Hamster Study Shows Nanofibers Knit Severed Neurons Together, Restore Vision

Image: COURTESY OF PNAS Brain injuries afflict more than five million Americans, ranging from head trauma to stroke. Currently, there is no way to restore lost function or recapture what can be a profound shift in ability and even personality. But new research suggests that nanofibers can help induce neurons to reconnect and restore vision in the process, at least in hamsters. A team of neuroscientists at the Massachusetts Institute of Technology and their colleagues at Hong Kong University purposefully wounded 53 newly born hamster pups. They cut a relatively deep gash--1.5 millimeters deep and two millimeters wide--through the optic tract in the brains of the young rodents. The wounds of 10 of the pups were then treated with 10 microliters of a solution composed of 99 percent water and 1 percent of a special ionic peptide. These short amino acids are capable of creating a molecular scaffold that can bridge such gaps.

Within 24 hours, the gash in the treated pups had begun to close (shown by the green area in the picture above, depicting regrowth), and by 30 days had completely closed. "We had never seen that before in any animals," says neuroscientist Rutledge Ellis-Behnke of M.I.T., who led the research. By placing a biological tracer in the hamsters' eyes the researchers also discovered that the neurons had actually grown back and reconnected through the center of the cut instead of routing around the wound--another first. None of the control animals showed any healing whatsoever.

The scientists then inflicted a similar wound on some adult hamsters to see if such connections could actually regenerate vision. By injecting 30 microliters of the solution, the scientists again healed the gaps in 30 days. And in subsequent behavioral tests, the animals had regained the ability to turn their eyes and heads toward a sunflower seed in their peripheral vision, though their turning response was slower than normal. The solution, which forms a tight-fitting fibrous gel in the wound, appears to have no long-term side effects, breaking down into its constituent amino acids and exiting the body through the bloodstream and urine. "We have healing of the brain, which we've never seen before. We have axons growing through the center of the cut, which we've never seen before, and we have axons connecting to the target tissue," Rutledge notes. "If we could use something like this to mitigate the damage caused by cutting the brain with a knife, that would be great." The research appears online this week in Proceedings of the National Academy of Sciences. --David Biello

Drug Found to Reverse the Ravages of Alzheimer's in Mice

Image: PHOTODISC Researchers have identified a compound that could significantly improve treatment of Alzheimer's disease. When administered to mice engineered to develop hallmarks of the disease, the drug reversed cognitive decline and reduced the two types of brain lesions--plaques and tangles--that occur in Alzheimer's patients. Frank M. LaFerla of the University of California at Irvine and his colleagues gave Alzheimer's mice and normal mice daily doses of the drug, known as AF267B, for eight weeks and then tested their ability to learn to locate a hidden platform in a tank of water. Alzheimer's mice that received AF267B, they found, performed significantly better on this test than untreated mice did. And normal mice showed no ill effects from the drug. But in a second memory test, wherein the animals had to learn to associate a dark chamber with a mild electric shock, the treated Alzheimer's mice did not outperform their untreated counterparts. Subsequent analyses of the brains of these animals revealed that the drug reduced the plaques and tangles in the hippocampus, which is known to play a key role in learning the water maze, but not in the amygdala, which figures importantly in the dark chamber test.

AF267B seems to work in part by enhancing the activity of receptors for the neurotransmitter acetylcholine. The compound binds to these so-called M1 receptors, and in so doing boosts the levels of an enzyme called alpha secretase. This enzyme blocks the production of beta-amyloid proteins, the accumulation of which into plaques is theorized to lead to Alzheimer's. The amygdala, it turns out, does not produce much alpha secretase, and even with the help of AF267B could not make enough to block the formation of beta-amyloid. Hence, the mice's poor performance on the dark chamber task.

The study also suggests that AF267B reduces the activity of an enzyme known as GSK3beta, which in turn prevents so-called tau proteins from clumping together into the disease's signature tangles. The team additionally found that suppressing the M1 receptors with a drug called dicyclomine exacerbated learning and memory impairment in the Alzheimer's mice, and led to the appearance of more of the plaques and tangles. These results, the scientists say, underscore the important role of M1 receptors in modulating these hallmarks of Alzheimer's disease. It remains to be seen whether AF267B is as effective in humans suffering from Alzheimer's as it is in the mice. But for their part, LaFerla and his collaborators are hopeful. "AF267B could be a tremendous step forward in the treatment of Alzheimer's disease," LaFerla observes. "Not only does it appear to work on the pathology of Alzheimer's and ease its symptoms, it crosses the blood-brain barrier, which means it does not have to be directly administered to the brain, a significant advantage for a pharmaceutical product. Although we cannot determine what the effects of AF267B will be in humans until clinical trials are complete, we are very excited by the results our study has yielded." The findings will be published tomorrow in the journal Neuron. --Kate Wong

Laotian Rodent Proves Living Fossil

Image: COURTESY OF MARK A. KLINGER When wandering through a hunter's market in Laos, Robert Timmins of the Wildlife Conservation Society happened upon a previously unknown rodent. Called kha-nyou by locals--or rock rat--the long-whiskered and furry-tailed rodent was reputed to favor certain limestone terrain. Western scientists named it Laonastes aenigmamus or stone-dwelling enigmatic mouse--partially because a live specimen has never been collected--and thought the rock rat represented a new family of mammals. But new research reported in today's Science proves that Laonastes actually represents a fossil come to life. Paleontologist Mary Dawson of the Carnegie Museum of Natural History and her team immediately recognized the strange rodent as a living member of a family thought to have been extinct for at least 11 million years: the Diatomyidae. Fossilized remnants of this group have been found throughout Asia with a distinctive jaw structure and molars. A new specimen of Diatomys discovered in June of last year in China bore an uncanny resemblance to Laonastes, including the same body size and tail span.

"It's the coelacanth of rodents," Dawson says, referring to the ancient fish believed extinct until a live specimen was hauled from the depths by South African fishermen. "One of the beautiful parts of this discovery was that we were able to correctly predict that Laonastes would have four roots in its molars just as in Diatomys."

The rock rat represents a rare opportunity to compare assumptions derived from the fossil record and an actual living specimen to determine overall accuracy of the techniques involved, the scientists argue. It also represents tantalizing support for the theory that many mammals evolved in Asia and later colonized other continents, as its closest living relative is the gundis--a guinea pig-like rodent of northern Africa. Ultimately, kha-nyou provides a compelling argument for preservation efforts in Southeast Asia, joining tree shrews, flying lemurs and tarsiers as remnant populations of ancient mammal families in the region. "Laonastes is not the only new organism to be discovered in southeastern Asia," Dawson adds. "The highest priority must be given to preserving this unique biota and especially Laonastes while it is still possible." --David Biello

Bacteria Turn Styrofoam into Biodegradable Plastic

Bacteria are everywhere, silently going about their business of breaking down cellulose, fermenting foods or fixing nitrogen in the soil, among a host of other activities. Given their ubiquity and diversity of functions, biotechnologists have been searching for new uses for different strains of the microscopic organisms, such as consuming oil spills or even capturing images. Now biologists at the University College Dublin in Ireland have found that a strain of Pseudomonas putida can exist quite happily on a diet of pure styrene oil--the oil remnant of superheated Styrofoam--and, in the process, turn the environmental problem into a useful, biodegradable plastic. Kevin O'Connor and his European colleagues turned the polystyrene into an oil through pyrolysis--a process that heats the petroleum-based plastic to 520 degrees Celsius in the absence of oxygen. This results in a chemical cocktail made up of more than 80 percent styrene oil plus low volumes of other toxicants. The researchers then fed this brew to P. putida CA-3, a special strain of a common soil microbe, fully expecting that the oil would have to be further purified in order to enable bacterial growth.

But the bacteria thrived on this new diet, turning 64 grams of undistilled styrene oil into nearly 3 grams of additional bacteria. In the process, the bacteria stored 1.6 grams of the energy of the styrene oil as a biodegradable plastic called polyhydroxyalkanoates, or PHA. This plastic can stand up to heat but also breaks down more naturally in the environment than petroleum-based products. Thus, though the biology-powered process results in some toxic byproducts such as toluene and requires significant energy to drive the pyrolysis, it fuels hopes that Styrofoam--and the polystyrene molecule that makes it--can become more environmentally friendly.

This would be good news for the U.S., which produced three million tons of polystyrene in 2000, according to the EPA, and threw away 2.3 million tons of the stuff, consigning the waste to rest for long years in landfills. The PHA from this process could be turned to more productive uses; it is already being used to make everything from forks to vitamins. And the process might not just be useful for getting rid of disposable cups. "Due to the general applicability of pyrolysis for plastic conversion to an oil and the large number of microorganisms capable of PHA accumulation from a vast array of molecules, the principle of the process described here can be applied for the recycling of any petrochemical plastic waste," the scientists claim in the paper presenting their findings in the April 1 issue of Environmental Science & Technology. Apparently, bacteria recycle, too. --David Biello

Cocoa Linked to Lower Risk of Disease

The Dutch have a long history with chocolate. Although native Mexicans and their Spanish conquerors first used the bitter bean--and reported on its tonic powers--a Dutchman was the first to extract modern cocoa and neutralize its bitterness with alkali. The modern chocolate bar was born. Now, results from a study of aging Dutch men have shown that cocoa consumers were half as likely to die from disease than those who did not eat the sweet treat. Brian Buijsse of the National Institute for Public Health and the Environment in Bilthoven and his colleagues measured the cocoa intake of 470 men between 1985 and 2000 as part of the Zutphen Elderly Study, a longitudinal look at nearly 1,000 Dutch men between 65 and 84 years of age. The nutrition experts identified 24 cocoa-containing foods that the elderly men ate, ranging from dark chocolate bars to chocolate spreads. They summed the total amount of cocoa each consumed and came up with a grams-per-day measurement, which they used to separate the men into three groups: those who ate little chocolate, a modest amount, and the most.

Among those who ate the most chocolate--averaging more than four grams a day--average systolic and diastolic blood pressure was 3.7 and 2.1 millimeters of mercury lower than their chocolate-spurning peers. This result did not hold true for other sweet foods nor did it vary among men who also smoked, were inactive or consumed a lot of alcohol. And, despite being strongly associated with greater intake of calories, chocolate lowered the overall risk of cardiovascular or any other disease by as much as 50 percent.

Although the chocolate definitely decreased blood pressure and prolonged life, the two were not statistically related, according to the researchers. This means that the exact mechanism by which chocolate helps remains a mystery. "Our findings, therefore, suggest that the lower cardiovascular mortality risk related with cocoa intake is mediated by mechanisms other than lowering blood pressure," the authors write in their report, published yesterday in the Archives of Internal Medicine. "Because cocoa is a rich source of antioxidants, it may also be related to other diseases that are linked to oxidative stress (e.g. pulmonary diseases, including chronic obstructive pulmonary disease and certain types of cancer)." --David Biello

Bioinformatics practical - Comparative Genomics

Use ClustalX to perform MSA and Jalview to edit your MSA results and create phylogenetic tree using programs from PHYLIP package with Patched proteins and PDGFRs as examples.

An Overview Collecting homologous protein sequences à Use a text editor to create a FASTA file (orthologs and paralogs) à Perform MSA using ClustalX à Edit MSA result using Jalview (save output file in both aln and (save edited file in aln format) phy format) à Reopen the edited MSA result à Use phylip seqboot to perform bootstrapping using ClustalX and save the file (phy file as input) into phy format à phylip protdist to calculate distance à phylip neighbor to calculate neighbor (“infile” as input file) à Use phylip drawgram to draw “rooted” tree (“intree” as input file)

Programs

ClustalX to perform multiple sequence alignment (MSA)

Jalview to edit MSA result

PHYLIP package to bootstrap and draw phylogenetic tree

Methods

Section A. Patched proteins

Patched proteins have been identified in a diverse group of organisms and function in Hedgehog signaling. At least two isoforms of patched proteins (patched 1 and 2) have been identified in mouse. In this section, we will examine the evolution of the patched proteins in various vertebrates.

Using one of the mouse Patched homologs (Q61115) to identify orthologs for Patched-1 and Patched-2 using blast search against SWISS-PROT database via a local server (http://sf01.bic.nus.edu.sg/blast/blast.html).
Extract the Patched-1 and Patched-2 sequences from the blast results and create a text file in FASTA format. Name this file as “Patched.fasta” using a text editor.
Launch ClustalX (KMenu à Education à ClustalX)
Load Patched.fasta file. Click on Alignment à Output Format Options, select Clustal format and PHYLIP format.
Do Complete Alignment.


Editing the MSA result

Jalview is used to edit the MSA result. Save the edited MSA result as Patched.aln to overwrite the aln file from previous MSA run. Use ClustalX to open this file and save as “Patched.phy” to overwrite the phy file from previous run. The “Patched.phy” will be the input file for PHYLIP package.


Generate Phylogenetic Tree using PHYLIP package

Download phylip_3.6.1-2_i386.deb from IVLE.
Click on “Penguin” à Root Shell
Type cd Desktop to change directory to Desktop
Type ls to list the file content on Desktop
Type dpkg -i phylip_3.6.1-2_i386.deb to depackage the PHYLIP files
Type /usr/bin/phylip to see the program options for PHYLIP package
By now you should have Patched.aln and Patched.phy on the Desktop


Bootstrap

Type phylip seqboot

You will see the following:

seqboot: can't find input file "infile"
Please enter a new file name>

Type Patched.phy and Enter. You will see something like this:

Bootstrapping algorithm, version 3.61

Settings for this run:
D Sequence, Morph, Rest., Gene Freqs? Molecular sequences
J Bootstrap, Jackknife, Permute, Rewrite? Bootstrap
% Regular or altered sampling fraction? regular
B Block size for block-bootstrapping? 1 (regular bootstrap)
R How many replicates? 100
W Read weights of characters? No
C Read categories of sites? No
S Write out data sets or just weights? Data sets
I Input sequences interleaved? Yes
0 Terminal type (IBM PC, ANSI, none)? ANSI
1 Print out the data at start of run No
2 Print indications of progress of run Yes

Y to accept these or type the letter for one to change

Type “Y” to accept the setting and Enter

Next you will see this:

Random number seed (must be odd)?

Enter any odd number e.g. 11 and Enter

You will see the following:

completed replicate number 10
completed replicate number 20
completed replicate number 30
completed replicate number 40
completed replicate number 50
completed replicate number 60
completed replicate number 70
completed replicate number 80
completed replicate number 90
completed replicate number 100

Output written to file "outfile"

Done.

Check Desktop for “outfile”. Rename the outfile as seqbootout.txt Double click on this file to see the content.


Calculate Distance

Type phylip protdist
You will see something like this

Protein distance algorithm, version 3.61

Settings for this run:
P Use JTT, PMB, PAM, Kimura, categories model? Jones-Taylor-Thornton matrix
G Gamma distribution of rates among positions? No
C One category of substitution rates? Yes
W Use weights for positions? No
M Analyze multiple data sets? No
I Input sequences interleaved? Yes
0 Terminal type (IBM PC, ANSI)? ANSI
1 Print out the data at start of run No
2 Print indications of progress of run Yes

Are these settings correct? (type Y or the letter for one to change)

Type “Y” to accept the setting and Enter

You will see something like

Computing distances:
O35595PT
Q9Y6C5PTC .
Q61115PTC ..
Q13635PT ...
Q90693PT ....
Q98864PT .....
Q09614PT ......
Q6T3U4NP .......

Output written to file "outfile"

Check “outfile” on Desktop and double clicking to see its content. Now rename “outfile” as “infile” which will be the input file to calculate neighbor using “neighbor” option in PHYLIP.

Calculate Neighbor

Under root shell, type phylip neighbor and Enter

You will see the following

Neighbor-Joining/UPGMA method version 3.61

Settings for this run:
N Neighbor-joining or UPGMA tree? Neighbor-joining
O Outgroup root? No, use as outgroup species 1
L Lower-triangular data matrix? No
R Upper-triangular data matrix? No
S Subreplicates? No
J Randomize input order of species? No. Use input order
M Analyze multiple data sets? No
0 Terminal type (IBM PC, ANSI, none)? ANSI
1 Print out the data at start of run No
2 Print indications of progress of run Yes
3 Print out tree Yes
4 Write out trees onto tree file? Yes


Y to accept these or type the letter for one to change

Type “Y” to accept the setting and Enter.

You will see

Protein distance algorithm, version 3.61

Settings for this run:
P Use JTT, PMB, PAM, Kimura, categories model? Jones-Taylor-Thornton matrix
G Gamma distribution of rates among positions? No
C One category of substitution rates? Yes
W Use weights for positions? No
M Analyze multiple data sets? No
I Input sequences interleaved? Yes

Protein distance algorithm, version 3.61
Settings for this run:
P Use JTT, PMB, PAM, Kimura, categories model? Jones-Taylor-Thornton matrix
G Gamma distribution of rates among positions? No
C One category of substitution rates? Yes
W Use weights for positions? No

Neighbor-Joining/UPGMA method version 3.61

Settings for this run:
N Neighbor-joining or UPGMA tree? Neighbor-joining
O Outgroup root? No, use as outgroup species 1
L Lower-triangular data matrix? No
R Upper-triangular data matrix? No
S Subreplicates? No
J Randomize input order of species? No. Use input order
M Analyze multiple data sets? No
0 Terminal type (IBM PC, ANSI, none)? ANSI
1 Print out the data at start of run No
2 Print indications of progress of run Yes
3 Print out tree Yes
4 Write out trees onto tree file? Yes


Y to accept these or type the letter for one to change

Type “Y” to accept the setting and then Enter. You will see something like

Cycle 5: species 1 ( 0.03614) joins species 2 ( 0.05739)
Cycle 4: species 3 ( 0.01320) joins species 4 ( 0.02322)
Cycle 3: node 3 ( 0.05218) joins species 5 ( 0.06119)
Cycle 2: species 7 ( 0.98182) joins species 8 ( 2.21938)
Cycle 1: node 3 ( 0.23822) joins node 7 ( 0.11636)
last cycle:
node 1 ( 0.26219) joins node 3 ( 0.05675) joins species 6 ( 0.17702)

Output written on file "outfile"

Tree written on file "outtree"

Done.


Check “outfile” and “outtree” on Desktop and double clicking on them to see their content.

Rename “outtree” as “intree” which will serve as input file for drawgram option in PHYLIP.

Tree Drawing

The “drawgram” option is used to generate a “rooted” tree.

Type phylip drawgram in root shell and Enter

You will see


DRAWGRAM from PHYLIP version 3.61
Reading tree ...
Tree has been read.
Loading the font ....
Font loaded.

Rooted tree plotting program version 3.61

Here are the settings:
0 Screen type (IBM PC, ANSI): ANSI
P Final plotting device: Postscript printer
V Previewing device: X Windows display
H Tree grows: Horizontally
S Tree style: Phenogram
B Use branch lengths: Yes
L Angle of labels: 90.0
R Scale of branch length: Automatically rescaled
D Depth/Breadth of tree: 0.53
T Stem-length/tree-depth: 0.05
C Character ht / tip space: 0.3333
A Ancestral nodes: Weighted
F Font: Times-Roman
M Horizontal margins: 1.65 cm
M Vertical margins: 2.16 cm
# Pages per tree: one page per tree

Y to accept these or type the letter for one to change

Type “Y” to accept the setting and Enter. You will see

Writing plot file ...

Plot written to file "plotfile"

Done.

A “drawgram” will appear. Examine the “drawgram”. Go to File à Plot

Check Desktop for “plotfile” and rename it as “plotfile.bmp” and double click to view.


Now insert the protein sequence with SWISS-PROT accession number Q13635 into the FASTA file created previously and repeat multiple sequence alignment, edit MSA result and draw tree as procedures you gone through above using ClustalX, Jalview, and PHYLIP, respectively.