Why working with Laboratory mice?

When I started my research career in 2004 I did not think much about laboratory mice. I remember talking with scientist specializing in human immunology about adopting and adapting on mouse cells the assay his lab developed. He asked me “do you think human and mouse immune system behave the same way?” I did not even pause a second, immediately replying “of course, they do”. Really?

Of course, I was always felt bad when harvesting lymphoid cells from experimental mouse that was a living breathing organism just few seconds earlier. Notwithstanding what laboratory animal husbandry suggests, sacrificing small animals through cervical dislocation, a method, believe it or not, considered to be the most humane way of euthanasia of laboratory mice, is still quite distressing. More frequently than not, I was able to accomplish a clean and swift cervical break, but in situations when I was not good, it felt terrible. Is it really worthy all of these experiments, especially on mouse?

National Institutes of Health (NIH) has many progressive guidelines on a paper. It actually mandates that before you conduct your first experiment on live animals, you need to justify that there are no other non-terminal, or non-invasive ways of accomplishing the experimental objectives. However, anyone can “easily” justify the necessity of in vivo experiments. Even if you just want to do purely in vitro cellular assays, how else can you harvest mouse cells except sacrificing the mouse first? Yes, you can harvest blood cells from tail vein but it will provide very few cells. So, what can be done?

First, people who approve protocols should ask themselves “is this model relevant for human disease”? Just because you can modify mice in such a way that it will develop asthma-like symptoms or can be infected with M. tuberculosis, it does not mean that the model has any relevance to humans. If mice works were so easily applicable for humans there would not be so many clinical failures in first place (TB, flu, dengue, ebola, all these pathogens do not replicate human diseases in mice, and still NIH is issuing grants for them).

I my view, NIH should make it obligatory that any new protocol requiring terminal or invasive procedures on laboratory mice to have strict human equivalency comparison. Moreover, NIH should mandate in silico modeling for any protocol. Advances in gene modification, like CRISPR-Cas9 technology, made it easier to convert normal human cells into any knockouts in vitro and this methods are accessible. It is much more relevant to conduct experiments on human cells rather than on mouse cells. Now you can study any molecular pathway in human cells you want. Why are we still clinging to mouse?

Some can say because FDA demands it. Not necessarily. FDA, like NIH, is a federal agency and maybe it is time to make some modification in its rules. Non-invasive, non-lethal experimentation in combination with in silico modeling is a better and more productive alternative to current state. It will force scientists to develop better computer modeling and better disease models.

posted by David Usharauli

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A tweet with valid URL does not show up under the Hashtag feed? Here is one solution

I recently joined Twitter. I thought I can use twitter to expand the audience and widen the exposure for my blog posts. I was totally unprepared to discover that any time I twitted about my newly published posts with the url link in it, twitter would not return it in search.

Initially I thought I was doing something wrong since I was a total newbie to twitter. Strangely, when I twitted anything without url links in it, I could easily found them in search (both in my home page and in public twitter search), but if the same tweet had url of any my blog posts added to it, it would appear only in my twitter home page and never be found in twitter search.

Now this made no sense at all. if not sharing tweets with link added to them, how else one can promote their blog posts or anything? As a new member of twitter community, obviously one does not have much of following. So how that can be true and what can be done about it?

This was my question. I wanted to find the solution to this ridiculous situation. In Google I found several discussions where people complained about the exact situation. One practical suggestion I found was to make tweets “not protected” in twitter setting so to allow tweets to be found in search and not just visible to your followers.

However, even with this modification, tweets with my blog post’s url added to them would not go into twitter search.

Again, I started to read more. I found that people who have reported the same issues, have done experiments and found that tweets without url would go into twitter search but the same exact tweets with url would not. More specifically, however, it turned out that tweets with the urls associated with big name websites would go into twitter search easily but when the same exact tweet was accompanied with one’s own blog post’s url or with not so well-known website urls, then it would not be found in twitter search.

Basically, when you are new to twitter and does not have much followers (obviously), your tweets would not be visible to anyone if you add url links not deemed to be authoritative links or prestigious enough for twitter to index it.

So, you can tweet anything you want as long as you add big name urls to your tweets, basically promoting others work. But who would like such restriction?

Here is what I did instead. I started adding well-known webpage urls to my tweets where I wanted to promote my blog post’s links. I called these urls CARRIER or Booster urls since they have no other function besides to be a “face control” for Twitter indexing algorithm. And surely, all my tweets with my links + CARRIER or Booster urls started to show up in twitter search.

Here is the example:

NOT found in Twitter search

New study suggests activity determines cell death. Review here

FOUND in Twitter search

New study suggests activity determines cell death. Review here

CARRIER

Since I writing mostly about science, I am using nature.com homepage url as a CARRIER or Booster url for blog post url. Remarkably, even homepage urls for journal Science or journal Cell was not good enough for Twitter to index my tweets. for some reason only nature’s homepage url worked as CARRIER out of these three urls tested.

Of course, with this method, up to 35 characters are lost for CARRIER or Booster urls, but it is worthy. In the end, at some point in the near future, I expect my twitter account to become more prestigious for Twitter indexing system and would allow tweeting with my blog post’s url alone.

Added on Feb 1, 2015: I found that if I attach photo to my tweet then tweets with my blog post’s url will be indexed in twitter’s public search without need of CARRIER urls.

posted by David Usharauli

Genuine confidence comes from experience

A lot of books are written about confidence. Confidence is a purely acquired, trained quality, not an inherited one. I remember reading a book about a true eyewitness account of one of the JFK’s speeches wherein during the process of evidently confident public speaking, JFK’s hands were trembling behind the podium. How revealing is that, just think.

If you are in a science field, the most dreaded task is giving presentations or articulating your opinions in front of your esteemed peers. You are continuously thinking: am I voicing something clever or stupid? How is my presentation perceived? Dull or exciting? You seek validation from number of questions asked to gauge the audience engagement level. Even the most seasoned scientists are nervous about it. My former science boss, for example, Polly Matzinger, probably the most brilliant mind in immunology since Niels Jerne, said to get GI tract upsets prior to every presentation, even though she would go on to mesmerize the audience afterwards.

Many science laboratories with 6+ postdoctoral researchers usually run science journal clubs to share recent research development in the field. If you are a PhD student or postdoc and your lab does not have such system, find a way to establish it. Go and ask your supervisor about it. In my opinion, journal clubs are must for young scientists. It is the only venue available where you do not have a personal standing in the work and can discuss and debate research results objectively, thus sharpening your skills as a thinker, not a doer.

I remember my first journal club presentation. I had no idea how to choose a paper for a journal club. I chose the paper that was published 2 years earlier, a clear indication of my lack of understanding how science research operates. I thought I had a confidence but this confidence was clearly based on ignorance rather than experience. I just have chosen the paper because I thought it was telling something “new”. I had no prior experience in lab environment and I did not receive any formal PhD training prior to my postdoctoral research. Honestly, it took me close to 3 years before I started to understand the principles of scientific reasoning relevant for the successful journal clubs.

I even started a blog, called NIHilist’s Immunology where I post my analyses of new research in immunology so young scientists who want to learn the principle of scientific reasoning can go and view my discussions and if it make sense to adopt them for their own purpose. I wish I had opportunity when I was doing my first steps in science.

A true measure of confidence comes when you are convinced and are able to convince others. Such feedback reinforcement happens only with the experience and multiple efforts, not naturally or out of blue.

posted by David Usharauli

Future of Medicine should not be Elysium

To have a long and healthy life was and is a dream of humankind since the inception of human civilization. Based on current pace, within 20-50 years medical science will achieve level where many difficult to manage diseases, like cancers and some infectious diseases become easily treatable.

Most recent example is hepatitis C virus story. Just 5 years ago, it seemed that there was no hope for people affected by Hep C. There was no approved vaccine or approved drugs capable of stopping this virus that causes extensive liver fibrosis. However now, if completely unexpectedly, we have anti-viral medicine that show up to 99% effectiveness rate (in some combination) and with less side effect (double benefit). No one could imagine this.

What about recent success in cancer therapy? Treatments with patient’s own tumor-specific re-engineered T cells or with antibodies to awaken the body’s own immune system had achieved an unprecedented level of success reaching up to 50% effectiveness rate.

These are all amazing news for anyone and especially for individuals with these medical conditions. However, even though the future seems bright, there is something unsettling in all this. It is the price tag.

Costs are so prohibitively high that almost 95% of world population would not able to afford it on their own (for example, tumor immuno-therapy with the patient’s own immune cells can cost more than 500,000$ , and Hep C treatment can cost between 50,000 – 120,000$ per treatment ).

More worrisome is the fact that unlike small molecules used in conventional pharmacology that can be easily manufactured in mass scale with minimal investment as generics, many advanced new treatments now days are based on large, complex molecules, called biologicals like humanized antibodies, or even more complex procedures like harvesting and re-engineering patient’s own T cells. These are completely different categories of medicine with no real way for mass scale production.

How can we afford this costly medicine to live longer and healthier?

Movie, Elysium, shows one such scenario. In this movie, society are divided into super rich and everyone else. Super rich live on a giant orbital station and are completely free of any disease thanks to medical technologies. These medical technologies are inaccessible to regular earthly people.

In my opinion, with the few rare exceptions, we do not control our medical health trajectory. Hence, every human should have an inherent right to have an access to the most advanced medical technologies. However, no federal or state budget can afford medical cost so high.

So what is the solution? how to make cutting edge medical technologies affordable to average person? The solution can lie in technology but in different kind of technology that is in infancy right now. It is gene therapy (gene silencing, cutting, replacing, modifying). Gene based medical technologies has two major advances over other type of biologicals: (a) gene therapy will be much cheaper (everyone who worked with PCR or RT-PCR can verify that costs for primer nucleotides are way cheap compared to antibodies), (b) gene therapy will treat the cause (genes) rather than outcome (proteins) of medical conditions.

Current medicine is based on repeated engagement with body’s proteins (receptors, cytokines, transcription factors). Gene therapy will be based on single or a few engagements with body’s DNA or RNA for long-term effect (for example, using CRISPR-Cas9 system). Such intervention can produce permanent fixes. An Overall cost will be low and majority will be able to afford it.

posted by David Usharauli

Genuine politeness should come out of respect not out of fear

Today’s public space is dominated by the idea of “political correctness”. This basically means that everything one says or does are extensively monitored and analysed. New rules of social interactions are developing and are introduced for the public use with such speed that it outpaces majority of human’s natural ability to adopt and adapt to it.

Many evolutionary conserved reactions to stressful situations such as expression of anger, yelling, shouting, even simple direct, blunt talking are considered socially unacceptable and inappropriate.

Interestingly, on the other hand, popular TV shows are popular exactly for opposite reasons for showing what society lacks and aspire to have: an opportunity for natural reactions to challenging social situations.

It is obvious that our society did not develop a heightened tendency for “political correctness” or “artificial politeness” out of genuine respect to a fellow human being as in Kant’s philosophy, but rather as a consequences of real, perceived or imaginary fear of getting ostracized, isolated, or even sued.

Fear, however, produces superficial interactions and artificial social norms with weak bonds. Moreover, since our natural reactions cannot be blocked, they must be expressed someway and somehow. In fact, rather than helping, such re-channeling of natural reactions further diminishes societal harmony by producing chronic “mean” behaviors.

In my opinion, a genuine politeness should be derived out of genuine respect. Genuine respect develops when there is a trust. Trust, in turn, develops when people’s verbal or non-verbal communications are matched with their actions.

posted by David Usharauli

Rise of autocratic science research

Advances in Science comes when there is a free exchange of accumulating knowledge. Thus, Science, by definition, should be a democratic institution by its nature.

As organization of family units is a foundation for modern states, organization of research laboratory units is a foundation for modern science. So analysis of laboratory units can give us a clue of how science advances or stumbles.

In contrast to popular belief, organization of modern research laboratory units is clearly and unequivocally autocratic in nature rather than democratic. Simply put, an absolute majority of research laboratories in both academia and industry do not and cannot contribute to the advancement of science, period. Of course, such labs do publish research articles at the end of the calendar year or submitting quarterly reports to justify their existence, but that all.

So a natural question is why is it the case? Almost everyone is starting their science journey as an idealist democrats and are ending up as a fearful autocrats. Why? This is because there is no separation between laboratory research and laboratory management. When the same Principal Investigator (PI) are required to conduct high quality scientific research as well as to procure the funds for the same research, there is a little tolerance to different ideas and opinions. Fear of losing funding prevent PIs to be courageous in science and follow their gut instincts. In the end, fear reduces the diversity and chances of great discoveries and such PIs become career scientists with more knowledge in bureaucracy than science.

This was a one reason why the US Government originally had created an intramural research labs with a secure funding where scientists were just asked to focus on science. The same idea was behind HHMI funding. When PIs are released from the fear of loosing financial support, and only requirement to them is that they will produce high quality research that can be published in journals like Nature or Science (or at least in top 5 journals in their subject field), then PIs would become open-minded and more democratic because high quality research needs a democratic environment to become a reality.

Actually, HHMI Investigators do publish in top journals. However, intramural biomedical research funded by the US Government did not fare well since Government did not put the requirement that the research should be of high quality, not just any kind of research conducted at leisure. Without such balancing approach, system becomes easily distorted.

posted by David Usharauli

Two myths I used to believe

Myth # 1. All scientists are brilliant or even clever or competent

If you grow up in a developing country where only way to “experience” science is through old books and occasional science journals published in English, you could easily start to imagine that people with a pipette in their hands who are working in the bio-hoods, with the richly stocked lab shelves in the background, are all professionals, basically geniuses in their field, especially if they are working in countries like USA (since you constantly hear that science awards, like Nobel Prize, are given to scientists for discoveries carried out in the USA).

You read or watch on TV the young people in the lab coats discussing how they love and enjoy doing science and how they want to make the difference. You actually believe that it is true until….

Until you end up as a postdoc yourself in a science lab in one of the most prestigious academic centers. Suddenly you realize you have no idea why some of the members of the lab have chosen to be here in the first place. They do not participate in science discussions, journal clubs, seminars or ask any questions. You wonder if they are capable of scientific reasoning and rationalization. Maybe not. They are just sitting there and after 5:00 pm leave for home. Nothing related to science excites them. And without excitement and true interest in experimental results, it is absolutely impossible for someone to do a 12 hour workday. Why are they occupying positions that could go to persons who at least are eager and excited about science?

I do not believe anymore that if someone is working in a science lab, even in the best labs, that they are smart, bright or even clever or competent. I do not believe in CV/Resumes either. I do not believe in personal interviews because it never works.

I only believe in how a person describes what he/she has done. In my experience people with a genuine interest in their science work have a wider vocabulary describing their own work and frequently use synonyms to easily adapt to the level of their audience, in contrast to people who are in science from reasons other than science itself.

Myth # 2. Private sector is more efficient than Government

I remember when I was working in the academic environment (that was a part of federal government), we had one scientist who joined the lab from the industry (one of the biotech company). He used to mention that he become disillusioned about the biotech industry since it allowed too much wastefulness. He was telling us how many of the company’s employees were not doing anything all day long, spending all of their time in the tea room in idle conversation with colleagues. Honestly, I did not believe him then. I was asking how come a private company was tolerating such behavior, did the company not care about its employees’ productivity? Why would the company keep such people? He would just laugh at me and tell that the company knew about these people but was doing nothing.

Now I know that he was absolutely correct. There is no difference between private, governmental or academic efficiency in science. Every lab has people who do nothing and this is tolerated. If you ask me why it is the case, I have no idea. I just know that it is true. It appears that majority of people prefer to pretend rather than have a constructive confrontation with their bosses, employees or colleagues. Many times, employers compensate the lack of productivity from such “do nothing” employees by dumping extra burden on other employees or hiring more people. Of course, in the long run, this will not work. No wonder that the company the guy came from to our lab does not exist anymore.

posted by David Usharauli

Serendipity in Science: Personal Observation

For a scientist reading review articles about his/her research topics is a quite boring task, since review articles usually contain summaries of what are already known. The reviews have no real scientific purpose except as a source of citations and cool looking figures.

There is one reason I found review articles to be very useful to read. Frequently, the authors of the review puts in the review unpublished data or discusses the results of experiment as an “unpublished observation” or a “personal communication”. These are data not available elsewhere. These can be so-called “negative data” that current publishing policies do not favor, though I think there is no such thing as negative data in science, if experiments were done correctly, of course.

Many times negative data can be a foundation for the scientific breakthrough. Here is one example from my personal experience.

Few years ago, I was working on one project related to in vitro Foxp3 induction in naive mouse T cells. At that time, Foxp3+ T cells were very popular. Almost every second paper in immunology was somehow related to Foxp3+ T regs. For me, the interest in Tregs has to do with the fact that current theoretical frameworks on how immune system operates can not tolerate the presence of negative regulatory populations. It is a strange idea but true. Neither self-nonself nor PAMP/DAMP based immune system requires Tregs to operate, theoretically speaking, of course.

My boss then, Polly, had an idea that Tregs are T cell subset driving different type (or class) of immune response rather that inhibiting anything. Since scientists did not measure all type of responses, they saw a down-regulation of a particular known immune type response as an evidence of inhibition rather than considering that system was shifted to unknown type (cytokine) not measured in the assay (for example, IgA or IL-???).

Unlike many laboratories working on Foxp3 induction using anti-CD3/anti-CD28 antibodies, I decided to use more physiological approach of Foxp3+ T cell induction using peptide pulsed DCs and naive peptide-specific CD4 T cells. Experiments went as expected that we were able to generate peptide-specific Foxp3+ CD4 T cells. During this same time period, new T cell subset, called Th17, has been discovered and characterized. It turned out that while Foxp3 induction required TGF-beta, one could generate Th17 simply by adding IL-6 in addition to TGF-beta (TGF-beta + IL-6). It was already shown by then that presence of IL-6 drove Th17 induction and while inhibiting Foxp3+ induction.

Since I was doing an assay with Foxp3+ T cells, I have decided, for no apparent reason, to add IL-6 in my assay. I was expected to see a decrease in Foxp3 expression and increase IL-17. Strangely, when I added IL-6 to Foxp3 induction milieu (TGF-beta, peptide, DCs, CD4 T cells) I noticed that percentage of Foxp3+ T cells went up rather than go down. Th17 induction was expected. I repeated this assay multiple times and over and over I saw that IL-6 was promoting rather than inhibiting Foxp3 induction in my hands.

Now, these results went completely opposite to what everyone else were reporting in the literature. I asked my fellow postdoc to repeat the same experiments to make sure that I was not doing something strange with my culture. Again, we saw the same: IL-6 promoted Foxp3+ T cell induction.

Using IL-4 or IFN-gamma KO mice on the same background, we observed that IL-6 effect on Foxp3 induction was abolished on IFN-gamma KO but not on IL-4KO background. Indeed, using anti-IFN-gamma antibodies with wild-type CD4 T cell assay confirmed that when IFN-gamma was blocked IL-6 lost its ability to increase Foxp3 expression. Parallel experiments showed that exogenous IFN-gamma inhibited Foxp3 expression and that IL-6 inhibited IFN-gamma expression, as expected from published literature.

In the end, after 5 months of continues work, we come up of new hypothesis that could explain our observation. Our finding suggested that IL-6 promoted Foxp3 expression by suppressing IFN-gamma expression in activated peptide-specific CD4 T cells. We reasoned that other laboratories failed to see the same effect since they were using anti-CD3/anti-CD28 activation mode rather more physiological, peptide activation mode. Indeed, in our hand too, IL-6 inhibited Foxp3 expression when anti-CD3/anti-CD28 antibodies were used to activate CD4 T cells.

So, we were very confident that we found something worthy of publication in one of prestigious journals.

Then, suddenly, we started to receive reports that mice from our mouse colony started to die in higher numbers than normally observed. People had no clue what was going on. In the end, after thorough investigation that took few months, it was revealed that new type, not yet described Streptococcus was causing mice death (one of clinical signs was a cardiomyopathy). The mouse colony was shut down, newly re-derived and repopulated.

As you may have already suspected, when we repeated our experiments with IL-6 on T cells from “clean” mice, we saw that now IL-6 was inhibiting Foxp3 expression rather than promoting it and this effect was seen both times when peptide or anti-CD3/anti-CD28 were used.

These are the type of experiences that make someone like me to appreciate why we do science. As to new Streptococcus we discovered, I did not have a chance to study it but I hope someone will be doing his/her PhD by studying this unusual bacteria and its unusual effects on immune system.

posted by David Usharauli

double-blind peer-review is a way to go for basic and pre-clinical studies

Today the most peer-review process in the prestigious journals is a single-blind, meaning that the reviewers know who the author(s) of the manuscript is(are) but not vice versa. Rarely, reviewers are allowed to disclose their own identities to the manuscripts’ authors, but in my experience and opinion, since reviewing is a process that require some degree of criticism and know one would prefer to be criticized by their own peers, an absolute majority of reviewers would not voluntarily disclose their own identity to the authors. So, double-open review process is less likely to be a viable option.

What is left is either currently operating single-blind or my favorite double-blind peer-review process. Current review process has two deficiencies: 1. not all editorially approved reviewers are of the same caliber, and 2. reviewers favor established names.

Double-blind evaluation would eliminate name bias favoritism.

Raising the quality of the reviewers, of course, would require conscious editorial decision. One solution would be for journal editors to send out potential reviewers names to researchers in the field and ask them to nominate the names. The names of the researchers selected to be reviewers should be published and be available for view. And I think it would helpful if the number of reviewers per submitted manuscript would be increased from current 3 to 5.

Many times I hear that double-blind peer review is impossible since the reviewer would be able to identify the manuscripts authors. Not really, in my view. The initial manuscripts will be submitted in two different formats, first regular one for the editor only and second one specific for the reviewers with no names, affiliations, acknowledgments, grant support information and statements likes “we”, “we showed before”, “as discussed in our previous” and so on. It is quite easy to accomplish. Now days scientific reagents, including commercial knockout mice strains, become so widely available that any research lab with sufficient resource can do a research indistinguishable from reagents point of view. The reviewers would not be able to identify the authors solely based on reagents and only criteria that would be left to examine will be the quality of the research manuscript itself.

Now, of course, established scientists would not like a double-blind review process since they will be the major subset that will be affected by elimination of bias and favoritism. However, double-blind review process would improve young scientists standing and would level the field to compete on the basis of objectivity and quality.

I do not even understand why there is still any discussion to implement or not double-blind review process for the scientific manuscripts. We are already using it in our clinical trials and no regulatory agency would approve any new drugs or biologicals without the results from double-blind trial. It is already overdue to introduce the similar level of scrutiny for basic and pre-clinical studies.

posted by David Usharauli

Online access to federally funded research publications must be free of charge from day 1

An absolute majority of the published scientific research are funded by tax payers’ money through the federal grants from NIH or NSF. Every scientist dreams to publish his/her work in the prestigious journals. Publication is the simplest way for a scientist to showcase the quality of his/her work.

The scientists, unlike social media followers, do not really care how many people will see their publication as long as it is seen by their colleague-scientists and could be submitted to the granting agencies. Furthermore, the absolute majority of the scientists would be OK if their research is freely avalable from the day 1 of its publication.

So, why are the journals still charging for online access to the new articles? some 30-35 years ago, when majority of scientific research were conducted at the University campuses by academic scientists, such fee for access was acceptable since Universities were able to afford multiple annual subscriptions to subject-matter journals.

However, these days, the small biotech companies conduct a big chuck of scientific research but still may lack additional funds to have annual subscriptions to many important subject-matter journals. Many times, when a new treatment fails in clinical trials, the source for a failure can be traced back to the lack of access to scientific literature at the earlier stages of treatment development.

Now days, there is 12 months delay before federally funded publication becomes open access. Can you believe that before 2007 legislation that made 12 months requirement the law, there was no such requirement at all.

But even 12 months is too long. Today research become accelerated due to improvement in computer technologies and the large data mining machinery and software. I just read news that beginning from Jan 1, 2017, Bill and Melinda Gates Foundation will require their grantees to make their research openly available immediately upon publication. Great decision, but I am still puzzled why not make it effective from Jan 1, 2015.

Journals should generate their revenue through an advertisement on their pages or through minimal submission fees. Subscription based revenue system is not viable in long run. Now days many science organizations stopped purchasing monthly hard copies of journals, including Nature and Science, and switched to online-only subscriptions to cut the cost. Eventually, the scientists will be asked to publish in a cheaper open access journals.

In the end, the prestige of any journal comes from the publication of high quality papers. Migration of high quality article submissions from today’s top journals to open access journals will raise the latter’s prestige and will benefit everyone else.

posted by David Usharauli