Identification of biochemical and putative biological role of a xenolog from Escherichia coli using structural analysis

YagE is a 33 kDa prophage protein encoded by CP4-6 prophage element in Escherichia coli K12 genome. Here, we report the structures of YagE complexes with pyruvate (PDB Id 3N2X) and KDGal (2-keto-3-deoxy galactonate) (PDB Id 3NEV) at 2.2A resolution. Pyruvate depletion assay in presence of glyceraldehyde shows that YagE catalyses the aldol condensation of pyruvate and glyceraldehyde. Our results indicate that the biochemical function of YagE is that of a 2-keto-3-deoxy gluconate (KDG) aldolase. Interestingly, E. coli K12 genome lacks an intrinsic KDG aldolase. Moreover, the over-expression of YagE increases cell viability in the presence of certain bactericidal antibiotics, indicating a putative biological role of YagE as a prophage encoded virulence factor enabling the survival of bacteria in the presence of certain antibiotics. The analysis implies a possible mechanism of antibiotic resistance conferred by the over-expression of prophage encoded YagE to E. coli.     

P2-P1' macrocyclization of P2 phenylglycine based HCV NS3 protease inhibitors using ring-closing metathesis

Macrocyclization is a commonly used strategy to preorganize HCV NS3 protease inhibitors in their bioactive conformation. Moreover, macrocyclization generally leads to greater stability and improved pharmacokinetic properties. In HCV NS3 protease inhibitors, it has been shown to be beneficial to include a vinylated phenylglycine in the P2 position in combination with alkenylic P1' substituents. A series of 14-, 15- and 16-membered macrocyclic HCV NS3 protease inhibitors with the linker connecting the P2 phenylglycine and the alkenylic P1' were synthesized by ring-closing metathesis, using both microwave and conventional heating. Besides formation of the expected macrocycles in cis and trans configuration as major products, both ring-contracted and double-bond migrated isomers were obtained, in particular during formation of the smaller rings (14- and 15-membered rings). All inhibitors had K(i)-values in the nanomolar range, but only one inhibitor type was improved by rigidification. The loss in inhibitory effect can be attributed to a disruption of the beneficial π-π interaction between the P2 fragment and H57, which proved to be especially deleterious for the d-phenylglycine epimers.     

Abundance of West Nile virus mosquito vectors in relation to climate and landscape variables

It is currently unclear if the potential for West Nile virus transmission by mosquito vectors in the eastern United States is related to landscape or climate factors or both. We compared abundance of vector species between urban and suburban neighborhoods of Henrico County, VA, in relation to the following factors: temperature, precipitation, canopy cover, building footprint, and proximity to drainage infrastructure. Mosquitoes were collected throughout the 2005, 2006, and 2007 seasons and tested for West Nile virus (WNV) in pools of 10–50. Test results of mosquito pools were compared to average site abundance from 37 sites in Henrico County, VA; abundance was then examined in relation to ecological variables. Urban infrastructure was positively correlated with the abundance of Culex pipiens L./Cx. restuans, and our findings implicate combined sewer overflow systems as large contributors to Culex vector populations. No measure of urbanization examined in our study was correlated with Aedes albopictus abundance. Our study showed that certain landscape variables identified using Geographic Information Systems are valuable for predicting primary WNV vector abundance in Virginia, and that temperature along with low precipitation are strong predictors of population growth. Our results support other regional studies that found WNV proliferates under drought conditions.     

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens^{1, 2}. The innate immune system of mosquitoes consists of several lines of defense ³. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut ⁴ enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens ^{5, 6}. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages ⁷. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis ⁸, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen ^{9, 10}. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively ¹¹. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues ¹¹. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.Download video file.^{(54M, mov)}     

Latency antigen α-crystallin based vaccination imparts a robust protection against TB by modulating the dynamics of pulmonary cytokines

Background

Efficient control of tuberculosis (TB) requires development of strategies that can enhance efficacy of the existing vaccine Mycobacterium bovis Bacille Calmette Guerin (BCG). To date only a few studies have explored the potential of latency-associated antigens to augment the immunogenicity of BCG.

Methods/Principal Findings

We evaluated the protective efficacy of a heterologous prime boost approach based on recombinant BCG and DNA vaccines targeting α-crystallin, a prominent latency antigen. We show that “rBCG prime - DNA boost” strategy (R/D) confers a markedly superior protection along with reduced pathology in comparison to BCG vaccination in guinea pigs (565 fold and 45 fold reduced CFU in lungs and spleen, respectively, in comparison to BCG vaccination). In addition, R/D regimen also confers enhanced protection in mice. Our results in guinea pig model show a distinct association of enhanced protection with an increased level of interleukin (IL)12 and a simultaneous increase in immuno-regulatory cytokines such as transforming growth factor (TGF)β and IL10 in lungs. The T cell effector functions, which could not be measured in guinea pigs due to technical limitations, were characterized in mice by multi-parameter flow cytometry. We show that R/D regimen elicits a heightened multi-functional CD4 Th1 cell response leading to enhanced protection.

Conclusions/Significance

These results clearly indicate the superiority of α-crystallin based R/D regimen over BCG. Our observations from guinea pig studies indicate a crucial role of IL12, IL10 and TGFβ in vaccine-induced protection. Further, characterization of T cell responses in mice demonstrates that protection against TB is predictable by the frequency of CD4 T cells simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and IL2. We anticipate that this study will not only contribute toward the development of a superior alternative to BCG, but will also stimulate designing of TB vaccines based on latency antigens.     

Enhanced striatal dopamine release during food stimulation in binge eating disorder

Subjects with binge eating disorder (BED) regularly consume large amounts of food in short time periods. The neurobiology of BED is poorly understood. Brain dopamine, which regulates motivation for food intake, is likely to be involved. We assessed the involvement of brain dopamine in the motivation for food consumption in binge eaters. Positron emission tomography (PET) scans with [(11)C]raclopride were done in 10 obese BED and 8 obese subjects without BED. Changes in extracellular dopamine in the striatum in response to food stimulation in food-deprived subjects were evaluated after placebo and after oral methylphenidate (MPH), a drug that blocks the dopamine reuptake transporter and thus amplifies dopamine signals. Neither the neutral stimuli (with or without MPH) nor the food stimuli when given with placebo increased extracellular dopamine. The food stimuli when given with MPH significantly increased dopamine in the caudate and putamen in the binge eaters but not in the nonbinge eaters. Dopamine increases in the caudate were significantly correlated with the binge eating scores but not with BMI. These results identify dopamine neurotransmission in the caudate as being of relevance to the neurobiology of BED. The lack of correlation between BMI and dopamine changes suggests that dopamine release per se does not predict BMI within a group of obese individuals but that it predicts binge eating.     

Resistance to friend virus-induced erythroleukemia in W/W(v) mice is caused by a spleen-specific defect which results in a severe reduction in target cells and a lack of Sf-Stk expression

The characteristic progression and specificity of Friend virus for the erythroid lineage have allowed for the identification of a number of host-encoded loci that are required for disease progression. Several of these loci, including the Friend virus susceptibility gene 2 (Fv2), dominant white spotting gene (W), and Steel gene (Sl), regulate the initial polyclonal expansion of infected erythroid progenitor cells. W and Sl encode the Kit receptor tyrosine kinase and its ligand, stem cell factor, respectively. W mutant mice are severely anemic, and earlier work suggested that this defect in erythroid differentiation is the cause for the resistance to Friend virus-induced erythroleukemia. Here we show that in bone marrow, W/W(v) mice have near normal numbers of target cells and the initial infection of bone marrow occurs normally in vivo. In contrast, spleen cells from W/W(v) mice infected both in vitro and in vivo with Friend virus failed to give rise to erythropoietin-independent colonies at any time following Friend virus infection, suggesting that mutation of the Kit receptor specifically affects target cells in the spleen, rendering the mutant mice resistant to the development of Friend virus-induced erythroleukemia. In addition, we show that the Kit+ pathogenic targets of Friend virus in the spleen are distinct from the pathogenic targets in bone marrow and this population of spleen target cells is markedly decreased in W/W(v) mice and these cells fail to express Sf-Stk. These results also underscore the unique nature of the spleen microenvironment in its role in supporting the progression of acute leukemia in Friend virus-infected mice.