Wednesday, December 31, 2014

Blogger's Desk#4- Probability in Science: Why


At the end of 3 years of blogging with 130+ posts and more than 131K views in total, I want to thank those people who have constantly questioned me and sometimes have asked me questions that left me running for answers. I have been able to answer a few of them, with a large chunk of reply attributed to the rescue statement "I'm not aware of any research or text in response to your question". But the path has been rewarding though. This blog page has been a product of my appreciation of research and researcher's alike.

However, there seems to be a question that hit me which is exquisitely simple, but to convincingly tell you an answer, seems to be so difficult. But lately, I think I have an answer and let me make an attempt. Let me re frame the question like this. "Why do scientists always talk about a finding in terms of 'probability' and not certainty? This question has also come up when I was data mining regarding understanding of science (Link) and its public outreach.

Let us consider a scenario. you are mixing a chemical X with Y to yield X2Y4 under specified conditions. If I say there is a statistical chance that you will not get the same reaction 15 times out of 100 attempts despite everything being same. What is your reaction? Absurd right. But that's the way Biology works, since it has variability. That means, at least simply by fluke you end up getting a deviated result, simply by chance. It is possible to quantify this degree of uncertainty and the possibility of chance to a certain extent by using standard bio statistical models. Here's is the first point why biologists talk in terms of probability, regarding any claims.

Second point, studying biology is a complicated process. There are many a times too many variables to consider, with each variable studied one at a time. This is called a reductionist approach. The puzzle is divided into multiple parts, with each individual part solved one at a time, and then the whole thing is stitched together. However, there is a catch here. There is nothing to tell you that the puzzle is complete and hence you cannot be sure. I want to quote from Richard Dawkins (I think it was in his book, "The greatest show on earth"). You could never prove anything with absolute certainty in science. Inability to disprove is a proof of correctness. This stands in line with the standard that any theory should be testable and falsifiable (Read more here).

Let me give you an example. Shamelessly, I'm replicating this from David Eagleman from his book "Incognito". Let us consider that a person who has never known anything about radio waves gets a radio which produces music. Having never seen such a thing he decides to scientifically deduce how it works. After years of research he figures out that by arranging circuit in certain way, he is able to generate the music. As per the science hypothesis would be, he is correct. But the actual way is there is a component which he has never studied and unknown to him. The telecasting radio waves. You see, despite everything being right there was a completely hidden component in the puzzle, but there is no way of knowing there is such an extra component. Now you get the point.

This provides an additional explanation of why with better technologies we come to know better and sometimes may even overturn a standard hypothesis based on fresh new understanding. In a summary, the extreme variance in biology and the inability to predict that a problem has been solved is the reason why a responsible sane scientist always talks about his results in terms of probability.

Wish You all a Happy New Year.

Tuesday, December 30, 2014

Blogger's Desk #3- Science awareness

More recently I have discovered from the internet that there is a great set of misinformation and lack of understanding of science, how it works and what makes it important. As a person into the science myself, I see deep problems with this. So I decided I could set up a survey and try to find some answers. The survey respondent has been far less than I expected it to be. I also talked about it to people who are not in science and tried to mine some additional data. 

The most striking feature that I found from the response is what does research mean? There was a universal agreement that science enhances our understanding of basic process and has enhanced our capabilities. More than a 50% declined to comment on a difference between Translational and basic research. Rest agreed that basic leads to translation. In reality there is no such thing as basic and translation. It’s a product of the highly competitive funding system (Link).

Fig 1: Preferred mode in updating and searching for literature
On an average the active researchers looking for science papers and materials, search and update their literature once in 3.20 days. That seems fine, till you see that majority of them use “Simple Google” approach. About 38% read review articles and almost 0.72% read original articles. And the big blow, a great deal of people agreed that the soundness of work was determined by the journal in which it has been published. It was found that majority of them don’t understand the article to a large degree; forget about critically evaluating the paper. For finding research paper of interest, PubMed followed by Google was the choice. I had expected that.

Towards science communication, there is quite a mixed signal. There was equal distribution in terms of how feasible was it to explain their science to layman and should scientists communicate more? Striking enough, too few people listen to materials such as podcast by journals such as Nature, Cell, Lancet, Science, NEJM etc. Some even don’t know they exist. In fact the aim of the podcast is simplifying the material, how far is the goal met, debatable. No wonder, “Google” is the choice. And I’m talking about one’s in science.

A chief complaint by majority of the researchers is that the published work is not available to read and you have to pay for individual papers. That forms the chief drive for Open access publications. But when asked if the paper is submitted to archives, very few agreed that they do in sites such as, arXiv, biorxiv or personal websites. A majority doesn't know it is legal to submit papers to repositories and a few denied they want to. Archiving should enable access to papers. 

The story is quite different from others perspective. Google is almost exclusively used for finding anything they want to know. They avoid reading scientific papers and rely heavily on news reports or webpages that looks promising (not necessarily true information). Their major problem is that scientists don’t communicate in an understandable layman language. Except when there is a ground breaking emergency or issue there is virtually no communication by the scientists to general public. Oh yes, most are not aware of authentic materials out there on web for general public, which attempts to communicate.

Box 1: The scenario.
The Box 1 is a depiction of the current science communication reality. I can quote an example. When scientists decided that they want to do an experiment to study H7N9 the hype of gain of function was needed to be addressed to public. Instead it was in a high tier journal (Link). Press picked up on it, and the tables turned around the wrong way. Some argued that scientists plan secretly and not open enough with public. Though the intention was to communicate and not cover the problem, the mode of doing it actually brought in mistrust. As said by Adel A. F. "The scientific justification presented for doing this work is very flimsy, to put it mildly, and the claims that it will lead to anything useful are lightweight”. People including scientists forgot what Gain of function and DURC is all about. 

Second, even the science specialists themselves have a problem. The most frequently encountered is the “authenticity in communication”. For example, when scientist say there is a “probability X that event Y will happen”, it sounds less authentic than media hyping, “Scientists claim they found Y will happen”. Even if the reader gets back to original paper and tries to understand it, chances are (Probability!!!) he won’t understand a word. As I have already shown relatively inexperienced scientists can find it difficult to understand modern complex high impact factor papers.

The original idea of scientific papers was to communicate science. Science had just started growing up and hence wasn't complex, easily understood. Today in journals like Nature, a original publication is a distillation of years of hard core science for years sometimes condensed into less than 4-5 pages. Naturally, a detail that seems to be universally known is not inked in. And here’s the catch. When you are at the very peak of knowledge, details that seem to be very complex can be perceived by you as a obvious fact. For others it isn't, and needs explanation. This phenomenon is called as “Knowledge paradox”. The resulting output is a very complex paper. The idea of abstract or author’s summary is supposed to curb this problem. However, the growing complexity of science literature makes this problem worse. Journal such as Elife aims to publish as much detail as possible. Such approach may alleviate the problem.

I by no means am discrediting the hard work of scientists nor does the public. The point is science understanding is currently low. In the ages of booming of physics, there used to be public lectures for the lay audience. In the newer age Podcasts and science shows could lead similar way.

Your opinion and thoughts?

Saturday, December 20, 2014

Ebola 2014: December updates


Fig 1: Ebola deaths in key areas.
Once again it is time for some updated news on Ebola 2014. As of he official reports, the total number of Ebola cases is 18,000, a figure more than all the cases combined, ever reported in past Ebola outbreaks. Also a recent outbreak discovered in Kono by the WHO, has brought in fresh concerns. The news report is that 87 bodies have been buried by 2 teams in past 11 days. The concern is Ebola outbreak hasn't completely leveled off. As Olushayo, an official in WHO's response Ebola team puts it, "Our team met heroic doctors and nurses at their wits end, exhausted burial teams and lab techs, all doing the best they could, but they simply ran out of resources and were overrun with gravely ill people". This underlines an important point. The total number of Ebola is probably under reported, as WHO agrees in their reports.

Other than the deaths of civilians, doctors killed in outbreak is also part of the headlines. Victor Willoughby, senior doctor involved in combating the outbreak, has died just when the experimental drug ZMab (Different from Zmapp) arrived from Canada on request. ZMab is also a monoclonal antibody preparation, targetting different epitopes- 1H3, 2G4, and 4G7. These monoclonal antibodies, raised in mice immunized with a VSV-based EBOV vaccine (VSVΔG-EBOVGP), recognizing the Ebola glycoprotein. The cocktail was tested recently in a Cuban doctor Felix Baez, who successfully recovered.

With favorable results from ChAd3 Ebola vaccine, in Phase I, plans to begin a Phase II trial has been initiated and currently under review by WHO. The trials are expected to begin by February 2015. In West Africa, trial has begun to check the efficiency of plasma exchange from a Ebola recovered donor (Link). A clinical trial is also underway, using a attenuated replication competent Ebola strain. The trial in Geneva has been halted a week after inception, based on four patients complaining of joint pain. 59 people have been vaccinated so far. Though it is expected that there would be side effects of mild nature, the replication competence of virus is to be considered. Hence the careful observation. The experts suggest it would be better to try and understand the reason behind side effects.

Fig 2: Action of FX-06. Source
A team from Frankfurt University Hospital, has published the use of a experimental drug on a A 38-year-old male doctor who had contracted Ebola. One of the major problems in EBOV is the vascular hemorrhage. The patient received a 3 day treatment course with FX06. The safety and efficacy of FX06 (The fibrin Bbeta chain-derived peptide) in the treatment of vascular leak syndrome has previously been explored in animal models of lipopolysaccharide induced and dengue haemorrhagic shock. FX-06 competitively binds to vascular endothelial (VE)-cadherin, thereby inhibiting leukocyte transmigration and initiating VE-cadherin-mediated signaling, which tightens the endothelial barrier and reduces capillary leakage. The sequence of FX-06 resembles that of a physiological peptide, and the compound has been well tolerated in clinical trials. On treatment, improvement was seen from an almost fatal case to improved respiratory and vascular function, reduction in viral load in blood. The virus was not detectable after 30 days.

With currently more than 53 different treatment regimens under R&D testing, the chances of developing a good vaccine and drug to combat Ebola Outbreak, the prospects appear good.
Audet J, Wong G, Wang H, Lu G, Gao GF, Kobinger G, & Qiu X (2014). Molecular Characterization of the Monoclonal Antibodies Composing ZMAb: A Protective Cocktail Against Ebola Virus. Scientific reports, 4 PMID: 25375093

Callaway, E. (2014). How do you test an Ebola vaccine? Nature DOI: 10.1038/nature.2014.16579

Timo Wolf, Gerrit Kann, Stephan Becker, Christoph Stephan, Hans-Reinhardt Brodt, Philipp de Leuw, Thomas Grünewald, Thomas Vogl, Volkhard A J Kempf, Oliver T Keppler, Kai Zacharowski. Severe Ebola virus disease with vascular leakage and multiorgan failure: treatment of a patient in intensive care. The Lancet; Published online December 19, 2014

Tuesday, December 09, 2014

One more add-on to brand of New Generation Antimalarials


I have previously blogged a few posts on efforts to fight malaria. Malaria has been a longstanding problem in tropics and efforts to control have achieved reasonable success, at least in some parts of the world. As recently noted, there is a growing need for better Anti-malarials (Link). More recently there is a headlines circulating over the web- "A new antimalarial compound". And this post is all about it.

Table 1: Drugs in development through MMV 
The original publication is based on a paper published in nature, in 2010. By using a high throughput phenotypic assay (The screening was done by identifying molecules that block the proliferation of Plasmodium falciparum strain 3D7 in co-cultures with human erythrocytes), 309474 unique compounds. The screen generated 1,134 valid hits. Of them 172 chemicals was closely evaluated. The database is available at St.Jude research. In nutshell, reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. One among the 3 series of potential chemicals was DHIQs (dihydroisoquinolones). (+)-SJ733 is a component of that class. There is a huge list of chemicals that are gaining popularity for use against plasmodium which are developed in partnership with MMV (Medicines for Malaria Venture). A list of some well known compounds is shown in Table 1.

Table 2: SJ733 
pfATP4 is a P-type cation-ATPase which serve as parasite plasma membrane protein with sequence similarities to Na+-ATPases. It is the target for many types of antimalarials such as Pyrazoleamide compounds, Spiroindolone etc. It counters the influx of Na+, thereby maintaining a low Na+ content. SJ733 or (+)-SJ557733 (both are same chemical), disrupts this action which leads changes in physical structure of RBC. The RBC is more shrunken, resembling a pattern of eryptosis. The RBC is seen as a damaged version in the reticuloendothelial system. The next step is obvious, immune system attacks the RBC thereby defeating the plasmodium inside. This is something similar to people who have G6PD deficiency. Plasmodium simply doesn't survive well cause of similar RBC elimination.

In a mouse model, the drug was administered as a single dose which cleared about 80 percent of malaria parasites within 24 hours. After 48 hours the parasite was not detectable. And here is the most interesting part. This target molecule is a heavily conserved system and mutation has a huge fitness cost. That means resistance can develop but in low level. To quote from Kiplin Guy (Source), "Our goal is to develop an affordable, fast-acting combination therapy that cures malaria with a single dose. These results indicate that SJ733 and other compounds that act in a similar fashion are highly attractive additions to the global malaria eradication campaign, which would mean so much for the world's children, who are central to the mission of St. Jude".

Oh of course, Clinical trials are now on the next "To Do" list.
Jiménez-Díaz M.B., Ebert D., Salinas Y., Pradhan A., Lehane A.M., Myrand-Lapierre M.E., O'Loughlin K.G., Shackleford D.M., Justino de Almeida M. & Carrillo A.K. & (2014). (+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium.PNAS PMID: 25453091

Guiguemde WA, Shelat AA, Bouck D, Duffy S, Crowther GJ, Davis PH, Smithson DC, Connelly M, Clark J, Zhu F, Jiménez-Díaz MB, Martinez MS, Wilson EB,Tripathi AK, Gut J, Sharlow ER, Bathurst I, El Mazouni F, Fowble JW, Forquer I, McGinley PL, Castro S, Angulo-Barturen I, Ferrer S, Rosenthal PJ, Derisi JL,Sullivan DJ, Lazo JS, Roos DS, Riscoe MK, Phillips MA, Rathod PK, Van Voorhis WC, Avery VM, Guy RK. Chemical genetics of Plasmodium falciparum. Nature. 2010 May 20;465(7296):311-5. PMID: 20485428

Monday, December 08, 2014

Salmonella- survival Inside Macrophage; How?


Isn't it interesting to know that that there are several regions of interest in research, that I have never talked about? Well considering that there is so much of information out there, this blog timeline is too short to have even covered the part of any. Often I get communications from readers, asking opinion on certain topics and ends with a "You need to blog it". Well the list is growing up, but I enjoy those discussions. Here is one such topic that I felt needs a share- Salmonella Typhi, etiological agent of typhoid fever.

More than the biology of Salmonella, it is the story behind it that seems to be popular. So popular that almost everyone has heard "Typhoid Mary". Perhaps only there is a handful of names where patient / carrier is famous as much as the disease. Mary Mallon ( Irish immigrant) was the first documented "healthy carrier" working as a cook in North America. She was banned from the cooking industry twice having infected more than 50 people. There is a radiolab podcast which you must listen to (Link) Salmonella is a mega group of organism, with more than 3000 members in it. Salmonella Typhi (Note, I wrote Typhi and not "typhi"). S Typhi, is in fact a serovariety. Its complete name is Salmonella enterica subsp enterica serovar typhi. You see why we should stick to the short name.

Pathogenesis of typhoid is one of the most fascinating research question. Salmonella Typhi needs to infect in a large dose (ID50 > 105). Most of the organisms are destroyed by gastric acidity. Once into intestine, Salmonella calls up macrophages near it an enjoy a free ride. In addition salmonella can use anaerobic respiration with tetrathionate as a terminal electron acceptor, which gives it a competitive advantage over other members of flora. The details are covered in splendid detail in a post by Elio (Link), in small things considered.

Fig 1: Salmonella inside host. Source

Once inside the macrophage, the bacteria can simply inject different proteins (Nearly 20 effector proteins) into host cell, two distinct type three secretion systems (T3SSs) located on Salmonella pathogenicity islands- 2. These proteins interfere with antigen presentation, Cytokine secretion and cell survival. Moreover, there is an ability to survive inside macrophages as a dormant, non replicating persister. In a earlier paper, it was shown that something stimulates PPARδ (Peroxisome proliferator-activated receptor- delta), a eukaryotic transcription factor involved in sustaining fatty acid metabolism, which increases glucose availability and enhances bacterial replication in macrophage model, and increases persistence. Another important point is once salmonella is inside a macrophage vacoule it probably messes with the pH through a mgtCBR leader sequence which senses cytosolic ATP levels, interacts with mgtCBR operon and is important in virulence.

This discussion came up when someone asked me how does salmonella survive inside a macrophage. As most of the times, as is true in science, it is very difficult to throw a complete definitive answer. That said, it makes an interesting question which I'm sure scientists are exploring.

There is a probable outbreak of salmonella in North Cumbria, with 10 laboratory confirmed cases and five probable cases. It is suspected that the origin is a food source from Penrith.
Forest CG, Ferraro E, Sabbagh SC, & Daigle F (2010). Intracellular survival of Salmonella enterica serovar Typhi in human macrophages is independent of Salmonella pathogenicity island (SPI)-2. Microbiology, 156 (Pt 12), 3689-98 PMID: 20817644

Helaine S, Cheverton AM, Watson KG, Faure LM, Matthews SA, & Holden DW (2014). Internalization of Salmonella by macrophages induces formation of nonreplicating persisters. Science, 343 (6167), 204-8 PMID: 24408438

Haraga A, Ohlson MB, & Miller SI (2008). Salmonellae interplay with host cells. Nature reviews Microbiology, 6 (1), 53-66 PMID: 18026123

Eisele NA, Ruby T, Jacobson A, Manzanillo PS, Cox JS, Lam L, Mukundan L, Chawla A, & Monack DM (2013). Salmonella require the fatty acid regulator PPARδ for the establishment of a metabolic environment essential for long-term persistence. Cell host & microbe, 14 (2), 171-82 PMID: 23954156

Tuesday, December 02, 2014

Neural code for immunity


A lot of time have I talked about the connection between immune system and nervous system. However, it never occurred to me question, as to why should there be a network sharing between immune and nervous system. As a part of routine, I was browsing through articles online and almost stumbled on NEJM article, that left me venturing.

Fig 1: A model of neuronal regulation.
Source: Kao etal
The idea is that whenever there is a local inflammation peripheral nervous system communicates the information to CNS. This helps in coordinating the response. It is argued that if this code of information can be understood, we could make use of it to our benefit. As Dr. Steinberg puts it, "Timely diagnosis and intervention are essential to minimize deaths and complications. If the neurons are reading this information from an infection in the blood or the liver and we can interrogate the nervous system, we can make a diagnosis in real time". I haven't been able to get the full article to digest the complete information. However, I thought that this probably isn't the first paper in literature.

The best method of studying a very complex system as immune and neural networking is a simple nematode Caenorhabditis elegans. They have a well characterized genome and a immune system representative enough to make a case. It has been shown with a C elegans model, innate response to a variety of pathogens elicit downstream immune regulators that the neuronal system can regulate. A model of this has been explained by Kao etal (See Fig 1). To cite an example, neural circuit involving NPR-1, which encodes a G protein–coupled receptor (GPCR), functions to suppress innate immune responses.

The leading hypothesis is that GPCRs participate in neural circuits that can recognize bacterial pathogens (probably via PAMPs) or receive inputs from non-neuronal infected cells which can then integrate them allowing coordination of appropriate immune responses.

Fig 2: Two way signalling between Neurons
and Immune system.
As a matter of fact, neurons express receptors which otherwise is classically associated with immune system such as toll like receptors, nucleotide binding oligomerization domain receptor etc. On the other hand immune cells express classic neuron related molecules especially neurotransmitter receptors. Clearly its a two way signalling as I have illustrated in Fig 2.

Fig 3: Components of Immunity.
It wouldn't require to convince you more that the same applies to the adaptive immunity, just as in the case of innate. In this light, I want to postulate that we can define 3 types of immunity (in contrast with classic 2 types- cellular and humoral; In reality this isn't my idea, I have already seen a published paper which advocates). The third addition is Neural immunity.

Perhaps in one simple sentence I could sum it up.. "Probably Neural systems are not the immune privileged type, they are the regulators.." More research will tell.
Kawli T, & Tan MW (2008). Neuroendocrine signals modulate the innate immunity of Caenorhabditis elegans through insulin signaling. Nature immunology, 9 (12), 1415-24 PMID: 18854822

Kao, C., Los, F., & Aroian, R. (2008). Nervous about immunity: neuronal signals control innate immune system Nature Immunology, 9 (12), 1329-1330 DOI:10.1038/ni1208-1329

Styer KL, Singh V, Macosko E, Steele SE, Bargmann CI, & Aballay A (2008). Innate immunity in Caenorhabditis elegans is regulated by neurons expressing NPR-1/GPCR. Science, 322 (5900), 460-4 PMID: 18801967

Steinberg BE, Tracey KJ, & Slutsky AS (2014). Bacteria and the neural code. The New England journal of medicine, 371 (22), 2131-3 PMID: 25427116

Andersson U, & Tracey KJ (2012). Neural reflexes in inflammation and immunity. The Journal of experimental medicine, 209 (6), 1057-68 PMID: 22665702