Mechanisms of Stage-Transcending Protection Following Immunization of Mice with Late Liver Stage-Arresting Genetically Attenuated Malaria Parasites

Malaria, caused by Plasmodium parasite infection, continues to be one of the leading causes of worldwide morbidity and mortality. Development of an effective vaccine has been encumbered by the complex life cycle of the parasite that has distinct pre-erythrocytic and erythrocytic stages of infection in the mammalian host. Historically, malaria vaccine development efforts have targeted each stage in isolation. An ideal vaccine, however, would target multiple life cycle stages with multiple arms of the immune system and be capable of eliminating initial infection in the liver, the subsequent blood stage infection, and would prevent further parasite transmission. We have previously shown that immunization of mice with Plasmodium yoelii genetically attenuated parasites (GAP) that arrest late in liver stage development elicits stage-transcending protection against both a sporozoite challenge and a direct blood stage challenge. Here, we show that this immunization strategy engenders both T- and B-cell responses that are essential for stage-transcending protection, but the relative importance of each is determined by the host genetic background. Furthermore, potent anti-blood stage antibodies elicited after GAP immunization rely heavily on F_C-mediated functions including complement fixation and F_C receptor binding. These protective antibodies recognize the merozoite surface but do not appear to recognize the immunodominant merozoite surface protein-1. The antigen(s) targeted by stage-transcending immunity are present in both the late liver stages and blood stage parasites. The data clearly show that GAP-engendered protective immune responses can target shared antigens of pre-erythrocytic and erythrocytic parasite life cycle stages. As such, this model constitutes a powerful tool to identify novel, protective and stage-transcending T and B cell targets for incorporation into a multi-stage subunit vaccine.     

Telemetric Blood Pressure Assessment in Angiotensin II-Infused ApoE-/- Mice: 28 Day Natural History and Comparison to Tail-Cuff Measurements

Abdominal aortic aneurysm (AAA) is a disease of the aortic wall, which can progress to catastrophic rupture. Assessment of mechanical characteristics of AAA, such as aortic distensibility, may provide important insights to help identify at-risk patients and understand disease progression. While the majority of studies on this topic have focused on retrospective patient data, recent studies have used mouse models of AAA to prospectively evaluate the evolution of aortic mechanics. Quantification of aortic distensibility requires accurate measurement of arterial blood pressure, particularly pulse pressure, which is challenging to perform accurately in murine models. We hypothesized that volume/pressure tail-cuff measurements of arterial pulse pressure in anesthetized mice would have sufficient accuracy to enable calculations of aortic distensibility with minimal error. Telemetry devices and osmotic mini-pumps filled with saline or angiotensin-II were surgically implanted in male apolipoprotein-E deficient (ApoE^-/-) mice. Blood pressure in the aortic arch was measured continuously via telemetry. In addition, simultaneous blood pressure measurements with a volume/pressure tail-cuff system were performed under anesthesia at specific intervals to assess agreement between techniques. Compared to controls, mice infused with angiotensin-II had an overall statistically significant increase in systolic pressure, with no overall difference in pulse pressure; however, pulse pressure did increase significantly with time. Systolic measurements agreed well between telemetry and tail-cuff (coefficient of variation = 10%), but agreement of pulse pressure was weak (20%). In fact, group-averaged pulse pressure from telemetry was a better predictor of a subject’s pulse pressure on a given day than a simultaneous tail-cuff measurement. Furthermore, these approximations introduced acceptable errors (15.1 ± 12.8%) into the calculation of aortic distensibility. Contrary to our hypothesis, we conclude that tail-cuff measures of arterial pulse pressure have limited accuracy. Future studies of aneurysm mechanics using the ApoE^-/-/angiotensin-II model would be better in assuming pulse pressure profiles consistent with our telemetry findings instead of attempting to measure pulse pressure in individual mice.     

Electron paramagnetic resonance and Mössbauer spectroscopy of intact mitochondria from respiring <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Mitochondria from respiring cells were isolated under anaerobic conditions. Microscopic images were largely devoid of contaminants, and samples consumed O₂ in an NADH-dependent manner. Protein and metal concentrations of packed mitochondria were determined, as was the percentage of external void volume. Samples were similarly packed into electron paramagnetic resonance tubes, either in the as-isolated state or after exposure to various reagents. Analyses revealed two signals originating from species that could be removed by chelation, including rhombic Fe³⁺ (g = 4.3) and aqueous Mn²⁺ ions (g = 2.00 with Mn-based hyperfine). Three S = 5/2 signals from Fe³⁺ hemes were observed, probably arising from cytochrome c peroxidase and the a₃:Cu_b site of cytochrome c oxidase. Three Fe/S-based signals were observed, with averaged g values of 1.94, 1.90 and 2.01. These probably arise, respectively, from the [Fe₂S₂]⁺ cluster of succinate dehydrogenase, the [Fe₂S₂]⁺ cluster of the Rieske protein of cytochrome bc ₁, and the [Fe₃S₄]⁺ cluster of aconitase, homoaconitase or succinate dehydrogenase. Also observed was a low-intensity isotropic g = 2.00 signal arising from organic-based radicals, and a broad signal with g _ave = 2.02. Mössbauer spectra of intact mitochondria were dominated by signals from Fe₄S₄ clusters (60–85% of Fe). The major feature in as-isolated samples, and in samples treated with ethylenebis(oxyethylenenitrilo)tetraacetic acid, dithionite or O₂, was a quadrupole doublet with ΔE _Q = 1.15 mm/s and δ = 0.45 mm/s, assigned to [Fe₄S₄]²⁺ clusters. Substantial high-spin non-heme Fe²⁺ (up to 20%) and Fe³⁺ (up to 15%) species were observed. The distribution of Fe was qualitatively similar to that suggested by the mitochondrial proteome.     

Does shrub invasion indirectly limit grass establishment via seedling herbivory? A test at grassland‐shrubland ecotones

Question: Does shrub invasion at ecotones indirectly limit grass establishment by increasing mammalian seedling herbivory? Location: Chihuahuan Desert, New Mexico, USA. Methods: We tested the hypothesis that herbivore‐related mortality of seedlings of the dominant perennial grass Bouteloua eriopoda would be highest in shrub‐dominated portions of grassland‐shrubland ecotones. We tested the hypothesis in two Chihuahuan Desert sites featuring similar shrub encroachment patterns but different shrub species, grass cover, and different abundances of small mammals. Within each site we transplanted B. eriopoda seedlings to grass‐dominated, middle, and shrub‐dominated positions of replicate ecotones during the time of year (mid‐summer) when they would naturally appear and monitored seedling fates. We estimated population size/activity of putative small mammal herbivores. Results: Seedlings were killed by mammals in greater numbers in shrubland than in grassland or middle ecotone positions at the site with large herbivore numbers. At the site with low herbivore numbers, most seedlings were killed in middle ecotone positions. The abundance patterns of herbivores did not parallel patterns of seedling herbivory across the ecotones or between sites. Conclusions: Seedling herbivory is an important process and is related to vegetation composition, but the mechanisms underlying the relationship are not clear. We speculate that variation in small mammal foraging behavior may contribute to seedling herbivory patterns. Restoration strategies in the Chihuahuan Desert need to account for the abundance and/or behavior of native herbivores.     

<Emphasis Type="Italic">Cleistocybe</Emphasis>, a new genus of Agaricales

A new clitocyboid genus of Agaricales, Cleistocybe, is described to accommodate the isolated evolutionary position of the new species Cleistocybe vernalis inferred by a previously published multigene phylogenetic study. Cleistocybe is distinguished from other clitocyboid lineages by a combination of morphological characters, including lamellae that become gray in age, an interwoven hymenophoral trama with divergent elements when young, strongly interwoven pileipellis with pigmented and encrusted hyphae, white spore deposit, a distinct or ephemeral fibrillose to submembranous partial veil, and smooth, inamyloid basidiospores that are inequilateral in profile view. Cleistocybe encompasses two species, C. vernalis and Clitocybe gomphidioides, based on morphological comparisons with C. vernalis. Clitocybe subvelosa is confirmed as conspecific with C. gomphidioides based on morphological and ITS sequence comparisons of type collections. Cleistocybe is known only from western North America in coniferous forests and appears most closely related to the ectomycorrhizal genus Catathelasma and the saprotrophic genera Callistosporium, Macrocybe, and Pleurocollybia based on nLSU-rDNA phylogenetic analysis. Together these lineages constitute the Catathelasma clade.     

Structure of acid beta-glucosidase with pharmacological chaperone provides insight into Gaucher disease

Gaucher disease results from mutations in the lysosomal enzyme acid beta-glucosidase (GCase). Although enzyme replacement therapy has improved the health of some affected individuals, such as those with the prevalent N370S mutation, oral treatment with pharmacological chaperones may be therapeutic in a wider range of tissue compartments by restoring sufficient activity of endogenous mutant GCase. Here we demonstrate that isofagomine (IFG, 1) binds to the GCase active site, and both increases GCase activity in cell lysates and restores lysosomal trafficking in cells containing N370S mutant GCase. We also compare the crystal structures of IFG-bound GCase at low pH with those of glycerol-bound GCase at low pH and apo-GCase at neutral pH. Our data indicate that IFG induces active GCase, which is secured by interactions with Asn370. The design of small molecules that stabilize substrate-bound conformations of mutant proteins may be a general therapeutic strategy for diseases caused by protein misfolding and mistrafficking.     

Human DNA ligase IV and the ligase IV/XRCC4 complex: analysis of nick ligation fidelity

In addition to linking nicked/fragmented DNA molecules back into a contiguous duplex, DNA ligases also have the capacity to influence the accuracy of DNA repair pathways via their tolerance/intolerance of nicks containing mismatched base pairs. Although human DNA ligase I (Okazaki fragment processing) and the human DNA ligase III/XRCC1 complex (general DNA repair) have been shown to be relatively intolerant of nicks containing mismatched base pairs, the human DNA ligase IV/XRCC4 complex has not been studied in this regard. Ligase IV/XRCC4 is the sole DNA ligase involved in the repair of double strand breaks (DSBs) via the non-homologous end joining (NHEJ) pathway. During the repair of DSBs generated by chemical/physical damage as well as the repair of the programmed DSB intermediates of V(D)J recombination, there are scenarios where, at least conceptually, a capacity for ligating nicks containing mismatched base pairs would appear to be advantageous. Herein we examine whether ligase IV/XRCC4 can contribute a mismatched nick ligation activity to NHEJ. Toward this end, we (i) describe an E. coli-based coexpression system that provides relatively high yields of the ligase IV/XRCC4 complex, (ii) describe a unique rate-limiting step, which has bearing on how the complex is assayed, (iii) specifically analyze how XRCC4 influences ligase IV catalysis and substrate specificity, and (iv) probe the mismatch tolerance/intolerance of DNA ligase IV/XRCC4 via quantitative in vitro kinetic analyses. Analogous to most other DNA ligases, ligase IV/XRCC4 is shown to be fairly intolerant of nicks containing mismatched base pairs. These results are discussed in light of the biological roles of NHEJ.