Monthly Archives: July 2011

Fluorescent Proteins in Imaging: Bright and Beautiful.

Hello there.

One of the things that I wish to convey when I write blog posts is not just the whys and hows of science and the technology that is derived from science but also how the results can be vitally important, in some circumstances, and very beautiful in others. This post is a little montage to fluorescent proteins. Fluorescence is a phenomenon where light of a particular wavelength is absorbed, and a different wavelength of light is emitted. There are several fluorescent proteins around, the most famous of which, perhaps is Aqueorin, more popularly called the Green Fluorescent Protein.

The discovery of the GFP won its discoverers the Nobel Prize in Chemistry in 2008. Since then, a wide variety of mutant proteins that fluoresce in different colours has been developed. However, this is not the only fluorescent protein around, several other proteins have been derived from other naturally occurring fluorescent proteins too.

Fluorescent Proteins palette

Since the primary post of this purpose is to highlight the beauty (and utility) of fluorescent proteins, I shall now rest my case with pictures and a brief caption.

Glofish

Glofish are fish that express fluorescent proteins.

Glow Mice (University of Pennsylvania)

and the critters above are Glow-mice.

It isn’t just organisms we can visualize in green, we could also look at internal structures.

Organ visualization

Here, a liver specific promoter has been used to express GFP only in liver cells, and the resulting fluorescence has enabled observations of the effect certain substances have on liver morphology.

We can also see tissues that make organs up…

Brainbow

In the above image, neurons of the dentate gyrus have been labelled using a technique called Brainbow.
and we can see cells…

Mouse intestinal epithelium

Above is a fluorescent micrograph of mouse intestinal epithelial cells, Image Credit – MIT.

If you think that is far as fluorescent tagging and visualization will go, think again. We can even peer inside cells to visualize and observe structures within…

Labelled Cell

Here, different organelles, including the nucleus and components of the cytoplasmic cytoskeleton have been labelled.
Image Credit – Harvard Medical School.

It isn’t just large regions such as the nucleus or the cytoplasm that can be labelled, relatively tiny structures can be labelled too…

Labelled spindle fibers

Here, Chromosomes, spindle fibers, and centrioles have been labelled, you may want to look at the post discussing the cell cycle for a brief overview of these structures.

Image Credit – Krendel et al, PLoS One, “mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures.”

We can also find out what stage of the cell cycle a cell is in by linking the expression of fluorescent proteins to genes that are specifically expressed at particular stages of the cell cycle. The method is commonly known as Fucci fluorescent visualization.

Here is a cool little video of it in action.

You can see how, as cells progress through the cell cycle, the fluorescent proteins they produce, and consequently the colour they attain change.

You can read about fluorescent microscopy here

I wish to close out with some more eye-candy.

Eye candy-one

Eye-candy Two

Eye-candy three

Image Credit – Invitrogen.

labelled stem cells

Image Credit – Invitrogen.

Image

Image Credit – Oxford Brookes University.

That is all I have this time round, happy ogling 😛

Cheers,
Ankur “Exploreable” Chakravarthy

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Top myths about brain and its ability

Recently, I was seeing cartoon network. Yes, I know it is cartoon network. Are you telling me you haven’t seen it recently? Boy, you are missing a lot!

Anyway, coming to the point, I saw Jerry whack Tom with a saucepan and Tom loses all his memories! Amnesia has really got the short end of the stick. I wanted to tell that doesn’t really happen to my father (Yes, I know! He was watching it with me). This gave me a brilliant idea. There are so many myths about the brain and its capacity. When it comes to the brain, everybody miraculously transforms into a neuroscientist. So, here is my article on the top myths about the brain and the reality behind it. Hope you enjoy it. Oh and by the way, don’t forget to catch up with Tom and Jerry.

We use only 10 percent of our brains. 
I have heard this time and again. For brain’s sake stop spreading this. It has been repeated in popular culture so many times, that people are compelled to believe this. The myth implies there is a huge reserve of untapped powers that can be utilized to perform extraordinary feats.

I want you to think about this before I give you the actuality behind this. Do you think evolution will be stupid enough to give humans a chunk of the brain without any use? Will it really allow us to carry around a mass of expensive tissue just in case we find a way to utilize it? In evolutionary terms, that 90% or whatever percent of brain which isn’t used is called as a vestigial organ.

There are multiple examples of vestigial organs in the human body. You can read more about this here.

Brain is an expensive organ. Not merely due to its activities but because it takes a huge amount of energy and complex compounds during foetal and childhood development to make it into the organ it is today. In that case, it would make no sense to completely utilize so much energy on a mass of tissue that may or may not be utilized in the course of the human life. Common, look around you, there are millions of people struggling to do normal stuff. How is it going to be for them to have 90% extra power in their hands? It is like giving a 5 year old kid keys to the Kohinoor diamond (Oh wait, has this been made into a movie? Movies these days, pfft)

Experiments using PET or fMRI scans show that much of the brain is engaged even during simple tasks, and injury to even a small bit of specified point in the brain can have profound consequences for language, sensory perception, movement or emotion.

True, we have some brain reserves. Autopsy studies show that many people have physical signs of Alzheimer’s disease (such as amyloid plaques among neurons) in their brains even though they were not impaired. Apparently we can lose some brain tissue and still function pretty well.

The older you get, your brain becomes inactive

This is partially true. Many of our cognitive skills decline as we age. Have you played a game of concentration against a 10 year old? Well, don’t! It isn’t worth the humiliation and you will have to be his slave for the next 100 years he is alive.

Young adults are faster than older adults to judge whether two objects are the same or different; they can more easily memorize a list of random words, and they are faster to count backward by sevens.

What we tend to forget is that the brain is responsible for every single thing we do. There are millions of things that old people can do that the youngsters aren’t very good at. For example, older people are better at vocabulary. They know more words and understand subtle linguistic distinctions. Given a biographical sketch of a stranger, they’re better judges of character. They score higher on tests of social wisdom, such as how to settle a conflict. And people get better and better over time at regulating their own emotions and finding meaning in their lives.

Brains are like computers. 

Repeat after me. The brain is not a computer.

We speak of the brain’s processing speed, its storage capacity, its parallel circuits, inputs and outputs. The metaphor fails at pretty much every level: the brain doesn’t have a set memory capacity that is waiting to be filled up; it doesn’t perform computations in the way a computer does; and even basic visual perception isn’t a passive receiving of inputs because we actively interpret, anticipate and pay attention to different elements of the visual world.

There’s a long history of likening the brain to whatever technology is the most advanced, impressive and vaguely mysterious. Descartes compared the brain to a hydraulic machine. Freud likened emotions to pressure building up in a steam engine. The brain later resembled a telephone switchboard and then an electrical circuit before evolving into a computer; lately it’s turning into a Web browser or the Internet. These metaphors linger in clichés: emotions put the brain “under pressure” and some behaviors are thought to be “connected like an electronic circuit.” Speaking of which…

The brain is hard-wired

This is one of the most enduring legacies of the old “brains are electrical circuits” metaphor. There’s some truth to it, as with many metaphors: the brain is organized in a standard way, with certain bits specialized to take on certain tasks, and those bits are connected along predictable neural and communicate in part by releasing ions (pulses of electricity).

But one of the biggest discoveries in neuroscience in the past few decades is that the brain is remarkably plastic. Brain plastic doesn’t mean brain is a fake piece of organ. It means it has the ability to change remarkably based on the situation.

In blind people, parts of the brain that normally process sight are instead devoted to hearing. Someone practicing a new skill, like learning to play the violin, “rewires” parts of the brain that are responsible for fine motor control. People with brain injuries can recruit other parts of the brain to compensate for the lost tissue.

A conk on the head can cause amnesia. 

Those bloody serials. How I would like to conk them in the head. Switching babies at birth and re-emerging from a tragic death. All shite that are shown on the tele nowadays. Along with that comes this: Someone is in a tragic accident and wakes up in the hospital unable to recognize loved ones or remember his or her own name or history. (The only cure for this form of amnesia, of course, is another conk on the head)

In the real world, there are two main forms of amnesia:

1. Anterograde (the inability to form new memories) and

2. Retrograde (the inability to recall past events).

Science’s most famous amnesia patient, H.M., was unable to remember anything that happened after a 1953 surgery that removed most of his hippocampus. He remembered earlier events, however, and was able to learn new skills and vocabulary, showing that encoding “episodic” memories of new experiences relies on different brain regions than other types of learning and memory do. Retrograde amnesia can be caused by Alzheimer’s disease, traumatic brain injury (ask that sports player who didn’t have enough marbles not enter the violent game to begin with) thiamine deficiency or other insults. But a brain injury doesn’t selectively impair autobiographical memory—much less bring it back.

“Flashbulb memories” are precise, detailed and persistent. 

We all have memories that feel as vivid and accurate as a snapshot, usually of some shocking, dramatic event—the assassination of a President/Prime Minister, the explosion of the space shuttle Challenger, the attacks of September 11, 2001.

People remember exactly where they were (On 9/11 I was sitting in my Grandma’s house), what they were doing (eating dinner), who they were with (with grandma duh), what they saw or heard (She asked me to change the channel so that she can catch on with her daily serial).

But several clever experiments have tested people’s memory immediately after a tragedy and again several months or years later. The test subjects tend to be confident that their memories are accurate and say the flashbulb memories are more vivid than other memories. Vivid they may be, but the memories decay over time just as other memories do. People forget important details and add incorrect ones, with no awareness that they’re recreating a muddled scene in their minds rather than calling up a perfect, photographic reproduction.

We have five senses. 

Sure, sight, smell, hearing, taste and touch are the big ones. But we have many other ways of sensing the world and our place in it.

Proprioception is a sense of how our bodies are positioned. Nociception is a sense of pain. We also have a sense of balance—the inner ear is to this sense as the eye is to vision—as well as a sense of body temperature, acceleration and the passage of time.

Compared with other species, though, humans are missing out. Bats and dolphins use sonar to find prey; some birds and insects see ultraviolet light; snakes detect the heat of warm blooded prey; rats, cats, seals and other whiskered creatures use their “vibrissae” to judge spatial relations or detect movements; sharks sense electrical fields in the water; birds, turtles and even bacteria orient to the earth’s magnetic field lines.

Happiness is in our hands we can do something to get it

In some cases we haven’t a clue. We routinely overestimate how happy something will make us, whether it’s a birthday, free pizza, a new car, a victory for our favorite sports team or political candidate, winning the lottery or raising children.

Money does make people happier, but only to a point—poor people are less happy than the middle class, but the middle class are just as happy as the rich. We overestimate the pleasures of solitude and leisure and underestimate how much happiness we get from social relationships.

On the flip side, the things we dread don’t make us as unhappy as expected. Monday blues? Oh please, there is a survey done and people predict that Monday mornings aren’t that unpleasant. So, suck it and get back to work.

Seemingly unendurable tragedies—paralysis, the death of a loved one—cause grief and despair, but the unhappiness doesn’t last as long as people think it will. People are remarkably resilient.

Our perception is always right 

We are not passive recipients of external information that enters our brain through our sensory organs.

Instead, we actively search for patterns (like a Dalmatian dog that suddenly appears in a field of black and white dots), turn ambiguous scenes into ones that fit our expectations (it’s a vase; it’s a face) and completely miss details we aren’t expecting.

In one famous psychology experiment, about half of all viewers told to count the number of times a group of people pass a basketball do not notice that a guy in a gorilla suit is hulking around among the ball-throwers.

We have a limited ability to pay attention (which is why talking on a mobiles while driving can be as dangerous as drunk driving), and plenty of biases about what we expect or want to see. Our perception of the world isn’t just “bottom-up”—built of objective observations layered together in a logical way. It’s “top-down,” driven by expectations and interpretations.

Men and women are different 

Some of the sloppiest, shoddiest, most biased, least reproducible, worst designed and most over interpreted research in the history of science purports to provide biological explanations for differences between men and women. Eminent neuroscientists once claimed that head size, spinal ganglia or brain stem structures were responsible for women’s inability to think creatively, vote logically or practice medicine. Today the theories are a bit more sophisticated: men supposedly have more specialized brain hemispheres, women more elaborate emotion circuits. Though there are some differences (minor and uncorrelated with any particular ability) between male and female brains, the main problem with looking for correlations with behavior is that sex differences in cognition are massively exaggerated.

Women are thought to outperform men on tests of empathy. They do—unless test subjects are told that men are particularly good at the test, in which case men perform as well as or better than women. The same pattern holds in reverse for tests of spatial reasoning. Whenever stereotypes are brought to mind, even by something as simple as asking test subjects to check a box next to their gender, sex differences are exaggerated.

Women college students told that a test is something women usually do poorly on, do poorly. Women college students told that a test is something college students usually do well on, do well. Across countries—and across time—the more prevalent the belief is that men are better than women in math, the greater the difference in girls’ and boys’ math scores. And that’s not because girls in Iceland have more specialized brain hemispheres than do girls in Italy.

Certain sex differences are enormously important to us when we’re looking for a mate, but when it comes to most of what our brains do most of the time—perceive the world, direct attention, learn new skills, encode memories, communicate (no, women don’t speak more than men do), judge other people’s emotions (no, men aren’t inept at this)—men and women have almost entirely overlapping and fully Earth-bound abilities.

I will leave you with some of my wisdom. This is a precious piece of information and I hope you treat it with the respect that it deserves. Watch Tom and Jerry and get enlightenment.

Peace

Avinash

A very basic introduction to gene therapy.

Please note – Right Clicking on an image and clicking “View Image” will bring up a larger, clearer version.

I’m writing this article on request after a friend asked me to, and what follows is a very simple introduction to what gene therapy entails, some notable examples and a very brief evaluation of the prospects of the field. I will also be looking at the challenges that must be met before gene therapy becomes an integral part of medicine.

At the outset, I would like to lay out a picture of disease pathology from a molecular biological perspective. In both genetic disorders, to which gene therapy is particularly relevant, and communicable disorders there is a certain degree of involvement of genes and genomes, and of transcription and translation. While extant chemotherapy usually ends up targeting the protein products of genes to prevent them from leading to disease, gene therapy focuses on correcting things well before translation even occurs.

With gene therapy, three approaches are possible, one is to introduce a properly functioning gene when there is none (and this is perhaps the most well-established principle) , one is to turn off genes that lead to an abnormal phenotype and the last one is to alter gene expression so that disorders can be circumvented. Where possible I will provide references to illuminating examples for all these approaches.

Before moving on, though, gene therapy can be classified broadly into ex-vivo or in-vivo gene therapy (ex-vivo gene therapy is administered to cells that are taken outside the body and in-vivo gene therapy takes place inside the body) and alternatively as somatic gene therapy and germline gene therapy (the former is not heritable, the latter is)

Classification of gene therapy strategies by location of modification.

Now I think it is time to move on to the approaches themselves, with the apposite examples included…

[I] Introducing properly functional genes when there are none.

There are lots of genetic disorders wherein a non-functional gene can cause real problems by affecting body function, the examples I shall use to illustrate the principles of gene therapy in this case involve two such examples, one is Cystic Fibrosis and the other is X-linked Severe Combined Immunodeficiency Syndrome.

Cystic Fibrosis

Cystic fibrosis is an autosomal recessive disorder (that is, both copies of the relevant gene need to be dysfunctional for symptoms to occur) that is caused by mutations that produce a non-functional CFTR (Cystric Fibrosis Transmembrane Conductance Regulator) gene. When both copies of this gene are abnormal it results in water balance in mucus not being properly regulated, which translates to extremely thick mucus that can block airways, impair breathing, affect digestion and endocrine function. The disease is also associated with a markedly higher propensity towards acquiring bacterial infections.

While there is no cure there are several management methods around, but usually lung transplants become necessary at some stage in life. Treating this disorder is a challenge that gene therapy tried to meet.

The technology is nascent and the processes are inefficient, but are nonetheless promising. The gene therapy trials that have been carried out to treat CF have involved the use of a specific kind of virus, called an adenovirus, to deliver functional copies of the CFTR gene to cells or alternatively the use of bubbles of fatty acid that contain the gene construct inside (we call these liposomes).

Cystic fibrosis gene therapy

Results are apparently promising from Phase I Clinical Trials. How things go in the future remains to be seen, but one thing is certain, we do know that targeting genes is a viable strategy at least in this case, we know that there are methods that can be used to an extent where clinical impact is possible, and we have a procedure and a set of methods that can be improved.

X-Linked SCID

This is a sex-linked disorder and involves dysfunctional versions of the Interleukin-2 receptor, which plays a very important role in how the body responds to infections. People who lack functional IL-2 receptors end up dying of infections due to compromised immunity in the early years of life. This is because IL-2 signalling is responsible for the maturation and differentiation of progenitors into T-Lymphocytes, functional B-lymphocytes and NK Cells. All of these are extremely vital components of the immune system and not having them opens the body to all kinds of pathogens.

SCID has been treated using ex-vivo gene therapy, where a lentiviral vector containing a copy of a fully functional IL-2 receptors was used to induce the gene expression required to rescue immune function.

The problem with using retroviral vectors is that they integrate randomly into host genomes, and this means it may affect normal gene expression and trigger cancers, for instance. In fact, this did happen and at least one baby in the original trial died, the rest though received chemotherapy and survived. See reference here

A similar approach has also been tried for versions of SCID which are due to a deficiency of functional Adenosine Deaminase, which leads to similarly dysfunctional immune systems.

You can find a lovely review of twenty years of SCID and gene therapy here

So there are two examples where people have attempted to fix genetic disorders by inserting functional copies of genes when there were none. What about the other two cases?

[II] Knocking out dysfunctional genes.

This approach to gene therapy relies on the use of agents to target genes directly and to knock them out. There are several methods that can be used to do this. RNAi (RNA interference) is one of these, the other is the use of antisense oligonucleotides and yet one more is the expression of antisense transcripts.

Ideal cases for the use of gene silencing (which is a form of gene therapy insofar I am concerned) would include viral infections and cancer. Examples of current use would include the use of RNAi in a clinical trial to treat a viral infection by RSV

However, the whole thing is very nascent and much of the work here has been in mouse models/cell lines. You may find references that are useful here (It is a Google Scholar Search List)

[III] Modifying gene expression.

The example I would like to give here is of Duchenne Muscular Dystrophy, which is a disorder in which muscles progressively die out and become weaker, eventually culminating in death. The modification of gene expression in this case is carried out using exon skipping.

So what happens is that Dystrophin, the protein whose mutant varieties are implicated in DMD, is synthesized from mRNA that is first cut from longer pre mRNA and then joined. Some people lack one of the exons and as a result the exons that follow cannot be joined, thus producing a short mRNA and a short, useless protein. However, not all exons have this problem, and some exons can be joined to later exons even if there is something missing. The idea is to block exons that truncate the mRNA using antisense oligomers so that exons that can bind to later exons are at the end. This way, the mutant, dysfunctional regions of dystrophin can be skipped over by blocking that particular region using an antisense oligomer. This prevents frameshifts from resulting in a dysfunctional protein and enables a functional protein to be expressed.

DMD Exon Skipping.

So that is a very brief (lol) introduction to what gene therapy is, what it entails and some of the things that demonstrate its potential. While there is great potential, we need to keep figuring out ways to improve gene expression using vectors, we must ensure that these vectors get safer, cheaper and can be produced more easily and most importantly of all, try to ensure that gene therapy does actually work when called upon, and when that point is reached I suspect the face of medicine may change forever, since we will be able to address diseases at their very roots.

That is all from me this time round, happy learning.

-Ankur “Exploreable” Chakravarthy