Health,Stem Cells, and Technology

Sunday, April 17, 2011

Continued Failure Of Genomics

The human genome was sequenced more than 10 years ago and never a week has gone by without some new genetic "breakthrough" being reported. Last week five new genes for Alzheimer's disease generated sometimes front-page coverage across the globe. But with a closer look the reality is very different.

Among all the genetic findings for widespread illnesses, such as heart diseases, cancers, and mental illnesses, only a handful are of genuine significance for human health. Faulty genes rarely cause, or even mildly predispose us, to disease, and as a consequence the science and medicine, and indeed the drug development, surrounding human genetics is facing a crisis.
The human genome sequencing project was based on a huge, but calculated, gamble. The leaders of the project believed that faulty genes inherited from our parents were probably the cause of most disease. Reasoning included that many rarer diseases were already known to be genetic. Therefore many thought it a small leap to suppose that inherited faulty genes would underlie common diseases, too.
The basis for their confidence was, however, flawed. Scientific evidence in humans supporting genes as causes of common disease was based on comparing disease rates in genetically identical twins against rates in non-identical twins (who share 50% of their DNA). These comparisons, called heritability studies, were designed to measure the relative contributions of genetic variation versus environmental variation.
Although these data were extremely widely used and cited, these studies were considered flawed by some geneticists. Dr. Helen Wallace at UC Berkeley and San Diego State University, and Dr. Richard Lewontin of Harvard University, for instance, called for alternative measures. Other critics pointed out that these experiments relied on the proposition that identical twins experienced no more identical environments than did non-identical twins, when it was abundantly clear that parents were treating their identical offspring more similarly than their non-identical twins. These arguments constituted a threat to the genome project, and were largely ignored.
In 2009, one of the few remaining scientifically active leaders of the original genome project, Dr. Francis Collins, published a review paper in the scientific journal Nature, along with 26 other prominent geneticists. The paper was titled Finding the Missing Heritability of Complex Diseases. In the paper, the authors acknowledged that, despite more than 700 genome-scanning publications and nearly $100bn spent, geneticists still had not found more than a fractional genetic basis for human disease.
Since the Collins eview paper was published nothing has happened to change that conclusion. It now seems that the original twin-study critics were more right than they imagined. Now, a plausible explanation for why genes for common diseases have not been found is that, with few exceptions, they do not exist.
As Dr. Jonathan Latham has argued, the failure to find meaningful inherited genetic predispositions is likely to become the most profound crisis that science has faced. Not only has the most expensive scientific project ever conceived failed to reach a goal it assured the world it would achieve, and led drug development down the wrong path (e.g. Maguire et al, 2006), but there is also the problem of why the weekly headlines have been so consistently discrepant with reality. As the failures to find significant, predictive genes have accumulated, geneticists have remained silent.
As Dr. Latham points out, there are still important decisions to be made. The Collins paper proposed a no doubt expensive and open-ended search among hitherto disregarded genetic locations. However, many now believe the likelihood that further searching might rescue the day for genomics appears slim. Perhaps therefore we should use phenotypic measurements instead of genomic measurements to study our commonest illnesses, and to search for means of prevention and treatment.

Third Harmonics Microscopy: New Methods For Non-Invasive Imaging Of Cornea And Brain

Further developments in the application of multi-photon microscopy have lead to a relatively new technology that promises to be a a new means for enabling better human diagnostic procedures and a better means for visualizing structures within the body, such as the brain, during surgical procedures. In the 1990s I collaborated with Scott Fraser's lab at Cal Tech using multi-photon microscopy to visualize the dynamics of synaptic movement in the living retina, which is a part of the brain (CNS) in the back of the eye. At the time, Fraser's multi-photon microscope was one of the few in the world. Now, not much more than a decade later, the technology is widespread and new variants are promising to change the way we visualize our world in many different applications, including human surgery.

Third Harmonics Generated (THG) microscopy is based on third harmonic light generated at the focal point of a tightly focused short-pulse laser beam. When the medium at the focal point is homogenous, the third
harmonic waves generated before and after the focal point interfere destructively, resulting in zero net THG. However, when there are inhomogeneities near the focal point, such asan interface between two media, the symmetry along the optical axis breaks and measurable amount of third harmonic is generated. Due to its nonlinear nature, the third harmonic light is generated only in a close proximity to the focal point. Therefore, high spatial resolution can be obtained, enabling THG microscopy to section the tissue and to construct three dimensional images of transparent samples. Because biological tissues have non-vanishing third order susceptibilities, THG microscopy can be utilized as a general-purpose microscopy technique, and is now being used as a surgical microscope in laboratory experiments. This means that different tissues, because of their varying optical properties, can be visualized with the THG microscope without the use of any staining procedures and can be performed in natural, live tissue. Below are two examples of relevant papers:

Opt Express. 2010 Mar 1;18(5):5028-40. doi: 10.1364/OE.18.005028. Harmonic microscopy of isotropic and anisotropic microstructure of the human cornea. Olivier NAptel FPlamann KSchanne-Klein MCBeaurepaire ELaboratory for optics and biosciences, Ecole Polytechnique ParisTech, CNRS, and INSERM U696, 91128 Palaiseau, France

Proc Natl Acad Sci U S A. 2011 Apr 12;108(15):5970-5. Epub 2011 Mar 28. Label-free live brain imaging and targeted patching with third-harmonic generation microscopy. Witte SNegrean ALodder JCde Kock CPTesta Silva GMansvelder HDLouise Groot M.
Biophysics Group, Institute for Lasers, Life, and Biophotonics Amsterdam, VU University, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands.

Monday, April 11, 2011

Tagging Rejuvenates Electron Microscopy

Modifying a protein from a plant much favored by science, researchers at the University of California, San Diego School of Medicine and colleagues have created a new type of genetic tag visible under an electron microscope, illuminating life in never-before-seen detail.
Led by Nobel laureate Dr. Roger Tsien, PhD, Howard Hughes Medical Institute investigator and UCSD professor of pharmacology, chemistry and biochemistry, a team of scientists radically re-engineered a light-absorbing protein from the cress plant Arabidopsis thaliana. When exposed to blue light, the altered protein produces abundant singlet oxygen, a form of molecular oxygen that can be made visible by electron microscopy (EM).
The work is published in the April 5 issue of the journal Public Library of Science(PLoS) Biology. Tsien was co-winner of the 2008 Nobel Prize in chemistry for his role in helping develop and expand the use of green fluorescent proteins (GFP), a tool widely employed in light microscopy to peer inside living cells or whole animals and observe molecules interacting in real-time.
Tsien said the development of the small, highly engineered Arabidopsis protein, dubbed “miniSOG,” may elevate the abilities of electron microscopy in the same way GFPs have made modern light microscopy in biological research much more powerful and useful.
The big advantage of EM is that it has always had much higher spatial resolution, that is you can see more detail, than light microscopy. You can achieve up to a hundred-fold higher useful magnification from EM than from light microscopy. The result has been extraordinarily detailed, three-dimensional images of microscopic objects at resolutions measuring in the tens of nanometers, tiny enough to meticulously render the internal anatomy of individual cells.
But current EM technologies do not distinguish or highlight individual proteins in these images. These can be tagged with GFP or other fluorescent proteins, but they are visible only with the limited resolution of light microscopy.
Existing EM images of cell structures are analogous to satellite or aerial photographs showing major landmarks and populations centers. Therefore with EM, difficulties arise when visualizing individual protein types and their location, just as most geographical maps do not show the location of individual classes of people, such as everyone with the surname Smith, or all of the orthodontists in an area. Tsien's new technique enables scientists to put beacons on just about any protein and visulaize a snapshot of the protein's location at the much higher resolution of EM.
To create this ability, the scientists began with a protein from Arabidopsis, a small flowering plant that has long used as a research model. The original protein absorbs incoming blue light, triggering biochemical signals that inform the plant how much sunlight it is receiving. Tsien rationally engineered the protein based on its atomic model so that it changes incoming blue light into a little bit of green fluorescence and a lot of singlet oxygen.
Established methods were then used to convert singlet oxygen production into a tissue stain that the electron microscope can see. The scientists tested the modified protein’s utility as an EM marker by first using it to confirm the locations of several already well-understood proteins in mammalian cells, nematodes and rodents, then used miniSOG to successfully tag two neuronal proteins in mice whose locations had not been known.
The UCSD scientists are optimistic that miniSOG will grant new powers to electron microscopy, permitting scientists to pursue answers to questions previously impossible to ask.
MiniSOG would be much appreciated by scientists who investigate cellular and subcellular structures including neuronal circuits at nanometer resolution in multicellular organisms, because previous methods have great difficulty in achieving both efficient labeling and good preservation of the structures under study.
However, EM will not replace light microscopy for a number of reasons.
When using miniSOG,  the tagged proteins plus the landmarks are used to navigating. On the other hand, EM has the disadvantage that it gives a snapshot of cells before we sacrificed them (to make the image), whereas light microscopy can show the dynamics in live cells. Each technique has different complementary strengths and weaknesses.
The new electron microscopy technique reveals the previously unknown locations of two neuronal proteins called SynCAM1 and SynCAM2. The first is an adhesion protein found at the synapse – or communications link – of neurons sending information. Its close relative, SynCAM2, is used by neurons receiving information. Neurons that send information are distinguishable because they contain synaptic vesicles, which are used to store neurotransmitters for communications use. In these images, the vesicles resemble small hollow circles.