Immunization with centrin-Deficient Leishmania braziliensis Does Not Protect against Homologous Challenge.

Immunization with various Leishmania species lacking centrin induces robust immunity against a homologous and heterologous virulent challenge, making centrin mutants a putative candidate for a leishmaniasis vaccine. Centrin is a calcium-binding cytoskeletal protein involved in centrosome duplication in higher eukaryotes and Leishmania spp. lacking centrin are unable to replicate in vivo and are non-pathogenic. We developed a centrin-deficient Leishmania braziliensis (LbCen-/-) cell line and confirmed its impaired survival following phagocytosis by macrophages. Upon experimental inoculation into BALB/c mice, LbCen-/- failed to induce lesions and parasites were rapidly eliminated. The immune response following inoculation with LbCen-/- was characterized by a mixed IFN-γ, IL-4, and IL-10 response and did not confer protection against L. braziliensis infection, distinct from L. major, L. donovani, and L mexicana centrin-deficient mutants. A prime-boost strategy also did not lead to a protective immune response against homologous challenge. On the contrary, immunization with centrin-deficient L. donovani (LdonCen-/-) cross-protected against L. braziliensis challenge, illustrating the ability of LdonCen-/- to induce the Th1-dominant protective immunity needed for leishmaniasis control. In conclusion, while centrin deficiency in L. braziliensis causes attenuation of virulence, and disrupts the ability to cause disease, it fails to stimulate a protective immune response.

Global insights into the fine tuning of human A2AAR conformational dynamics in a ternary complex with an engineered G protein viewed by NMR.

G protein-coupled receptor (GPCR) conformational plasticity enables formation of ternary signaling complexes with intracellular proteins in response to binding extracellular ligands. We investigate the dynamic process of GPCR complex formation in solution with the human A2A adenosine receptor (A2AAR) and an engineered Gs protein, mini-Gs. 2D nuclear magnetic resonance (NMR) data with uniform stable isotope-labeled A2AAR enabled a global comparison of A2AAR conformations between complexes with an agonist and mini-Gs and with an agonist alone. The two conformations are similar and show subtle differences at the receptor intracellular surface, supporting a model whereby agonist binding alone is sufficient to populate a conformation resembling the active state. However, an A2AAR "hot spot" connecting the extracellular ligand-binding pocket to the intracellular surface is observed to be highly dynamic in the ternary complex, suggesting a mechanism for allosteric connection between the bound G protein and the drug-binding pocket involving structural plasticity of the "toggle switch" tryptophan.

Behavioral thermoregulation in the fasted C57BL/6 mouse.

Under relatively cool ambient temperatures and a caloric deficit, mice will undergo daily torpor - a short-term regulated reduction in metabolic rate with a concomitant drop in body temperature. Mice can alternatively achieve metabolic savings by utilizing behavioral changes, such as seeking a warmer environment. However, there is a lack of knowledge about the behavioral interaction between torpor utilization and thermotaxis. That is, if a fasted mouse is faced with a choice between a warm environment not conducive for torpor, and a cool environment that will induce torpor, which scenario will the fasting mouse choose? Here, the temperature preferences of fasted mice were studied using a temperature gradient device that allows a mouse to freely move along a gradient of temperatures. C57BL/6 mice were implanted with temperature telemeters that recorded location, core temperature (Tb), and activity concurrently over a 23-h period in the thermal gradient. When the gradient was on, mice preferred the warm end of the gradient when fed (71 ± 4% of the time) and even more so when fasted (84 ± 2%). When the gradient was on, the fasted minimum Tb was significantly higher (34.4 ± 0.3 °C) than when the gradient was off (27.7 ± 1.6 °C). Further, fasted mice lost significantly more weight when the gradient was off despite maintenance of a metabolically favorable lower minimum Tb in this condition. These results indicate that fasted mice not only prefer warm ambient temperatures when given the choice, but that it is also the pathway with more favorable metabolic outcomes in a period of reduced caloric intake.