Shotgun proteomic profiling of five species of New Zealand Pachycladon

The genus Pachycladon consists of ten species of alpine plants, nine of which are endemic to New Zealand. The species are closely related to the model plant Arabidopsis thaliana with respect to their sequence divergence and chromosome synteny, occupy distinct geographical habitats in terms of both latitude and altitude, and display a range of morphologies. We have performed label‐free quantitative shotgun proteomic analysis of five different species of Pachycladon, namely P. cheesemanii (CH), P. exile (EX), P. fastigiatum (FA), P. enysii (EN) and P. novae‐zelandiae (NZ). The total non‐redundant data set for all five species contained 1489 proteins. The numbers of proteins identified reproducibly in each species ranged from 629 for CH to 987 for NZ, with 681 for EN, 741 for EX and 934 for FA. Previous metabolite‐based studies have shown that FA hydrolyzes glucosinolates completely to isothiocyanates while EN converts glucosinolates to nitriles. In this study, we observed high expression of ESP (At1g54040, epithiospecifying senescence regulator protein) and myrosinase 2 (At5g25980, glycosyl hydrolase family protein), which result in production of nitriles and epithionitriles, in EN and NZ, and we also observed higher expression of ESM1 (At3g14210, GDSL esterase/lipase), which mediates the formation of isothiocyanate, in FA.     

A polymorphism in the splice donor site of ZNF419 results in the novel renal cell carcinoma-associated minor histocompatibility antigen ZAPHIR

Nonmyeloablative allogeneic stem cell transplantation (SCT) can induce remission in patients with renal cell carcinoma (RCC), but this graft-versus-tumor (GVT) effect is often accompanied by graft-versus-host disease (GVHD). Here, we evaluated minor histocompatibility antigen (MiHA)-specific T cell responses in two patients with metastatic RCC who were treated with reduced-intensity conditioning SCT followed by donor lymphocyte infusion (DLI). One patient had stable disease and emergence of SMCY.A2-specific CD8+ T cells was observed after DLI with the potential of targeting SMCY-expressing RCC tumor cells. The second patient experienced partial regression of lung metastases from whom we isolated a MiHA-specific CTL clone with the capability of targeting RCC cell lines. Whole genome association scanning revealed that this CTL recognizes a novel HLA-B7-restricted MiHA, designated ZAPHIR, resulting from a polymorphism in the splice donor site of the ZNF419 gene. Tetramer analysis showed that emergence of ZAPHIR-specific CD8+ T cells in peripheral blood occurred in the absence of GVHD. Furthermore, the expression of ZAPHIR in solid tumor cell lines indicates the involvement of ZAPHIR-specific CD8+ T cell responses in selective GVT immunity. These findings illustrate that the ZNF419-encoded MiHA ZAPHIR is an attractive target for specific immunotherapy after allogeneic SCT.     

A call for standardized naming and reporting of human ESC and iPSC lines

Human embryonic and induced pluripotent stem cell lines are being generated at a rapid pace and now number in the thousands. We propose a standard nomenclature and suggest the use of a centralized database for all cell line names and a minimum set of information for reporting new derivations.     

Increased diacylglycerols characterize hepatic lipid changes in progression of human nonalcoholic fatty liver disease; comparison to a murine model

Background and Aims

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and progression to cirrhosis. While differences in liver lipids between disease states have been reported, precise composition of phospholipids and diacylglycerols (DAG) at a lipid species level has not been previously described. The goal of this study was to characterize changes in lipid species through progression of human NAFLD using advanced lipidomic technology and compare this with a murine model of early and advanced NAFLD.

Methods

Utilizing mass spectrometry lipidomics, over 250 phospholipid and diacylglycerol species (DAGs) were identified in normal and diseased human and murine liver extracts.

Results

Significant differences between phospholipid composition of normal and diseased livers were demonstrated, notably among DAG species, consistent with previous reports that DAG transferases are involved in the progression of NAFLD and liver fibrosis. In addition, a novel phospholipid species (ether linked phosphatidylinositol) was identified in human cirrhotic liver extracts.

Conclusions

Using parallel lipidomics analysis of murine and human liver tissues it was determined that mice maintained on a high-fat diet provide a reproducible model of NAFLD in regards to specificity of lipid species in the liver. These studies demonstrated that novel lipid species may serve as markers of advanced liver disease and importantly, marked increases in DAG species are a hallmark of NAFLD. Elevated DAGs may contribute to altered triglyceride, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) levels characteristic of the disease and specific DAG species might be important lipid signaling molecules in the progression of NAFLD.     

Deletion of UCP2 in iNOS deficient mice reduces the severity of the disease during experimental autoimmune encephalomyelitis

Uncoupling protein 2 is a member of the mitochondrial anion carrier family that is widely expressed in neurons and the immune cells of humans. Deletion of Ucp2 gene in mice pre-activates the immune system leading to higher resistance toward infection and to an increased susceptibility to develop chronic inflammatory diseases as previously exemplified with the Experimental Autoimmune Encephalomyelitis (EAE), a mouse model for multiple sclerosis. Given that oxidative stress is enhanced in Ucp2-/- mice and that nitric oxide (NO) also plays a critical function in redox balance and in chronic inflammation, we generated mice deficient for both Ucp2 and iNos genes and submitted them to EAE. Mice lacking iNos gene exhibited the highest clinical score (3.4+/-0.5 p<0.05). Surprisingly, mice deficient for both genes developed milder disease with reduced immune cell infiltration, cytokines and ROS production as compared to iNos-/- mice.     

Opposing roles for CD34 in B16 melanoma tumor growth alter early stage vasculature and late stage immune cell infiltration

Tumor growth and metastasis are determined by the complex interplay of factors, including those intrinsic to tumor cells and extrinsic factors associated with the tumor microenvironment. Our previous work demonstrated key roles for CD34 in the maintenance of vascular integrity and eosinophil and mast cell homing. Since both of these functions affect tumor development, we characterized the effect of CD34 ablation on tumor growth using the B16F1 melanoma model. Intriguingly, we found that CD34 plays a biphasic role in tumor progression. In early growth, both subcutaneous-injected tumors and intravenous-injected lung metastases grew more slowly in Cd34(-/-) mice. This correlated with abnormal vessel morphology and increased vascular permeability in these mice. Bone marrow transplantation experiments confirmed that this reflects a non-hematopoietic function of CD34. At later stages, subcutaneous tumor growth was accelerated in Cd34(-/-) mice and surpassed growth in wildtype mice. Bone marrow chimera experiments demonstrated this difference was due to a hematopoietic function for CD34 and, correspondingly we found reduced intra-tumor mast cell numbers in Cd34(-/-) mice. In aggregate, our analysis reveals a novel role for CD34 in both early and late tumor growth and provides novel insights into the role of the tumor microenvironment in tumor progression.     

Delineation of stage specific expression of Plasmodium falciparum EBA-175 by biologically functional region II monoclonal antibodies

Background

The malaria parasite Plasmodium falciparum EBA-175 binds its receptor sialic acids on glycophorin A when invading erythrocytes. The receptor-binding region (RII) contains two cysteine-rich domains with similar cysteine motifs (F1 and F2). Functional relationships between F1 and F2 domains and characterization of EBA-175 were studied using specific monoclonal antibodies (mAbs) against these domains.

Methods and Findings

Five mAbs specific for F1 or F2 were generated. Three mAbs specific for F2 potently blocked binding of EBA-175 to erythrocytes, and merozoite invasion of erythrocytes (IC₅₀ 10 to 100 µg/ml IgG in growth inhibition assays). A mAb specific for F1 blocked EBA-175 binding and merozoite invasion less effectively. The difference observed between the IC₅₀ of F1 and F2 mAbs was not due to differing association and disassociation rates as determined by surface plasmon resonance. Four of the mAbs recognized conformation-dependent epitopes within F1 or F2. Used in combination, F1 and F2 mAbs blocked the binding of native EBA-175 to erythrocytes and inhibited parasite invasion synergistically in vitro. MAb R217, the most potent, did not recognize sporozoites, 3-day hepatocyte stage parasites, nor rings, trophozoites, gametocytes, retorts, ookinetes, and oocysts but recognized 6-day hepatocyte stage parasites, and schizonts. Even though efficient at blocking binding to erythrocytes and inhibiting invasion into erythrocytes, MAb R217 did not inhibit sporozoite invasion and development in hepatocytes in vitro.

Conclusions

The role of the F1 and F2 domains in erythrocyte invasion and binding was elucidated with mAbs. These mAbs interfere with native EBA-175 binding to erythrocyte in a synergistic fashion. The stage specific expression of EBA-175 showed that the primary focus of activity was the merozoite stage. A recombinant RII protein vaccine consisting of both F1 and F2 domains that could induce synergistic activity should be optimal for induction of antibody responses that interfere with merozoite invasion of erythrocytes.     

Cost-effectiveness of 2009 pandemic influenza A(H1N1) vaccination in the United States

Background

Pandemic influenza A(H1N1) (pH1N1) was first identified in North America in April 2009. Vaccination against pH1N1 commenced in the U.S. in October 2009 and continued through January 2010. The objective of this study was to evaluate the cost-effectiveness of pH1N1 vaccination.

Methodology

A computer simulation model was developed to predict costs and health outcomes for a pH1N1 vaccination program using inactivated vaccine compared to no vaccination. Probabilities, costs and quality-of-life weights were derived from emerging primary data on pH1N1 infections in the US, published and unpublished data for seasonal and pH1N1 illnesses, supplemented by expert opinion. The modeled target population included hypothetical cohorts of persons aged 6 months and older stratified by age and risk. The analysis used a one-year time horizon for most endpoints but also includes longer-term costs and consequences of long-term sequelae deaths. A societal perspective was used. Indirect effects (i.e., herd effects) were not included in the primary analysis. The main endpoint was the incremental cost-effectiveness ratio in dollars per quality-adjusted life year (QALY) gained. Sensitivity analyses were conducted.

Results

For vaccination initiated prior to the outbreak, pH1N1 vaccination was cost-saving for persons 6 months to 64 years under many assumptions. For those without high risk conditions, incremental cost-effectiveness ratios ranged from $8,000–$52,000/QALY depending on age and risk status. Results were sensitive to the number of vaccine doses needed, costs of vaccination, illness rates, and timing of vaccine delivery.

Conclusions

Vaccination for pH1N1 for children and working-age adults is cost-effective compared to other preventive health interventions under a wide range of scenarios. The economic evidence was consistent with target recommendations that were in place for pH1N1 vaccination. We also found that the delays in vaccine availability had a substantial impact on the cost-effectiveness of vaccination.     

The yeast prions [PSI+] and [URE3] are molecular degenerative diseases

The yeast prions [URE3] and [PSI] are not found in wild strains, suggesting they are not an advantage. Prion-forming ability is not conserved, even within Saccharomyces, suggesting it is a disease. Prion domains have non-prion functions, explaining some conservation of sequence. However, in spite of the sequence being constrained in evolution by these non-prion functions, the prion domains vary more rapidly than the remainder of the molecule, and these changes produce a transmission barrier, suggesting that these changes were selected to block prion infection. Yeast prions [PSI] and [URE3] induce a cellular stress response (Hsp104 and Hsp70 induction), suggesting the cells are not happy about being infected. Recently, we showed that the array of [PSI] and [URE3] prions includes a majority of lethal or very toxic variants, a result not expected if either prion were an adaptive cellular response to stress.Key words: [URE3], [PSI⁺], prion, Sup35p, Ure2pfMammalian prions are uniformly fatal, but a lethal yeast prion would not be detected by the usual procedure, which requires growth of a colony under some selective condition. As a result, the prion variants commonly studied are quite mild in their effects. This circumstance has led to the suggestion that yeast prions actually benefit their host. Sup35p, the translation termination subunit whose amyloid becomes the [PSI⁺] prion, is essential for growth and Ure2p, the nitrogen regulation protein whose amyloid constitutes the [URE3] prion, is important for growth, with ure2 mutants showing noticeably slowed growth.When yeast prions were discovered,¹ we assumed they were diseases, by analogy with the mammalian diseases and the many non-prion amyloid diseases. Inactivating the essential Sup35p or the desireable Ure2p did not seem like a useful strategy. While control of either protein''s activity might be advantageous, and Ure2p activity control is the key to regulation of nitrogen catabolism, prion formation is a stochastic process, so it makes control of activity of these proteins random instead of appropriate to the circumstances. The [Het-s] prion changed that picture.² Here was a prion necessary for a normal function, heterokaryon incompatibility, and we suggested that it was the first beneficial prion.³