In our search for understanding the evolution of blood sucking by arthropods, we realize the methodology used has drastically changed in the past few years. Traditionally, the research process first identified a biological activity in saliva or salivary gland homogenates of a particular organism, and then proceeded to isolate that activity as a relatively pure entity to allow its molecular identification. In the case the activity derived from a protein, peptide fingerprinting allowed the design and use of nucleotide probes to clone the coding mRNA (in the form of a cDNA) and final identification of the peptide sequence;the clone also allowed the manufacture of recombinant protein for further studies. Nowadays, the process has reverted. cDNA libraries are constructed from salivary glands of blood sucking arthropods and mass sequenced. Bioinformatic analysis reveals the salivary transcriptome of these organisms, which contains many unique protein families with unknown properties. We then proceed to select clones for expression, bioassay screening and characterization. Accordingly, there are two processes used in our lab, first, the construction and analysis of salivary gland cDNA libraries, and second, the recombinant expression and characterization of these proteins. We have also been developing bioinformatic capabilities in the form of specific software to help direct our studies. Sialotranscriptome discovery projects: Because host hemostasis (the physiological process that prevents blood loss, consisting of platelet aggregation, blood clotting and vasoconstriction) is a complex and redundant phenomenon, the salivary glands of blood sucking arthropods consist of a magic potion with diverse chemicals that in a redundant way counteract host mechanisms to prevent blood loss, allowing the fast acquisition of a meal. Salivary transcriptome made in the past few years indicate that the magic potion consists of 70-100 different proteins in the case of mosquitoes, for example, to over 400 in the case of ticks (Ticks feed for several days and have to disarm host immune reactions, in addition to the hemostatic system). Transcriptome studies also show that the salivary proteins of blood sucking arthropods are at a very fast pace of evolution, perhaps explaining why every genera studied so far has several unique protein families. Indeed there are unique proteins found at the subgenus level. Given we can now describe in detail the sialotranscriptome (from the Greek word sialo = saliva) of a single organism, we can ask now what is the universe of salivary proteins associated to blood feeding, the so called sialoverse. There are near 19,000 species of blood sucking arthropods in 500 different genera. If we find (minimally) 5 novel protein families per genus (within the 70-500 proteins in each sialome), there are at least 2,500 novel proteins to be discovered, each one with an interesting pharmacological property. We have so far explored less than a dozen genera of blood sucking arthropods, and it is our goal to extend sialotranscriptome discovery to map this pharmacological mine for future studies, and in the process learn the paths taken by genomes in their evolution to blood feeding, and identify proteins with pharmacological and vaccine potential. In the current fiscal year (2011), we produced a total of 21 papers and one patent previously applied was granted by the USPO. Two of the papers describe sialomes, including the sialome of the black fly Dipetalogaster maxima, the first so far produced for this genus of kissing bug vectors of Chagas disease and for Amblyomma variegatum, an African species that is of veterinary and public health importance in Africa and the Mediterranean regions. Functional sialomic studies: We advanced our knowledge regarding the function of several salivary proteins, as well as discovering novel salivary properties. We identified the structure and function of a major protein from the saliva of malaria vectors, characterized the function of a triatomine protein that inhibits platelet aggregation by binding to Thromboxane A2, characterized functionally a salivary anticlotting of the mosquito Aedes albopictus, and characterized the crystal structure of cysteine proteinase inhibitors of ticks. A novel family of platelet aggregation inhibitors from tabanid flies was characterized functionally, and a previously described anticlotting from Rhodnius was tested as an anticoagulant that inhibits arterial thrombosis but does not impair hemostasis. The saliva of the tick Rhipicephalus sanguineus, the common dog tick and a vector of Rickettsia in the USA, has abundant adenosine and prostaglandin E2 in its saliva, adenosine being for the first time identified in tick saliva. Two review articles were produced by members of the section. Expertise capabilities spin off: Our bioinformatic capability lead to collaboration with diverse studies, helping annotating secreted proteins of parasitic worms;to discover novel transposable elements in Anopheles gambiae, to annotate a gene expression database from the yellow Fever mosquito, to identify genes expressed in the ovary of the Chagas disease vector Rhodnius prolixus and to identify novel proteins and peptides expressed in the semen of Aedes aegypti. A patent applied previously in 2008 was awarded last June (Valenzuela, J.G., Belkaid, Y., Kamhawi, S., Sacks, D. and Ribeiro, J.M.C. Anti-artrhpods vector vaccines, methods of selecting and uses thereof. US Department of Commerce, Patent and Trademark Office. Patent number 7,964,576 issued on June 21, 2011).
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