Testing Webservices

Debugging (Photo credit: mikemol)

Last night I added 2 more webservices to DermKnowledgeBASE. Debugging webservices is a nightmare. (At least it was, until the wee hours of the morning). It always returns SoapFault exception mentioning that "looks like we got no XML document". After several wasted hours of hard work I found the way to use the instruction here. The best answer is rather cryptic. So I have included the code I used to display the request and response so that debugging becomes much easier.

<?php $option=array('trace'=>1); $client=new SoapClient('http://gulfdoctor.net/dermbase/wsdiffs11.php?wsdl',$option); try{ $client->getDiffs("LichenPlanus"); }catch(SoapFault $fault){ echo 'Request/Response : <br/><xmp>', $client->__getLastRequest(), $client->__getLastResponse(), '</xmp><br/><br/> Error Message : <br/>', $fault->getMessage(); } ?>

Change the wsdl path and the function call. Hope this saves some time for someone!

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The great anti-ageing mantra

Matrix metalloproteinases (MMPs) are important in dermatology as they are involved in remodelling extracellular matrix (ECM). It is a big family of enzymes, all having a common domain structure with a pro-peptide region, catalytic domain, hinge region and the haemopexin-like C-terminal domain.  They are involved in a variety of biological functions. MMP1 is involved in the premature ageing in smokers.[1] Inhibition of certain MMPs have anti-ageing effect while inhibition of others promote hyperproliferative disorders and scars. However because of the structural similarities, rational design of specific inhibitors are very difficult. Most of the rationally designed MMP inhibitors like  marimastat (BB-2516) and  cipemastat (Ro 32-3555) have performed poorly in clinical studies.

This is a Graph showing the mutual activation of matrix metallo-proteinases. (Photo credit: Wikipedia)

This paper [2] proposes a new approach wherein regulatory sites hidden and scattered over different locations on the protein’s surface are targeted instead of the catalytic site using specially designed branched amphiphilic molecules. They have demonstrated that MMP family possess a unique set of such sites making it possible to target specific MMPs without affecting others. If this technology can be successfully developed it may have several applications including many dermatological uses.

References:

1. Lahmann, Christine et al. "Matrix metalloproteinase-1 and skin ageing in smokers." The Lancet 357.9260 (2001): 935-936.

2. Udi, Yael et al. "Unraveling hidden regulatory sites in structurally homologous metalloproteases." Journal of Molecular Biology (2013).

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Rational Peptide Design

A hexapeptide mimic of the N-terminal end of SNAP-25 which competes with this protein for a position in the SNARE complex is being used as a cosmetic peptide to eliminate facial lines resulting from muscular activity. However industry standard for the same indication is Botulinum Neurotoxin A that can enter the neuron by SV2 mediated endocytosis. It is not known whether the hexapeptide mimic actually enters the neuron in sufficient concentration to give the proposed effect. Read the clinical implications of the story here.

One of the earliest studies on this Acetyl Hexapeptide - 3 gives a ‘rational’ explanation for this limitation. Let me quote from the article:[1]

Gp41 coiled coil hexamer 1aik topview (Photo credit: Wikipedia)

Sequence and structure analysis of the N-terminal of SNAP-25 revealed the sequence EMQRR that display a high propensity to acquire an alpha-helical Structure along with a pronounced coiled-coil probability. AGADIR, a programme that estimates helical propensity of peptides, predicted a remarkable 12 % probability for this small peptide. These properties suggest that a peptide patterned after this sequence may modulate Calcium dependent  exocytosis, Similar to those peptides derived from the C-terminal of SNAP-25.

I have the following questions about the rational explanation:

1. How can a hexapeptide have coiled-coil probability? (A minimum of 20 amino acids are needed for a coiled coil)[2]
2. Is 12% probability remarkable?
3. How is a N-terminal mimic be similar to a C-terminal mimic?

Is this an attempt to suggest that the structural peculiarities mediate an HIV virus like receptor mediated endocytosis. If only we knew the truth. If only....

References:


1. Blanes‐Mira, C et al. "A synthetic hexapeptide (Argireline) with antiwrinkle activity." International journal of cosmetic science 24.5 (2002): 303-310.

2. Rose, A. "Genome-Wide Identification of Arabidopsis Coiled-Coil Proteins and ..." 2004. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC389916/>

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Best peptides for women

In continuation with my previous posts here and in skin deep, let me add few more things I found recently.

Peptide Synthesis Deprotection (Photo credit: Beige Alert)

The article titled “Systematic Discovery of New Recognition Peptides Mediating Protein Interaction Networks “ published recently in PLOS Biology ( full text at : http://dx.plos.org/10.1371/journal.pbio.0030405 ) describes the signalling by short peptide segments. Short peptide segments interact with globular protein domains that share a common sequence pattern (e.g., SH3 binding to PxxP). They also point out that sequence comparison experiments are unlikely to discover the optimum short motif. They recommend using data from genome-scale interaction studies. So the methodology adopted by the designers of peptide-21 may not be robust.

OK, now I am going to do something here that I rarely ever do on my blogs: I am going to shamelessly self promote me without actually giving away much information. My plan is to get noticed by the cosmetic tycoons and make some money out of this incessant babbling about beauty peptides :-)

English: Example of mechanism of direct penetrating peptide (Photo credit: Wikipedia)

I used a newly published algorithm (not mine) for bioinformatics analysis and found some interesting information.

The most commonly occurring tetra peptide in collagen is in fact GXXG and PXXP the commonest being GPPG.

The commonest tetra peptide repeats were GERG, GEKG, GFPG, GENG, GPRG, GHRG.

This is what terapeptide-21 is based on and there is nothing new till here.

But here comes the most surprising part!

The highest scoring short peptide of probable biological (signal) function does not belong to the above list!!

Hey, Do I hear my phone ringing?????

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Beauty born out of Bioinformatics

The beauty and the anti-ageing industry is focused on finding ways to augment the fibrillar proteins collagen and elastin embedded in an extracellular matrix in the dermal layer of the skin. A pentapeptide KTTKS, within the carboxy terminal propeptide of type I collagen is extensively used as an anti-ageing cosmetic ingredient. The proposed mechanism of action is tricking the body into believing that there is excessive collagen degradation so that collagen biosynthetic pathway is stimulated. I have blogged about the potential limitations in Skin Deep .

B0005968 Collagen fibril forming in vitro (Photo credit: wellcome images)

Yet another peptide called tetrapeptide - 21 is claimed to be superior to KTTKS in anti-ageing properties. They claim to have discovered this by using bioinformatics methodology.

Let me quote from a product brochure:

We used bioinformatic methods to identify highly repetitive amino acid motifs in several human ECM proteins to identify sequences with inherent anti-aging activities.  Using the amino acid sequences of collagen I, II, III, IV, V, elastin, and proelastin, tetra-peptide sequences were identified by their frequency of occurrence. 

Beauty box1886 (Photo credit: Wikipedia)

The methodology has never been published. Their patent application states that they found GXXG and PXXP as the common motif among all collagens and elastin. They tried various combinations and found GEKG as the most biologically active one. At the sequence level all collagens are quite similar. It is also well known that the most common motifs in all types of collagen is GPX. So finding GXXG as the commonest tetrapeptide motif is hardly surprising.

So dear biochemicians:

1. Where does the MMPs cleave collagen?
2. How many amino acid residues are there in collagen degradation fragments?
3. Do you think GXXG can mimick collagen fragment?

and dear bioinformaticians:
1. If we consider Collagen I & III into consideration (more relevant for skin) which are the commonest penta and hexa peptide motifs. Do they converge?

Susan Anton (Photo credit: Wikipedia)

2. How does adding an H to the carboxy terminal of such a penta/hexapeptide affect the polarity? Will it increase the skin penetration as described here? http://www.ncbi.nlm.nih.gov/pubmed/22452335

and finally dear cell biologists:
1. Do all collagen degradation fragments stimulate UVB induced hyaluronic acid degradation as described here: http://www.ncbi.nlm.nih.gov/pubmed/21454612

I have added a wiki page for this to summarise the findings. Feel free to add your thoughts here.

Resources:

collagen alpha-1(I) chain preproprotein [Homo sapiens] : http://www.ncbi.nlm.nih.gov/protein/NP_000079.2

collagen alpha-2(I) chain precursor [Homo sapiens]
http://www.ncbi.nlm.nih.gov/protein/NP_000080.2

collagen alpha-1(III) chain preproprotein [Homo sapiens]
http://www.ncbi.nlm.nih.gov/protein/4502951

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Importing ONTODerm Ontology

Map of Colombia with Departments (Photo credit: Wikipedia)

Two engineering students from Colombia are using ONTODerm for a noble cause. They are planning to take dermatology to the poor and the underprivileged. They started a teledermatology project, but discontinued it because of lack of support. Now they are working on an Ontology based diagnostic application using semantic web technologies.

One of the problems they have encountered may be important to be addressed here. They could not import ONTODerm into Sesame. The URL provided in the ONTODerm home page is protege specific. However you can export the latest ONTODerm version in the native format from the project page on knoodl. Just request for a free membership to ONTODerm community.

Please keep me posted if you are using ONTODerm or DermKnowledgeBASE for any such projects and I wish both of them all the very best.

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A Biochemical challenge!

Dear Reader

I am back again with a challenge this time. My last challenge got excellent response and I finally published the findings here. This challenge is probably more to do with biochemistry and I hope the Biochemicians facebook group under Prof. PTS would find this problem interesting.

Before I define the problem, please read the posts here to know about the clinical context.

1. The Big Bang Theory

2. Beauty of dark hair.

Molecule Man (Photo credit: rizkapb)

In brief, Afamelanotide is a peptide drug being developed by Clinuvel pharmaceuticals for tanning. It is a synthetic alpha-MSH analog. (Click here for details)

A new patented molecule claims to have similar mechanism of action. (Click here for details)

The challenge is to find out:

1. How similar is the patented molecule to Afamelanotide?

2. Do you think the new molecule is designed for binding to cysteine rich keratin family in the hair cuticle? Can we improve this binding further by structural alterations?

3. Is the structure optimized for cutaneous penetration? In other words, what is the polarity and other relevant chemical and physical properties of the new molecule?

Please refer to the patented molecule as 'The bang' in your comments.

Thanks

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