Identification, characterization, and expression of a unique secretory lipase from the human pathogen Leishmania donovani

Lipases have been implicated to be of importance in the life cycle development, virulence, and transmission of a variety of parasitic organisms. Potential functions include the acquisition of host resources for energy metabolism and as simple building blocks for the synthesis of complex parasite lipids important for membrane remodeling and structural purposes. Using a molecular approach, we identified and characterized the structure of an LdLip3-lipase gene from the primitive trypanosomatid pathogen of humans, Leishmania donovani. The LdLip3 encodes a ~33 kDa protein, with a well-conserved substrate-binding and catalytic domains characteristic of members of the serine lipase-protein family. Further, we showed that LdLip3 mRNA is constitutively expressed by both the insect vector (i.e., promastigote) and mammalian (i.e., amastigote) life cycle developmental forms of this protozoan parasite. Moreover, a homologous episomal expression system was used to express an HA epitope-tagged LdLip3 chimeric construct (LdLip3::HA) in these parasites. Expression of the LdLip3 chimera was verified in these transfectants by Western blots and indirect immuno-fluorescence analyses. Results of coupled immuno-affinity purification and enzyme activity experiments demonstrated that the LdLip3::HA chimeric protein was secreted/released by transfected L. donovani parasites and that it possessed functional lipase enzyme activity. Taken together these observations suggest that this novel secretory lipase might play essential role(s) in the survival, growth, and development of this important group of human pathogens.     

Relationships between uterine culture, cytology and pregnancy rates in a Thoroughbred practice

Endometrial cytology and culture specimens (n=2123) were collected concurrently with a guarded uterine culture instrument from 970 mares (738 barren, 1230 foaling and 155 maiden mares) during three breeding seasons (2001-2004). Results were compared to the 28-d pregnancy rate for the cycle from which the samples were taken. Cytological smears were evaluated for inflammation at x100 and graded as: not inflammatory (0-2 neutrophils/field), moderate inflammation (2-5 neutrophils/field), severe inflammation (>5 neutrophils/field), or hypocellular (scant epithelial cells and no neutrophils). Uterine culture swabs were plated within 6h, incubated for 72 h and results determined at 24, 48, and 72 h. Approximately, 20% (n=423) cytology samples were positive for inflammation (>2 neutrophils), whereas approximately 11% (n=231) of cultures had microorganisms recovered. A majority (64%) of the positive cultures (147/231) had inflammation on cytology smears. Streptococcus equi subsp. zooepidemicus was associated with more positive cytology results than coliforms (P<0.01). Mares with positive cytology or culture had lower pregnancy rates than mares with normal findings (P<0.01). Lowest pregnancy rates were recorded for mares with severe endometrial inflammation (21%, versus moderate inflammation 48%). Isolation of a microorganism from mares with endometrial inflammation was not associated with a further reduction in pregnancy rates. In barren, foaling and maiden mares, cytology was positive in 28, 17, and 5%, respectively, and culture was positive in 12.2, 11.1, and 3.2%. Foaling and maiden mares had higher pregnancy rates than barren mares (62, 69, and 44%, respectively, P<0.001). In conclusion, a positive cytology was twice as common as a positive culture, and isolation of microorganisms was associated with reduced pregnancy rates, even in the apparent absence of inflammation.     

Influence of language and ancestry on genetic structure of contiguous populations: A microsatellite based study on populations of Orissa

Background

Although cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the severity of disease is highly variable indicating the influence of modifier genes. The intestines of Cftr deficient mice (CF mice: Cftr ^tm1Unc ) are prone to obstruction by excessive mucus accumulation and are used as a model of meconium ileus and distal intestinal obstruction syndrome. This phenotype is strongly dependent on the genetic background of the mice. On the C57Bl/6 background, the majority of CF mice cannot survive on solid mouse chow, have inflammation of the small intestine, and are about 30% smaller than wild type littermates. In this work potential modifier loci of the CF intestinal phenotype were identified.

Results

CF mice on a mixed genetic background (95% C57Bl/6 and 5% 129Sv) were compared to CF mice congenic on the C57Bl/6 background for several parameters of the intestinal CF phenotype. CF mice on the mixed background exhibit significantly greater survival when fed dry mouse chow, have reduced intestinal inflammation as measured by quantitative RT-PCR for marker genes, have near normal body weight gain, and have reduced mucus accumulation in the intestinal crypts. There was an indication of a gender effect for body weight gain: males did not show a significant improvement at 4 weeks of age, but were of normal weight at 8 weeks, while females showed improvement at both 4 and 8 weeks. By a preliminary genome-wide PCR allele scanning, three regions were found to be potentially associated with the milder phenotype. One on chr.1, defined by marker D1Mit36, one on chr. 9 defined by marker D9Mit90, and one on chr. 10, defined by marker D10Mit14.

Conclusion

Potential modifier regions were found that have a positive impact on the inflammatory phenotype of the CF mouse small intestine and animal survival. Identification of polymorphisms in specific genes in these regions should provide important new information about genetic modifiers of the CF intestinal phenotype.     

Robustness and fragility in immunosenescence

We construct a model to study tradeoffs associated with aging in the adaptive immune system, focusing on cumulative effects of replacing naive cells with memory cells. Binding affinities are characterized by a stochastic shape space model. System loss arising from an individual infection is associated with disease severity, as measured by the total antigen population over the course of an infection. We monitor evolution of cell populations on the shape space over a string of infections, and find that the distribution of losses becomes increasingly heavy-tailed with time. Initially this lowers the average loss: the memory cell population becomes tuned to the history of past exposures, reducing the loss of the system when subjected to a second, similar infection. This is accompanied by a corresponding increase in vulnerability to novel infections, which ultimately causes the expected loss to increase due to overspecialization, leading to increasing fragility with age (i.e., immunosenescence). In our model, immunosenescence is not the result of a performance degradation of some specific lymphocyte, but rather a natural consequence of the built-in mechanisms for system adaptation. This “robust, yet fragile” behavior is a key signature of Highly Optimized Tolerance.     

Arbuscular mycorrhizal fungi associated with Populus-Salix stands in a semiarid riparian ecosystem

This study examined the activity, species richness, and species composition of the arbuscular mycorrhizal fungal (AMF) community of Populus-Salix stands on the Verde River (Arizona, USA), quantified patterns of AMF richness and colonization along complex floodplain gradients, and identified environmental variables responsible for structuring the AMF community. Samples from 61 Populus-Salix stands were analyzed for AMF and herbaceous composition, AMF colonization, gravimetric soil moisture, soil texture, per cent organic matter, pH, and concentrations of nitrate, bicarbonate phosphorus and exchangeable potassium. AMF species richness declined with stand age and distance from and elevation above the channel and was positively related to perennial species cover and richness and gravimetric soil moisture. Distance from and elevation above the active channel, forest age, annual species cover, perennial species richness, and exchangeable potassium concentration all played a role in structuring the AMF community in this riparian area. Most AMF species were found across a wide range of soil conditions, but a subset of species tended to occur more often in hydric areas. This group of riparian affiliate AMF species includes several not previously encountered in the surrounding Sonoran desert.     

Multitaxonomic diversity patterns along a desert riparian-upland gradient

Riparian areas are noted for their high biodiversity, but this has rarely been tested across a wide range of taxonomic groups. We set out to describe species richness, species abundance, and community similarity patterns for 11 taxonomic groups (forbs & grasses, shrubs, trees, solpugids, spiders, scarab beetles, butterflies, lizards, birds, rodents, and mammalian carnivores) individually and for all groups combined along a riparian-upland gradient in semiarid southeastern Arizona, USA. Additionally, we assessed whether biological characteristics could explain variation in diversity along the gradient using five traits (trophic level, body size, life span, thermoregulatory mechanism, and taxonomic affiliation). At the level of individual groups diversity patterns varied along the gradient, with some having greater richness and/or abundance in riparian zones whereas others were more diverse and/or abundant in upland zones. Across all taxa combined, riparian zones contained significantly more species than the uplands. Community similarity between riparian and upland zones was low, and beta diversity was significantly greater than expected for most taxonomic groups, though biological traits explained little variance in diversity along the gradient. These results indicate heterogeneity amongst taxa in how they respond to the factors that structure ecological communities in riparian landscapes. Nevertheless, across taxonomic groups the overall pattern is one of greater species richness and abundance in riparian zones, coupled with a distinct suite of species.     

Visualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors

Glutamine plays a central role in the metabolism of critical biological molecules such as amino acids, proteins, neurotransmitters, and glutathione. Since glutamine metabolism is regulated through multiple enzymes and transporters, the cellular glutamine concentration is expected to be temporally dynamic. Moreover, differentiation in glutamine metabolism between cell types in the same tissue (e.g. neuronal and glial cells) is often crucial for the proper function of the tissue as a whole, yet assessing cell-type specific activities of transporters and enzymes in such heterogenic tissue by physical fractionation is extremely challenging. Therefore, a method of reporting glutamine dynamics at the cellular level is highly desirable. Genetically encoded sensors can be targeted to a specific cell type, hence addressing this knowledge gap. Here we report the development of F?ster Resonance Energy Transfer (FRET) glutamine sensors based on improved cyan and yellow fluorescent proteins, monomeric Teal Fluorescent Protein (mTFP)1 and venus. These sensors were found to be specific to glutamine, and stable to pH-changes within a physiological range. Using cos7 cells expressing the human glutamine transporter ASCT2 as a model, we demonstrate that the properties of the glutamine transporter can easily be analyzed with these sensors. The range of glutamine concentration change in a given cell can also be estimated using sensors with different affinities. Moreover, the mTFP1-venus FRET pair can be duplexed with another FRET pair, mAmetrine and tdTomato, opening up the possibility for real-time imaging of another molecule. These novel glutamine sensors will be useful tools to analyze specificities of glutamine metabolism at the single-cell level.     

On the Role of CD8?T Cells in the Control of Persistent Infections

The control of pathogen density during infections is typically assumed to be the result of a combination of resource limitation (loss of target cells that the pathogen can infect), innate immunity, and specific immunity. The contributions of these factors have been considered in acute infections, which are characterized by having a short duration. What controls the pathogen during persistent infections is less clear, and is complicated by two factors. First, specific immune responses become exhausted if they are subject to chronic stimulation. Exhaustion has been best characterized for CD8 T cell responses, and occurs through a combination of cell death and loss of functionality of surviving cells. Second, new nonexhausted T cells can immigrate from the thymus during the infection, and may play a role in the control of the infection. In this article, we formulate a partial-differential-equation model to describe the interaction between these processes, and use this model to explore how thymic influx and exhaustion might affect the ability of CD8 T cell responses to control persistent infections. We find that although thymic influx can play a critical role in the maintenance of a limited CD8 T cell response during persistent infections, this response is not sufficiently large to play a significant role in controlling the infection. In doing so, our results highlight the importance of resource limitation and innate immunity in the control of persistent infections.     

Phylogeny of the M superhaplogroup inferred from complete mitochondrial genome sequence of Indian specific lineages

Background  

Phylogenetic analysis of human complete mitochondrial DNA sequences has largely contributed to resolving phylogenies and antiquity of different lineages belonging to the majorhaplogroups L, N and M (East-Asian lineages). In the absence of whole mtDNA sequence information of M lineages reported in India that exhibits highest diversity within the sub-continent, the present study was undertaken to provide a detailed analysis of this haplogroup to precisely characterize the lineages and unravel their intricate phylogeny.