Identification and Characterization of a Leucine-Rich Repeat Kinase 2 (LRRK2) Consensus Phosphorylation Motif

Mutations in LRRK2 (leucine-rich repeat kinase 2) have been identified as major genetic determinants of Parkinson's disease (PD). The most prevalent mutation, G2019S, increases LRRK2's kinase activity, therefore understanding the sites and substrates that LRRK2 phosphorylates is critical to understanding its role in disease aetiology. Since the physiological substrates of this kinase are unknown, we set out to reveal potential targets of LRRK2 G2019S by identifying its favored phosphorylation motif. A non-biased screen of an oriented peptide library elucidated F/Y-x-T-x-R/K as the core dependent substrate sequence. Bioinformatic analysis of the consensus phosphorylation motif identified several novel candidate substrates that potentially function in neuronal pathophysiology. Peptides corresponding to the most PD relevant proteins were efficiently phosphorylated by LRRK2 in vitro. Interestingly, the phosphomotif was also identified within LRRK2 itself. Autophosphorylation was detected by mass spectrometry and biochemical means at the only F-x-T-x-R site (Thr 1410) within LRRK2. The relevance of this site was assessed by measuring effects of mutations on autophosphorylation, kinase activity, GTP binding, GTP hydrolysis, and LRRK2 multimerization. These studies indicate that modification of Thr1410 subtly regulates GTP hydrolysis by LRRK2, but with minimal effects on other parameters measured. Together the identification of LRRK2's phosphorylation consensus motif, and the functional consequences of its phosphorylation, provide insights into downstream LRRK2-signaling pathways

Ecological and Control Techniques for Sand Flies (Diptera: Psychodidae) Associated with Rodent Reservoirs of Leishmaniasis

Background:Leishmaniasis remains a global health problem because of the substantial holes that remain in our understanding of sand fly ecology and the failure of traditional vector control methods. The specific larval food source is unknown for all but a few sand fly species, and this is particularly true for the vectors of Leishmania parasites. We provide methods and materials that could be used to understand, and ultimately break, the transmission cycle of zoonotic cutaneous leishmaniasis.Methods and Findings:We demonstrated in laboratory studies that analysis of the stable carbon and nitrogen isotopes found naturally in plant and animal tissues was highly effective for linking adult sand flies with their larval diet, without having to locate or capture the sand fly larvae themselves. In a field trial, we also demonstrated using this technique that half of captured adult sand flies had fed as larvae on rodent feces. Through the identification of rodent feces as a sand fly larval habitat, we now know that rodent baits containing insecticides that have been shown in previous studies to pass into the rodents\u27 feces and kill sand fly larvae also could play a future role in sand fly control. In a second study we showed that rubidium incorporated into rodent baits could be used to demonstrate the level of bloodfeeding by sand flies on baited rodents, and that the elimination of sand flies that feed on rodents can be achieved using baits containing an insecticide that circulates in the blood of baited rodents.Conclusions:Combined, the techniques described could help to identify larval food sources of other important vectors of the protozoa that cause visceral or dermal leishmaniasis. Unveiling aspects of the life cycles of sand flies that could be targeted with insecticides would guide future sand fly control programs for prevention of leishmaniasis

Arthropod Surveillance Programs: Basic Components, Strategies, and Analysis

Job file for the creation/design of stained glass from either the Charles J. Connick Studio (1912-1945) or the Charles J. Connick Associates studio (1945-1986). The job file contains a job number, location information, date of completion, size, contact information, price, and a description of the project. This particular job file contains information on a job located at: Holyoke, Massachusetts. Second Congregational Church

Diagram of the use of stable isotope analysis to link the plants used for forage by jirds to the feces produced by these jirds and to sand flies that had fed on these feces as larvae.

<p>The δ¹³C and δ¹⁵N values of 100.0% of sand flies fed jird feces as larvae in the lab and 50.0% of sand flies captured at the study site matched the field collected jird feces. Stable isotope analysis also indicated the remaining 50.0% of sand flies captured at the study site had fed as larvae on one of two distinct alternative (and currently unknown) food sources other than jird feces.</p

Stable carbon and nitrogen isotope ratios (δ¹³C and δ¹⁵N) of feces of wild jirds, tissue of the plant fed on by jirds, and individual wild adult sand flies that had been collected near jird burrows or reared in the laboratory as larvae on field-collected jird feces.

<p>Hierarchical cluster analysis identified 3 significantly different groups of samples based on δ¹³C and δ¹⁵N values. The cluster circled in red contained feces of wild jirds, tissue of the plant fed on by jirds, individual sand flies reared in the laboratory as larvae on field-collected jird feces, and 50.0% of the sand flies captured near the jird burrows. The clusters circled in blue and yellow contained 36.4% and 13.6% of the sand flies captured near the jird burrows, respectively.</p

Shaw's jird feeding on bait that had been placed at a study site.

<p>Shaw's jird feeding on bait that had been placed at a study site.</p

The mean number of females of <i>P.</i><i>papatasi</i> captured per night and the mean number of sand flies that was positive for the presence of rubidium at sites treated with rodent bait containing rubidium plus ivermectin or rubidium alone.

<p>The mean number of females of <i>P.</i><i>papatasi</i> captured per night and the mean number of sand flies that was positive for the presence of rubidium at sites treated with rodent bait containing rubidium plus ivermectin or rubidium alone.</p