bacterial Vaccine Epitopes Using a Radio- Protective Mn2+-Peptide Complex from Deinococcus".

     In addition to the problem posed above there is another problem that authors highlight in the introduction. With decreasing genome-size, the dose of irradiation needed to hit an organism increases, the hurdle posing the most in case of viruses. So, if you could find a protector of proteins, at high radiation assaults, then you have a solution. The authors hit open this hypothesis with recent findings that Deinococcus radiodurans, a bacterium which has an extreme resistance to ionizing radiation. The observation was that the proteins of D radiodurans could sustain proteins but not DNA damage at same high radiation levels. The implied mechanism was  Mn2+ complexes which specifically protect proteins from ROS damage.

Fig 3: The radiation response mechanism of Deinococcus radiodurans (Source)

      The authors, reconstituted a Mn2+ and a decapeptide (H-Asp-Glu-His-Gly-Thr-Ala-Val-Met-Leu- Lys-OH), in orthophosphate (Pi) buffer (henceforth referred as Mn-DP-Pi). To test the capability, dodecameric enzyme glutamine synthetase which is deactivated at 150 Gy was used. Mn-DP-Pi mixture had retained 50% activity of the enzyme at 50,000 Gy. By the way Gy is a unit of radiation. The question as to if Mn2+ is a non specific ROS inactivator was solved by replacing with Mg2+, Ca2+, Fe2+, Ni2+, Cu2+or Zn2+ ions. But the best contender was mixture of 3 mM DP and 1 mM Mn2+ in 25 mM Pi. I have an interjection here. At this point I didn't really understand, if they have tried using only Mn2+, only decapeptide and then looking for protective efficiency.

    They also looked into the ability of the protector, to save bacteriophage gamma. The study looked into bacteriophage lambda survival at various doses. At this point they have shown the effects with nly Mn2+, only decapeptide. That answered my question. (See fig 4). As you can see from their survival plot, indeed the combination effect is high.

Fig 4: Bacteriophage lambda survival

    The main question is the phage might survive, but sustaining how much of a damage. Fig 5 says it all. Bacteriophage gamma DNA damage assessed by Southern blotting using a 32P-labeled gamma DNA probe and gamma protein damage assessed by SDS-PAGE. The Protection of antibody binding epitopes of gamma phage was assessed by western blotting, showing a good protection. 

Fig 5: Damage assessment with Southern blotting for DNA damage and SDS-PAGE for protein damage. 

     A small digression here. Science has a flow. the first step in science is to take up the observations and make sense out of it by constructing a theory. Once a theory is in place it is tested by various parameters for accuracy and the results matched to observation. At this point, a hypothesis becomes sufficiently a theory. But the main test of theory comes from a prediction that the theory makes. This prediction is then sought and finding it implies affirmation.

            Once the players of this paper knew that they had a strong base they moved on to another organism-  Venezuelan equine encephalitis virus (VEEV). The V3526 strain was a attenuated strain that which couldn't make it to vaccines, because it couldn't tolerate radiation. The effect of radiation was loss of important epitopes. With Mn-DP-Pi, they could well preserve viral epitopes, at high radiation levels. With the same strategy they were also able to preserve immunogenicity of MRSA. These radiation levels were much higher than the levels otherwise required to achieve sterility. Given the fact that there are no well defined marketed vaccines for VEEV or MRSA, this really is a starting point.

      I would have stopped at this point excited. But the authors really went a step ahead. they evaluated, if the MRSA rescued antigens was of any good. For this, they used MRSA USA300 that had been exposed to 25 kGy with or without protecting agent. After radiation they removed the Mn-DP-Pi and resuspended in Complete Freund's adjuvant. (See Fig 6 on left). The Fig 6 contrasts Anti-S. aureus IgG titers in serum from mice immunized in complete Freund’s adjuvant (CFA) with MRSA USA300 irradiated in the absence (IRS) or presence (MnDP-IRS) of Mn-DP-Pi.

         They subsequently also used a mouse model to evaluate the ability of MnDP-IRS immunization to elicit protective immunity. Of note, Mn-DP-Pi in itself was not shown to have any antibody eliciting capacity. And with some immunologic studies they were able to conclude MnDP-IRS immunization elicits antibody- and Th17 cell-mediated immunity.

       In short, the study shows that a decapetide in combination with Mn2+ avoids some serious damage to the important antigenic epitopes. This can be of help in creating a vaccine quickly, by strongly radiating a pathogen and administering the whole set. Looks attractive. I want to leave a final figure of this post that sum up the facts.

Fig 7: Contrast between damages with or without Mn-DP-Pi (Source)
Gaidamakova, Elena. (2012). Preserving Immunogenicity of Lethally Irradiated Viral and Bacterial Vaccine Epitopes Using a Radio- Protective Mn2+-Peptide Complex from Deinococcus Cell Host & Microbe DOI: 10.1016/j.chom.2012.05.011

Further reading:
  1. Deinococcus radiodurans. Microbewiki (Link)
  2. Misra CS, Appukuttan D, Kantamreddi VS, Rao AS, Apte SKRecombinant D. radiodurans cells for bioremediation of heavy metals from acidic/neutral aqueous wastes. Bioeng Bugs. 2012 Jan 1;3(1):44-8. doi: 10.4161/bbug.3.1.18878. (Link)

Monday, July 16, 2012

Shigella and salmonella messing with mTOR

Good to see you back,

Fig 1:Vaccines combine to produce new virus strain (Image from science news)
        As always, this week too had a lot of microbiology going on. The one which has beaten the hell out of people is a report from University of Melbourne, that a veterinary vaccine has backfired. The paper is by Sang-Won Lee and others titled "Attenuated Vaccines Can Recombine to Form Virulent Field Viruses". This would have been an excellent topic for me to talk about, but i couldn't get hold of the paper from Science. However, i could find an interesting story on the same well written in science news. And since its well covered by the science news, i guess i could just leave this matter for the time now, with a link to the story.

Fig 2: TEM of GFAJ-1 (Source)
            A yet another news that am not blogging about here, is the debate on arsenic based life forms. But that's still worth a mention here. The paper first published in 2010 reported that  bacteria GFAJ-1 can survive in high arsenic concentration by using arsenic instead of phosphorous met with strong controversy. The study was challenged by yet another study which argued the other way. Though most of the scientists have concluded that phosphorous cannot be replaced, the original contenders of the story are still trying to latch on to their claim. (Link to the controversial story here and here). Maybe we have more twists and turns to come up in this story. Anyways, keep your senses open for any news.

      The third story that i thought i will post in the blog is about "Galectin-8", and its association with autophagy. But then I came across a post in ASM blog that talks about it. (Oh man, that cover story also well covered up). In fact only after reading that post i came to know there's a journal attributed to discuss only Autophagy. That means, the topic has incredible amount of importance more than what i had thought about. But then i haven't lost a blog story totally after all. At least not after reading the article from "Cell Host & Microbe". I didn't find a blog on that so guess i can talk about it.

So here goes the story.

     Let me give you a analogy of autophagy. The best way to evade an intruder from getting into a house is to guard it well with defence (Immune system in this case). But with modern weaponry (Virulence factors), the intruder just manages to get into the system and takes good control. Now you are left with 2 choices, one abandon the battle (Don't expect the immune system to do that) or set of a detonator, that blows up itself from inside the house and blows up everything (Not a bad option, when it comes to cells. You can regenerate one).

  Interruption!! Did i forget to tell you what paper am i talking about? The paper is titled "Amino Acid Starvation Induced by Invasive Bacterial Pathogens Triggers an Innate Host Defense Program" by Ivan Tattoli and others.

      The innate defense mechanism of autophagy is regulated by a molecule called as mTOR which is a metabolic checkpoint kinase. The authors main aim of investigation was to give  deeper look into the how of the whole situation. Autophagy is a very conserved process that allows the cells to remove old non functional or highly damaged tissue elements to be digested and then removed from existence, that paves way to new ones. The process is highly regulated. The Fig 3 shown below depicts how the mTOR is invloved with autophagy. However it should be mTOR has many identified roles such as its importance in Influenza replication (Source) and in normal cellular translation process (Shown in Fig 4; Source)

Fig 3: Coordinated regulation of nutrient sensing by mTOR and AMPK (Source)

Fig 4: Importance of mTOR in normal cellular process
     The first thing that the authors did was speculate. They argued that probably the intracellular infections messed up with cellular metabolism and thereby with mTOR signaling.

      The study used Shigella (They didn't talk about the species or type, but still makes sense cause all 4 are nearly the same) and infected the HeLa cells, and they were able to demonstrate that there is a down-regulation of the phosphorylation of S6K1 and 4EBP1, with important interaction with mTOR. Rapamycin which is a potent inhibitor of mTOR (Source) was also used to demonstrate the reduction of phosphorylation. It was also noted that Shigella infection resulted in a strong induction of AKT phosphorylation.
Fig 5:  HeLa cells left unstimulated (CTR) or infected with Shigella for 4 hr, analyzed by IF with antibodies against mTOR and LAMP2.
Fig 6: Stress response
    Then to give a more detailed look, like all other scientists would do, they looked directly (I mean to say fluorescence based studies). With IF (immunofluorescence), it can be seen that Shigella infection resulted in a cytosolic relocalization of mTOR in infected cells (See Fig 5 on right). If you are thinking why they labelled LAMP 2 (Lysosomal-associated membrane protein 2), the full form should tell you why. Then by playing around a bit with raptor they could conclude that "Shigella infection down regulates mTORC1 activity by displacing mTOR from LAMP2+ LE/Ly vesicles, at the level of Raptor targeting to Rag GTPases".

    They could show that intracellular amino acid sensor GCN2 was rapidly phosphorylated in Shigella-infected cells mean to indicate that there is a Amino acid starvation. To confirm that this was actually the case a liquid chromatography/mass spectrometry (LC/MS) analysis was done on 2 amino acids L-leucine and L-isoleucine (Since they are supposedly highly influential with the mTOR pathways). Result? Shigella caused a reduction of cytosolic L-leucine/L-isoleucine levels. With these, they were able to elucidate and support stress response cascade (See Fig 6).

      The authors set to identify at genetic level. By comparing RNA expression patterns, from the one's in Shigella-infected cells and rapamycin-stimulated cells a large group of 87 genes were found. But as they looked for high expression patterns they found transcription factor ATF3 and its target CHAC1, were highly upregulated. The kinetic studies showed that ATF3 was induced as early as 1 hr. So thats how they came across the Fig 6, a classic GCN2/ eIF2a / ATF4/ ATF3 integrated stress response (ISR).

     When i had finished reading this much the first thing that struck me was "This should be a broad phenomenon". And to answer this ? (I mean the authors also had the same question) they did the study with  Salmonella Typhimurium. But they found that in this case the Salmonella-induced amino acid starvation was transient. And they did many other studies such as lentiviral-mediated RNA interference to look into other aspects (It didn't grab my attention much).

From these points i can conclude one thing. The amino acid starvation response, TOR pathway, and autophagy are highly audited metabolic connections that seems to define our understanding of the importance of battle inside the cells.

*Please note that the entire paper has not been discussed. I have taken only the upper half that i felt had enough interesting catch. But the paper dwells more deeper into various aspects and interested people should read the paper. The theme of this blog was to generate interest and i guess by leaving some part undiscussed here am giving you a room to explore certain areas yourself. Or maybe with enough request a episode 2 of this will be written.
Ivan Tattoli, Matthew T. Sorbara, Dajana Vuckovic, Arthur Ling, Fraser Soares, Leticia A.M. Carneiro, Chloe Yang, Andrew Emili, Dana J. Philpott, & Stephen E. Girardin1 (2012). Amino Acid Starvation Induced by Invasive Bacterial Pathogens Triggers an Innate Host Defense Program Cell Host & Microbe DOI: 10.1016/j.chom.2012.04.012

Further Reading:
  1. Qian Yang and Kun-Liang Guan. Expanding mTOR signaling. Cell Research (2007) 17:666–681. doi: 10.1038/cr.2007.64 (Link)
  2. Mathieu Laplante and David M. Sabatini. mTOR signaling at a glance. J Cell Sci October 15, 2009 122, 3589-3594. doi: 10.1242/​jcs.051011 (Link)

Monday, July 09, 2012

Hey "SAMHD1", You better stay out of nucleus

Hello fellows,

        "Science" is at the top of the world in past few weeks. Thats what i can say in brief. The  jury of H1N5 had just been settled, and a news wooped up claiming the virus is just a few mutations away, getting into humans. (Ooooh.... Didn't scare me though; Link). Scientists working at CERN finally give a verdict that Higgs Boson is a truth (Considered as a ground shaking discovery!; Link) Some serious evidence of dark matter (Huh, the news of Higgs boson blew this news away; Link) O my, i just could go with the list...... By the way, If you are thinking that I'm about to talk about any of these above you are wrong. I just couldn't resist saying this. The Photo posted on the left is that of a Large Hadron Collider at CERN (Image Source). And i cannot talk about Higgs boson (Or any of the other boson for that matter), cause i know only know as much as you know.

      Alright am not going to bluff more. So what do i have in blog pocket today. Hmmm, let me see. Oh yeah, Till the moment i was about to ink this blog, i was very much about to talk about  "Purinergic receptors and its important role in Human macrophage infection" influenced by the paper Hazleton JE etal publication in Journal of Immunology (Published in May2012; Link). The second paper that heavily caught my attention was a review article by Robert A. Britton and Vincent B. Young titled "Interaction between the intestinal microbiota and host in Clostridium difficile colonization resistance" (Link). But then another paper on HIV caught my attention.

For a background reading on HIV please refer to my previous post on HIV (Link), and have a look at the ppt below. So, the paper under discussion here is from BMC- Retrovirology Journal. 


    SIV stands for Simian immunodeficiency virus, or more interestingly known as African Green Monkey virus is thought to be parental type of HIV. Most of our understanding of HIV comes from experiments with this virus. The vast literature of HIV suggests that human cells have four major restriction factors to attack retroviruses. But as you would expect, the virus has armour evolved to protect itself. This is summarized in below table.

Photo: African Green Monkey (Source)

Table 1: Restriction factors against HIV/SIV (Source).

        APOBEC3G/F is a cytidine deaminase known to involve in antiretroviral immunity. Vif recruits cellular Elongin B/C-Cullin5 E3 ubiquitin ligase to target the host antiviral protein APOBEC3G (A3G) for proteasomal degradation. TRIM interacts with capsid of the HIV in cytoplasm. Tetherin ( Bst-2 CD317 ) is a cell surface protein, induced by IFNα, which can inhibit the HIV viral particle HIV overcomes this restriction by deploying vpu. The action is speculated to be mediated by recruiting SCF-E3 ubiquitin ligase complex, through an interaction between the β-TrCP protein and conserved phospho-serine residues in Vpu's cytoplasmic tail. If that is one part of the story, SAMHD1 is the other part of the story.

Fig 1: Vpx, SAMHD1 and viral replication (Source).

        SAMHD1 is a short version of SAM domain and HD domain 1, located in 20 pter-q12. The function of this protein is not well understood but is often speculated to possess a role in immunity and and cerebral vascular hemeostasis (Read more). Biochemically, it has a deoxynucleoside triphosphate triphosphohydrolase activity. Mutations with the function of this gene is known to be associated with Aicardi-Goutieres syndrome. (Click here to learn more about gene SAMHD1). It is a known fact that HIV-1 replicates poorly in human dendritic cells, monocytes and, and to an extent in macrophages. Th restriction factor was then traced to SAMHD1, the arch enemy of reverse transcriptase. And the counter force is applied by vpx. By the way, SAMHD1 localizes to the nucleus in human fibroblasts.

Fig 2: Ribbon representation of the SAMHD1 dimer with the major lobe (blue), minor lobe (grey) and C-terminal region (red). (Source)

Fig 3: HeLa cells expressing the indicated GFP4 fusion constructs
  The first experiment done by the authors was to fuse the SAMHD1 to a GFP4. HeLa cells expressing the GFP fusion constructs (green) was imaged by fluorescence microscopy. The cellular nuclei were stained by using DAPI (blue). By doing the same experiment, fusing the GFP at various sites, they were able to show that the Nuclear localization signal (NLS) was present in first 150 amino acids. A co-localization with fibrilarin suggested that SAMHD1 is in the nucleoplasm, but not in the nucleolus.

        With a site directed mutagenesis experiment, they further nailed down the location of NLS to peptide 11 KRPR 14 starting at position 11. To further prove that 11 KRPR 14 was indeed the NLS, they incorporated the implicated peptide sequence to N terminus of a GFP protein fused to the viral protein muNS. The muNS which is from avian reovirus that localizes to the cytoplasm. IF the KRPR is the signal then the muNS which is found in cytoplasm  is now expected to move into nucleus. And the results (Shown below, fig 4) was obvious. It moved.

Fig 4: Transferrability of NLS

      Now, lets see what happens in the virus vs SAMHD1 in battle field. It is stated that nuclear localized SAMHD1 blocks retroviral infection before reverse transcription. I have never understood this concept. My understanding is that reverse transcription happens in cytoplasm (Remember! RT interfered by APOBEC3G/F. So how exactly a person sitting in nucleus is messing with reverse transcription?). But answer seems to be there here. The authors went on to test, if SAMHD1 needs to sit in nucleus to cause effect? And they found answer is no. Am relieved.

       Now the question is what's the counter force of virus? Its shown that Vpx assembles with the CUL4A-DDB1 ubiquitin ligase through DCAF1 recruitment. This was presumed to cause erosion of restriction.

Fig 5: Presumed mode of action of HIV-2 Vpx in macrophage infection (Source).

      The authors hypothesized that nuclear localization of SAMHD1 is required for the ability of Vpx to induce its degradation and overcome restriction. To answer the question they just measured the ability of nuclear and cytoplasmic Vpx proteins to induce degradation of SAMHD1 variants that localized to the cytoplasm. They found that Vpx nuclear alleles required a  nuclaer localized SAMHD1 so as to induce its degradation and to overcome restriction.

       So I conclude second important thing. (the first important thing was 11 KRPR 14  is the NLS). The SAMHD1 should be present in cytoplasm to attack reverse transcriptase but for the vpx to attack SAMHD1, should be in nucleus. That means, if the SAMHD1 stays in cytoplasm cell has competitive edge. But if the SAMHD1 goes to nucleus, now the virus has the say. Ooops, thats wonderful isn't it. It should be noted that the vpx encounters the SAMHD1 in nucleus (O come on what do you expect, SAMHD1 to just sit and wait for a retrovirus to come when it has got the NLS to get into nucleus). Now note that if SAMHD1 is brought to cytoplasm for proteosomal degradation then virus is in trouble. So its safe to assume that the virus degrades the SAMHD1 at least partially, brings it to cytoplasm and then targets it to proteosomal degradation. Now for a min assume that the virus may have a mechanism to increase the SAMHD1 going to nucleus (Pathogens always possess these wooing to grave yard kind of strategy. Just guessing anyway).

       So what is the take home message. I would like to say i want to exploit this. Tell the SAMHD1 to be there in cytoplasm. Block the NLS...
Alberto Brandariz-Nuñez, Jose Carlos Valle-Casuso, Tommy E White, Nadine Laguette, Monsef Benkirane, Jurgen Brojatsch, & Felipe Diaz-Griffero (2012). Role of SAMHD1 nuclear localization in restriction of HIV-1 and SIVmac Retrovirology DOI: 10.1186/1742-4690-9-49

Further reading:
  1. Nadine Laguette etal. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature 474, 654–657. Link
  2. Kasia Hrecka etal. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature 474, 658–661. Link

Monday, July 02, 2012

The saga of Hush Bush continues..

Hello audience,

        Its not long ago since i had blogged about "Influenza lab created virus", that set stage for historical debate of the year in Science community. I dont know if i should call this post a follow up or just just yet another insight to the whole issue. Anyways, if you have not read my previous post on influenza A/H5N1 transmissibility, i recommend you first read the post (Link) and then get back to this page.

Photo 1: Avian Influenza virus

Photo 2: Dr Ron Fouchier

       Also i have a couple of links to some shows that i recommend you watch or listen to before going ahead. The first is the meeting held by NSABB (Link) that discussed on the publication of the paper, and the second is a podcast by TWiV 182 (Link). These episodes should give you a complete picture of how the story has twisted and turned in the last few months. And ultimately when the paper published, the most important point that was taken home was "Only 5 mutations are crucial for an avian influenza of H5N1 to cross species barrier and invade the human territorial lines". And suddenly there was a hush bush created. For people who are still in doubt about the risks and benifits of influenza research you must read a the science paper in June 2012 issue by Anthony S. Fauci and Francis S. Collins (Link)

      Thats the recap of the story (If i could call it so). As the legend goes, hot science stories never crumble to an end quickly. And here it makes the news again (Don't get me wrong on this. I just mean to dramatise, to say that there's more insight into the story of Influenza A H5/N1 transmissibility).

      So with all the links, and all the background let me dive into the topic. The paper in discussion, in this blogpost is a paper by Colin A. Russell etal titled "The Potential for Respiratory Droplet–Transmissible A/H5N1 Influenza Virus to Evolve in a Mammalian Host".

      The paper starts with a statement that says it all "A/Indonesia/5/2005 avian A/H5N1 influenza virus may require as few as five amino acid substitutions, and the A/Vietnam/1203/2004 A/H5N1 influenza virus requires four substitutions and reassortment to become transmissible between ferrets via respiratory droplets". The leucine at 222 and serine at 224 in the hemagglutinin (HA) is known to provide human like alpha-2-6–linked sialic acid.

         Ok, So what has been done in this study is to look at various sequences of Influenza A H5N1 virus collected over the time and, circled out the ones that comes close to the required amino acid substitutions. Now, here's the catch. Many viruses that might have evolved with this change in birds will be simply negatively selected, because they are still in birds. So thats why probably they haven't made into humans in a notifiable bang as of yet. The second most important point that struck me hard is the data of samples from Nepal, Mongolia, Japan, and Korea (Time period 2009-11), belonging to the clade They were the closest ones.Of the 94 strains they have sequenced 17, but none yielded a E627K substitution. E627K substitution is connected with mammalian polymerase adaptation. To me that makes sense again, because they are still in birds. Colin Russell, said: “Viruses that have two of these mutations are already common in birds, meaning that there are viruses that might have to acquire only three additional mutations in a human to become airborne transmissible. The next key question is ‘is three a lot, or a little?’"

          Let me digress a bit here. I was curious to know if the H5N1 has already made big news anywhere. So i fished out the WHO data (Link). And I didn't find Nepal, Mongolia, Japan or Korea in the list where cases of humans were reported. Indonesia was leading reported area. A total of 606 cases were confirmed with 357 deaths. Thats aprox 58.91%! So now i see the source why people think H5N1 is fatal. The fact is that the 606 are cases of known apparent infection. The asymptomatic ones is not accounted for denominator in this case. Fig 1 is a study by Smith etal, which was looking for predominance of  H5N1 influenza variant in China. They were able to isolate a good number of them, but their serological conversion was not so good. 

Fig 1: Comparison of H5N1 influenza virus isolation rate (%) in chicken (A), duck (B) and goose (C) from southern China, July 2004 to June 2006. (Source)

Smith etal also made a clear statement in the paper, "The predominance of this virus over a large geographical region within a short period directly challenges current disease control measures." The highlighted region says it all. But they were mainly looking for viruses not its sequences.

So back to the paper what we i was blabbing about. The authors go on to make a note that surveillance has detected humans with A/H5N1 viruses four nucleotide mutations and three mutations away in HA. "So if the species barrier jump is possible for this virus?", that was their question. This gets me to lit bit of confusion here. H5N1 infections are reported (See WHO link above). So instead of going into the mathematical model, i would have tried looking into the sequences of the viruses that already has caused infection and then see are they the same that "Ron Fouchier" had projected for ferrets (Either i haven't caught hold of some point or this angle of looking has flaws!! I don't know, or rarely maybe am absolutely right).

Fig 2: Documented Avian Influenza infections in humans (Click here for source)

So with a mathematical model approach (At this point am doomed. Am so bad with math u see, and i just believe what they are saying here). And the fig 3 is what they came up with after considering random mutation, positive selection and long infection.

Fig 3: Expected absolute numbers and proportions of respiratory droplet–transmissible A/H5N1 virions within a host initially infected by strains that require five (blue), four (green), three (orange), two (red), or one (purple) mutation (or mutations) to become respiratory droplet–transmissible.

So they concluded that the factors that may play major contributions, positively for gaining those important mutations include Random mutation (Any given virus has enough of those), Positive selection, Long infection, Functionally equivalent substitutions (Given that virus is good at mutations selection pressure should ease this factor)Diversity in the within-bird virus population and Transmission also accounts. The factors likely to negatively regulate were an effective immune response, Deleterious substitutions and order of acquiring those mutations. (Additional Source).

Derek Smith had to say about this "With the information we have, it is impossible to say what the exact risk is of the virus becoming airborne transmissible among humans. However, the results suggest that the remaining three mutations could evolve in a single human host, making a virus evolving in nature a potentially serious threat".

So, if you had asked me, my views are so. First, the paper makes an attempt to answer the question "If its possible?". But just as any other biological property the percentage of chance is very unpredictable. Second, the paper should have gathered more sequences. They have studied only 18% of mutations and say E627K substitution was not detected. And if that could have come from the sequence of the ones who were in the clinic it would have been a strong proof. (Maybe they will do it in future. Am not a flu virologist, but that data would impress me). But then the paper do suggest that possibility is not nil. As Prof Smith puts it "The situation is similar to assessing the risk of an earthquake or tsunami". So guess we need to have something like we have for earthquake or tsunami. Probably stock pile some vaccine.
Colin A. Russell, Judith M. Fonville, André E. X. Brown, David F. Burke, David L. Smith, Sarah L. James, Sander Herfst, Sander van Boheemen, Martin Linster, Eefje J. Schrauwen, Leah Katzelnick, Ana Mosterín, Thijs Kuiken, Eileen Maher, Gabriele Neumann, Albert D. M. E. Osterhaus, Yoshihiro Kawaoka, Ron A. M. Fouchier, & Derek J. Smith (2012). The Potential for Respiratory Droplet–Transmissible A/H5N1 Influenza Virus to Evolve in a Mammalian Host Science (New York, N.Y.) : 10.1126/science.1222526

Further reading:

1. Influenza Hush Bush, Link

2. Anthony S. Fauci and Francis S. Collins. Benefits and Risks of Influenza Research: Lessons Learned. Science 22 June 2012: Vol. 336 no. 6088 pp. 1522-1523. Link

3. Bruce Alberts, H5N1. Science 22 June 2012: Vol. 336 no. 6088 p. 1521. Link