Large scale synchronisation of biosensors using synergistic coupling

One of the major hurdles in synthetic biology has been the construction of bio-sensors that are accurate over a large scale and that can be monitored without the aid of sophisticated microscopes. Getting a bacteria to signal in one way or another when it encounters an environmental toxin (or something else) is one thing but getting a colony of bacterial cells to signal together in synchrony is a whole lot more difficult. Orchestrating thousands of colonies of bacterial cell is an entirely different ball-game. Yet this is exactly what a group of scientists from the University of California, San Diego have achieved. They report their findings in an article published in the Advanced Online Publications section of Nature. And this is the article that I want to briefly review over here.

To synchronise the bacterial cells within a colony they tapped into a natural mechanism used by bacteria to communicate among themselves, quorum sensing. However, quorum sensing is only effective over a  range of a few micrometers. So they used a different communication molecule, one that is in the gaseous phase and hence can diffuse quickly to longer distances. But gaseous  molecules are weak and short-lived, being in the vapour phase, they disperse a lot quicker. However when these two communicating mechanisms were synergistically coupled, the results were quite surprising.

To achieve the intracellular coupling they used quorum sensing involving acyl-homoserine lactone (AHL). The principle here is quite simple, each bacterial cell synthesises a certain amount of AHL which acts as an inducer. When this inducer binds with a receptor it causes the expression of certain genes which among other products also include the genes for synthesizing the inducer itself. Thus the inducer is technically inducing its own production, rendering it an autoinducer. However the amount of this inducer molecule, AHL in this case, produced by any bacterial cell is not enough to initiate this positive feedback loop. It is only when that the colony reaches a certain size and when a certain critical concentration of the autoinducer has been achieved, that the expression of the downstream proteins occur. As a result, if you insert fluorescent proteins among them, you get a colony of bacterial cells fluorescing in unison. But mind you, this only gets you a coordinated bacterial colony over a very small range. You need something else to scale this up.

So they put a copy of a gene coding for NADH dehydrogenase ll (ndh) under the control of another lux promoter. Now NADH dehydrogenase II is a respiratory enzyme that produces low levels of H₂O₂   and superoxide. Now H₂O₂  being a in the vapour state is able to pass between the 25 micron thick PDMS walls that is used to separate the colonies. This H₂O₂ , being a reactive oxygen species, initiates a defence mechanism in bacterial cells once it enters them. Among the different global regulatory systems that mediate this defence mechanism is one called the ArcAB system, which (lo and behold!) has a binding site in the lux promoter region. Under normal conditions, ArcAB is partially active, rendering the lux promoter partially repressed. But once H₂O₂  enters the cell, ArcAB swings into action, thus activating the lux promoter and hence initiating the same quorum sensing mediated communication system and hence synchronising this new colony. Now the researchers did a lot of tests to confirm that it was the external H₂O₂ , and not endogenously produced molecules, which brought about the desired effects. Describing them is out of scope here but the results confirmed their hypothesis.

So what it is that they finally achieved? Synchronisation of approximately 2.5 million cells over a distance of about 5 mm, exhibiting consistent oscillation with a temporal accuracy of 2 minute compared to the 5-10 minute accuracy of a single oscillator!

The paper includes a bit more about the construction of an arsenic detecting biosensor using this technique but I don’t think that’s entirely relevant here. For a brief description of the experiment and the potential applications of it, watch the video:

 

 

Merry Mythmas and Happy New Year to all our readers!

– Debayan

 

A little tribute to Hitchens.

Hiya there, Ankur here.

Sometimes, people turn a blind eye to dogmatism, cruelty and oppression. Hitchens wasn’t one of them. I shall remember his for the passionate and indomitable demeanour with which he stood up for the oppressed, and for his clarion calls against the evils of dogmatism and all the misery it wrought.

I shall remember him for his sharp and acerbic wit, his dulcet tones he brought forth as he spoke, his lucid analysis of situations and events, and for his immense courage, for he countered first hand, fascists in Lebanon and ended up being assaulted for standing up to them, and underwent waterboarding personally and revised his stance on the technique, acknowledging that it is torture and nothing less and because he exposed the sinister facade of the appalling, inhuman attitudes that were espoused by “Mother” Teresa and her contemptible Missionaries of “Charity”. I know that no words that I express shall be able to express the magnitude of the influence he’s had on me, or to express how much I admire him and his work, but I shall try nonetheless, for words must flow and resisting is futile; as should tears, plentiful that they are. I shall remember him for the way he promptly, with wit and the wielding of facts as they comprised a razor, ripped apart the Catholic Church’s pretensions to being a force for good in that famous debate.

His demise is sadder than anything else, but it is also a moment to reflect upon what he brought to the table, his writings, his oratory, and pursuit of freedom were, and are, a joy to behold still, and it is a slight consolation, perhaps, that he will live on through his works and that he will live on through the thoughts and spirits of the people he influenced through his work, which was always marked by finesse, elegance and utter class.

I am slightly glad that I had the opportunity to write to him thanking him for his work with fellow comrades-in-free-thought, and I shall let an excerpt from that letter do my bidding here insofar the significance of his life to me is concerned. “We thank you by avowing our own commitments to critical thought and analysis combined with fearless free speech, which represent humanity at its very best and which have led to some of our biggest leaps as a species in terms of genuine progress. We will carry on defending those values which each of us shares with you and which you have so effectively expounded throughout your inspiring career.”

Here’s to a celebration of his life and to pensive mourning of his passing. It was a life well lived, courageous, influential, and one that has left behind a strong legacy. I can only fervently hope that it will live on, as we seek to earn for the oppressed the freedoms we enjoy but they do not, and to free people from the irrational shackles of society that constrain their freedoms so that they may live full, happy lives free of oppression and suffering.

Christopher, my good fellow, here’s a toast to you, you will be remembered.

In which my appreciation of science art continues.

Just another quick one today. I would like to introduce y’all to some rather brilliant scientific artwork by a friend.
Lucy Walsh is an artist who specializes in painting pictures of galaxies and nebulae and I think they look quite gorgeous. Her work is usually acrylic on canvas and has a crisp and vivid feel to it.

Some samples of her work follow…

An abstract piece called “Fusion”

V838 Monocerotis Eruptive Star.

Pluto and its 3 moons.

Flame Nebula

Arp 24

If you like her work, please do check out her official page on DeviantArt Gallery at http://lucyjain.deviantart.com/
or her official facebook page at https://www.facebook.com/pages/Lucyjains-Space-Art/130069343727953

Quite a few of these pieces have prints available from DeviantArt, too.

That is all from me this time round.

Cheers.
Exploreable.

Deadly Organism of the Fortnight: Lonomia obliqua.

This fortnight’s selection is a vicious caterpillar from the rainforests of Brazil & its neighbours.

Lonomia obliqua.

As you can already see, this critter has vivid aposematic colouration (Bright colours can indicate danger to predators, especially in caterpillars) and lots of urticating hairs. Now most caterpillars with urticating hairs can be a major hazard insofar major dermal irritation and itching is concerned, but Lonomia obliqua is exceedingly lethal due to its venom.

Envenomation often occurs when people accidentally brush against a group of these caterpillars on tree bark or on the floor et cetera, the results aren’t pretty; Lonomia obliqua envenomation is due to a venom with haemolytic properties; post-onset, recently healed wounds may re-open and start to bleed, there is massive internal bleeding that manifests in haematuria (blood in the urine) and out of other orifices, there can also be brain haemorrhages to compound the misery of anyone who experiences envenomation from these caterpillars.

The venom contains fibrinolytic proteases that breaks down fibrin (which is essential in the coagulation of blood) and also causes bleeding elsewhere by sequestering clotting factors. Light was shed on what the venom contained and the relevant cDNA sequences after someone did a little study. I hope you will find the paper illuminating insofar understanding the mechanics of Lonomia envenomation is concerned. You can find the paper here.

The paper also has in significant detail a census of the protein families involved and putative targets in the human clotting cascade that stand to be affected. Happy reading.

That paper is slightly outdated, but still provides valuable insights with respect to cataloging the overall composition of Lonomia venom. A newer paper, which I’ll link to later in the post, explains the points of action of components of the venom wrt the human clotting cascade.

Rather paradoxically, Lonomia obliqua venom works by activating the blood clotting system, which consumes the resources needed by the body to prevent bleeding elsewhere, and consequently leads to the massive bleeding described above. A protein called Losac activates Factor X and a protein called Lopap activates Prothrombin.

The blood clotting cascade is as follows, and may help you locate the points of action of the aforementioned proteins.

Blood clotting cascade; should help you locate points of action of L.obliqua venom.

The paper I mentioned is a case study, and may be found here.

That is all from me with respect to this fortnight’s deadly organism. I’ll leave you with some particularly nasty Ecchymoses (large bruises under the skin) from the case study.

Ecchymoses from Lonomia envenomation , Image credit; Canadian Medical Association

Neurosciences behind Ecstasy

For a long time, I have been a shy person, an introvert (to be exact). I preferred to keep my thoughts to myself and never expressed an opinion on anything!

Then, I slowly developed this whole new image of the “wild child”. Not exactly drugs and booze, just that I am open to talk and experience a whole lot of things that aren’t necessarily conventional. No, I am not talking about sneaking a cigarette or drowning a bottle of Tequila. I am talking about things like expressing my views on any topic that are considered to be taboo in India.

Recently, I was talking to my friends about this blog that I wanted to write. About human sexual activities and love (you can read about love here). Not in the adult film kind of way, but the neurosciences behind it. I know most of you started reading this blog post with a certain image in mind. I won’t be pasting pictures of naked women and men. I think there are enough sites to see that. Here I am going to write about the neurosciences and psychology behind that ultimate experience humans go through – Orgasm.

A case study published by a team of Taiwanese neurologists reported a most unusual set of circumstances.

One of their 41 year-old female patients, diagnosed with epilepsy, had a seizure every time she brushed her teeth. Seizures in response to external stimulation are not unusual – flashing lights are a well known source – and other sorts of stimulation are not uncommon triggers. A recent case-report even involved seizures induced by vacuum cleaner use.

So the unusual aspect for the Taiwanese case was not the trigger, but the effect of the seizure. The woman had seizures when she brushed her teeth, and had an orgasm every time she had a seizure, shortly before losing consciousness.

Although probably doing wonders for her dental health, the condition has left neurologists rather puzzled. Because so little is known about sex and the brain, her doctors had very little to go on when they tried to explain what was happening.

Ecstasy? Agony? Euphoria? Surprisingly little is known about what happens in the brain at the very peak of our sexual experience. A Dutch team have done the unthinkable and scanned the brains of men and women during orgasm.

Neuroscientists trying to untangle the riddle of desire and sexual pleasure in the brain have discovered something that turns conventional wisdom on its head. During orgasm, men experience heightened activity in the emotion-processing centres of the brain. But women’s brains, say researchers, are shut down in emotion-processing regions during arousal and orgasm.

Physiologist Dr Gemma O’Brian from the School of Biological, Biomedical and Molecular Sciences at the University of New England also has a keen interest in the neurobiology of the orgasm, as fleeting an experience as it can be sometimes.

Gemma O’Brien: And this is one of the problems with trying to look inside the brain during something like orgasm. Orgasm’s a fairly brief event. We enjoy the response for an extended period of time, the resolution phase, but the actual brain changes are fairly brief so finding a technique that can see what’s happening in the brain.

The whole orgasm process is divided into three stages by Masters and Johnson: stage of arousal plateau, sexual climax and resolution. And it’s the sexual climax part that many people use the word ‘orgasm’ for.

So, is it all chemicals or is it all in the brain?

In men who have a high spinal cord injury may have ongoing capacity to use the reproductive tract so they can manually stimulate or tactilely stimulate to erection and all the way through to ejaculation but not detect anything by brain, not orgasm. They have to actually look or feel by hand to see whether anything happened. But they don’t actually have the sensations of euphoria that come with it.

Both male and female sexual reward releases beta-endorphin (Dictionary of Pharmaceutical Medicine, Springer Vienna, 2009) in the brain, which in experiments caused tremor.

[Looking at the brains of orgasming men using a PET scanner] scientists also saw heightened activity in brain regions involved in memory-related imagery and in vision itself, perhaps because the volunteers used visual imagery to hasten orgasm. The anterior part of the cerebellum also switched into high gear. The cerebellum has long been labeled the coordinator of motor behaviors but has more recently revealed its role in emotional processing. Thus, the cerebellum could be the seat of the emotional components of orgasm in men, perhaps helping to coordinate those emotions with planned behaviors. The amygdala, the brain’s center of vigilance and sometimes fear, showed a decline in activity at ejaculation, meaning, men are oblivious to their surroundings during an orgasm.

To find out whether orgasm looks similar in the female brain, [neuroscientist Gert] Holstege’s team asked the male partners of 12 women to stimulate their partner’s clitoris—the site whose excitation most easily leads to orgasm—until she climaxed, again inside a PET scanner. Not surprisingly, the team reported in 2006, clitoral stimulation by itself led to activation in areas of the brain involved in receiving and perceiving sensory signals from that part of the body and in describing a body sensation—for instance, labeling it “sexual.”

But when a woman reached orgasm, something unexpected happened: much of her brain went silent. Some of the most muted neurons sat in the left lateral orbitofrontal cortex, which may govern self-control over basic desires such as sex. Decreased activity there, the researchers suggest, might correspond to a release of tension and inhibition. The scientists also saw a dip in excitation in the dorsomedial prefrontal cortex, which has an apparent role in moral reasoning and social judgment—a change that may be tied to a suspension of judgment and reflection.

Brain activity fell in the amygdala, too, suggesting a depression of vigilance similar to that seen in men, who generally showed far less deactivation in their brain during orgasm than their female counterparts did. “Fear and anxiety need to be avoided at all costs if a woman wishes to have an orgasm; we knew that, but now we can see it happening in the depths of the brain,” Holstege says. He went so far as to declare at the 2005 meeting of the European Society for Human Reproduction and Development: “At the moment of orgasm, women do not have any emotional feelings.”

You cannot basically conclude that these parts of the brains are shutting down because of the intense experience that the body is going through. It is like a busy road, it maybe because there is a sale in the local supermarket, or there might be a circus performance, or there might be a cat fight going on. Come on, you are interested to see two cats fight viciously right?

Brain scanning just finds associations, but to find out whether an area is causally involved in a particular function or whether it is necessary for the function, research with brain injured patients is one of the most powerful methods.

For example, if you think a brain area is necessary for orgasm, or a certain component of orgasm, a person with damage to that area should not experience what you’ve predicted.

Sexual neuroscience is one of the most under-researched areas in the human sciences. A quick search of PubMed (the international database of medical research) shows that we know more about the neuroscience of hiccups than we do about orgasm.

Part of the problem is practical. fMRI scanners, some of the most useful and popular tools in cognitive neuroscience, involve lying in a tube while scanning takes place and need the head to be completely still. Add the fact that you’re being watched by neuroscientists and none of this makes for relaxed coupling, or even self-stimulation.

So, if you have a way of testing people going through this, then tell me. Maybe I can do some mind boggling errr research. (I solemnly swear I am up to only good)

Peace

Avinash

Say hello to Julietooo.

Hello there,

This is a slightly belated announcement and welcome, seeing that she’s already made a post, but I’d like to introduce you to Juliet, who is an anthropologist and a skeptic with a penchant for sharp, incisive and lucid writing, as her first post will have already hopefully illustrated.
You can expect to see lots of very educational and informative posts in due course of time.

Welcome to the blog, Juliet, and happy blogging.

– Ankur “Exploreable” Chakravarthy

Essay writing tips for Scientists in the making

1.  Understanding the Question – Comprehensive analysis of the title or question:

• Read it several times
• How many sections are there?
• Break up into key words/segments – Underline/highlight key words
• What do they mean?
• How can they be interpreted?
• How does it link to course lectures?
• What else do I need to find out?
• Discuss with others

2. Research

Make a list of:

• All the questions ideas that have arisen from analysing question/title
• All the information that needs to be found
• Where to get the answers: notes, books, library …

3. Read quickly to find information

• Use contents page, index, headings, sub headings
• Read first sentence, summary, conclusion
• Scan for key words, phrases

4. Read carefully to improve understanding of the information

• Pay attention to what you are reading
• Try to prevent your mind from wandering
• Re-read ‘missed’ material
• Be an active reader – Make outline notes, draw sketches, boxes, doodles to organise ideas

5.  Tips on Note-taking

• Develop good note-taking
• Note sources for referencing
• Put quotation marks round direct quotes
• Have a system for keeping notes ( I use endnote. Easy way to organise your references)

6. Analyse the Information

• Find links, themes, differences etc
• Cross reference the information: use a map, coloured post-its’, coloured highlights
• Draw graphs, bar charts if relevant
• Be critical – ask: which information is important, is the question being answered? – only use relevant information
• Look at the bigger picture
• Be independent and draw your own conclusions

7. Writing the Essay

• Do a rough draft – Don’t expect to it get right first time
• Use own words – Keep direct quotes to a minimum
• Include references to ALL work that is not your own
• Draw diagrams/graphs to illustrate and explain, where appropriate
• Proof read draft and edit – more than once if necessary

8. How to Acknowledge your Sources

• Word-for-word:  quotation marks and reference [i.e. make a footnote, endnote or put information in brackets if using Harvard system].
• Author’s idea in your own words (paraphrase):  no quotation marks, but reference.
• No need to reference things that are common knowledge

Concept – here comes RNA Interference, just as promised.

The behaviour of genes can be affected by other genes and other elements that interact with genes, ranging from transcription factors to antisense transcripts that operate post-transcriptionally, and today the focus of my post will be something called RNA Interference (RNAi in short)

If one were to look at what happens to genes in terms of the central dogma of molecular biology, you will find that the biochemistry of genes can be written down as follows.

DNA ————-> RNA (Transcription) ———–> Protein (Translation)

RNAi acts on genes following transcription but before translation, and it does so by breaking down mRNA that would have been translated otherwise, and this is mediated by the RNA Interference pathway, something that is conserved in Eukaryotes (with the exception of certain fungal lineages, the interesting thing is that the loss appears to be independent in fungal lineages) and is, as I will elaborate later in this post, of much practical utility.

The next part of my post will deal with the RNAi pathway per se. But before we venture there, it would be prudent to have a brief outline of the components required, to put it extremely simply, we will need double stranded RNA and a set of enzymes to use double stranded RNA as the reference for destroying target RNA and then something to cleave the target RNA.

Double stranded RNA does the trick well, and basically long chains of double stranded RNA are cut into smaller double stranded RNA fragments  of a length that is 20-25 base pairs long by an enzyme called, funnily enough, Dicer. These shorter double stranded fragments, that come with a two base overhang, are called siRNA (short interfering RNA).

siRNA then binds to Argonaute proteins to form what is called an RISC, a multi-protein complex that is involved in the final part of RNAi, the identification of complementary mRNA and the breakdown thereof. RISC stands for RNA Induced Silencing Complex, by the way.

A very good video of the process can be found below.

Organisms may use RNAi for gene regulation through the function of miRNA (microRNA) , here a long noncoding chain of RNA (pre-miRNA to be precise) is produced first by transcription, and this undergoes self-base pairing within the ssRNA strand to form a structure with loops and stems, further processing by enzymes like Drosha and Pasha help in the conversion of the long transcript into a double stranded form, which is mature miRNA, after which the RNAi pathway functions as described above.

That is about the jist of what RNAi is, but what about the implications? And what empirical standing do the proposed applications of this system have?

Well, we already know that RNAi can turn genes off, which means it is great when we are looking to target the mRNA of viruses et cetera, identify target sequence, find sites that are unique only to the viral mRNA, design dsRNA, introduce that into cells, and watch as the RNAi apparatus goes about marmalizing the viral genome. The very fact that some regions of viral genomes are conserved, even in highly variable strains like Influenza viruses means that broad spectrum therapies could be developed utilizing this process.

Want to study gene function and what happens if you knock a gene out? Same principle more or less, put in your dsRNA sequence, watch as the gene is silenced, observe for phenotypic changes, the very fact that one is now able to silence genes directly instead of generating knockout organisms by traditional mutagenesis and breeding is certainly very attractive.

Want to study or control gene regulation? Not a problem, you can just add in extra copies of miRNA or target miRNA for cleavage, either using other antisense approaches (antagomirs) or using complementary dsRNA. I hope you are now able to appreciate the scope of this technique.

There have been recent advances, too, such as the development of a technique called dicer substrate RNAi , wherein, instead of using long dsRNA which has to be processed by dicer, it is possible to introduce 25-28 nucleotide long double stranded RNA directly for integration with Argonaute, and this method, according to reports in the scientific literature, can greatly increase the efficacy of RNAi.

I am not sure that we have many RNAi therapies in active clinical use at the moment, but it sure does look promising. You may want to peruse the following resources for further information.

Wikipedia article on RNA interference.
RNAi Therapeutics: Principles,Prospects & Challenges (peer reviewed paper)
RNAi as a means of controlling lipid levels (peer reviewed paper)
Science : RNAi Collection (A collection of peer reviewed research papers)
Science: miRNA Collection (A collection of peer reviewed research papers)

Antagomirs et cetera and case studies of RNAi application will be parts of future posts somewhere down the line, until then, have a happy Interfering time :P.

PS – work on RNAi won the 2006 Nobel for Dr.Andrew Fire and Dr.Craig Mello, the apposite press release can be found here
& Dr.Fire’s Nobel lecture is here and Dr.Mello’s Nobel Lecture is here

These are always entertaining to watch because they give you an opportunity to ‘connect’ with these researchers in a way that research papers themselves do not. Happy viewing.