Genetic mapping of quantitative trait loci controlling fruit size and shape in papaya

Papaya (Carica papaya L.) is a pan-tropical tree that bears fruit exhibiting a wide range of size and shape. Depending on variety and environment, papaya fruit may weigh from 0.2 kg up to 10 kg. Papaya fruit shape is a sex-linked trait ranging from spherical to ovate, cylindrical or pyriform. An F2 mapping population, produced from a cross between the Thai variety Khaek Dum, bearing 1.2 kg, red-fleshed fruit, and variety 2H94, a Hawaii Solo type bearing a 0.2 kg, yellow-fleshed fruit, was used to identify quantitative trait loci (QTLs) that influence papaya fruit characters including weight, diameter, length and shape. Fruit phenotype data, collected from two subpopulations planted in successive growing seasons, showed striking differences by year indicating significant genotype × environment interactions. Fourteen QTL with phenotypic effects ranging from 5 to 23% were identified across six linkage groups (LGs) with clusters of two or more QTL on LGs 02, 03, 07 and 09. These loci contain homologs to the tomato fruit QTL ovate, sun and fw2.2 regulating fruit size and shape. The papaya fruit QTL provide a starting point for dissecting the genetic pathways leading to extreme fruit size and shape and may prove useful for papaya breeders attempting to tailor new varieties to specific consumer markets.     

IL-17 Mediates Immunopathology in the Absence of IL-10 Following Leishmania major Infection

Leishmaniasis, resulting from infection with the protozoan parasite Leishmania, consists of a wide spectrum of clinical manifestations, from healing cutaneous lesions to fatal visceral infections. A particularly severe form of cutaneous leishmaniasis, termed mucosal leishmaniasis, exhibits decreased IL-10 levels and an exaggerated inflammatory response that perpetuates the disease. Using a mouse model of leishmaniasis, we investigated what cytokines contribute to increased pathology when IL-10-mediated regulation is absent. Leishmania major infected C57BL/6 mice lacking IL-10 regulation developed larger lesions than controls, but fewer parasites. Both IFN-γ and IL-17 levels were substantially elevated in mice lacking the capacity to respond to IL-10. IFN-γ promoted an increased infiltration of monocytes, while IL-17 contributed to an increase in neutrophils. Surprisingly, however, we found that IFN-γ did not contribute to increased pathology, but instead regulated the IL-17 response. Thus, blocking IFN-γ led to a significant increase in IL-17, neutrophils and disease. Similarly, the production of IL-17 by cells from leishmaniasis patients was also regulated by IL-10 and IFN-γ. Additional studies found that the IL-1 receptor was required for both the IL-17 response and increased pathology. Therefore, we propose that regulating IL-17, possibly by downregulating IL-1β, may be a useful approach for controlling immunopathology in leishmaniasis.     

Role of Cortactin Homolog HS1 in Transendothelial Migration of Natural Killer Cells

Natural Killer (NK) cells perform many functions that depend on actin assembly, including adhesion, chemotaxis, lytic synapse assembly and cytolysis. HS1, the hematopoietic homolog of cortactin, binds to Arp2/3 complex and promotes actin assembly by helping to form and stabilize actin filament branches. We investigated the role of HS1 in transendothelial migration (TEM) by NK cells. Depletion of HS1 led to a decrease in the efficiency of TEM by NK cells, as measured by transwell assays with endothelial cell monolayers on porous filters. Transwell assays involve chemotaxis of NK cells across the filter, so to examine TEM more specifically, we imaged live-cell preparations and antibody-stained fixed preparations, with and without the chemoattractant SDF-1α. We found small to moderate effects of HS1 depletion on TEM, including whether the NK cells migrated via the transcellular or paracellular route. Expression of HS1 mutants indicated that phosphorylation of HS1 tyrosines at positions 222, 378 and 397 was required for rescue in the transwell assay, but HS1 mutations affecting interaction with Arp2/3 complex or SH3-domain ligands had no effect. The GEF Vav1, a ligand of HS1 phosphotyrosine, influenced NK cell transendothelial migration. HS1 and Vav1 also affected the speed of NK cells migrating across the surface of the endothelium. We conclude that HS1 has a role in transendothelial migration of NK cells and that HS1 tyrosine phosphorylation may signal through Vav1.     

Assessing the Residual Risk for Transfusion-Transmitted Infections in the Philippine Blood Supply

Due to a USAID-funded study on blood banks, a national policy was instituted in 1994 that set standards for Philippine blood services, promoted voluntary donation, and led to a ban on commercial blood banks. In this follow-up study, we assess the safety of the supply by determining the residual risk for transfusion-transmitted infections (syphilis, hepatitis B and C, HIV). We also identified unsafe facility practices and generated policy recommendations. A 1992 study found that transfusion-ready blood was not safe using the LQAS method (P > 0.05). We found that the 2012 residual risk became 0 to 0.9 percent attributable to the national policy. We noted poor to fair adherence to this policy. We identified unsafe practices such as use of rapid tests and lack of random blood retesting. Training and use of regional networks may improve safety. Despite improvement in safety, facilities complain of funding and logistical issues regarding compliance with the policy.     

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography ¹ or Activity Based Protein Profiling ². Trifunctional Capture Compounds (CCs, Figure 1A) ³ are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-_L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" ⁴ or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules). S-Adenosyl-_L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature ^{5, 6}. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA ⁷, RNA ⁸, proteins ⁹, or small molecules ¹⁰. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor ¹¹. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability ¹², SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5α of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.Download video file.^{(106M, mov)}     

Recombinant bacteriophage lysins as antibacterials

With the increasing worldwide prevalence of antibiotic resistant bacteria, bacteriophage endolysins (lysins) represent a very promising novel alternative class of antibacterial in the fight against infectious disease. Lysins are phage-encoded peptidoglycan hydrolases which, when applied exogenously (as purified recombinant proteins) to Gram-positive bacteria, bring about rapid lysis and death of the bacterial cell. A number of studies have recently demonstrated the strong potential of these enzymes in human and veterinary medicine to control and treat pathogens on mucosal surfaces and in systemic infections. They also have potential in diagnostics and detection, bio-defence, elimination of food pathogens and control of phytopathogens. This review discusses the extensive research on recombinant bacteriophage lysins in the context of antibacterials, and looks forward to future development and potential.     

Enterococcus faecalis Endocarditis Severity in Rabbits Is Reduced by IgG Fabs Interfering with Aggregation Substance

Background

Enterococcus faecalis is a significant cause of infective endocarditis, an infection of the heart endothelium leading to vegetation formation (microbes, fibrin, platelets, and host cells attached to underlying endothelial tissue). Our previous research determined that enterococcal aggregation substance (AS) is an important virulence factor in causation of endocarditis, although endocarditis may occur in the absence of AS production. Production of AS by E. faecalis causes the organism to form aggregates through AS binding to enterococcal binding substance. In this study, we assessed the ability of IgGs and IgG Fabs against AS to provide protection against AS⁺ E. faecalis endocarditis.

Methodology/Principal Findings

When challenged with AS⁺ E. faecalis, 10 rabbits actively immunized against AS⁺ E. faecalis developed more significant vegetations than 9 animals immunized against AS⁻ E. faecalis, and 9/10 succumbed compared to 2/9 (p<0.005), suggesting enhanced aggregation by IgG contributes significantly to disease. IgG antibodies against AS also enhanced enterococcal aggregation as tested in vitro. In contrast, Fab fragments of IgG from rabbits immunized against purified AS, when passively administered to rabbits (6/group) immediately before challenge with AS⁺ E. faecalis, reduced total vegetation (endocarditis lesion) microbial counts (7.9×10⁶ versus 2.0×10⁵, p = 0.02) and size (40 mg versus 10, p = 0.05). In vitro, the Fabs prevented enterococcal aggregation.

Conclusions/Significance

The data confirm the role of AS in infective endocarditis formation and suggest that use of Fabs against AS will provide partial protection from AS⁺ E. faecalis illness.     

Proteome Analysis of the Surface of Trichomonas vaginalis Reveals Novel Proteins and Strain-dependent Differential Expression

The identification of surface proteins on the plasma membrane of pathogens is of fundamental importance in understanding host-pathogen interactions. Surface proteins of the extracellular parasite Trichomonas are implicated in the initial adherence to mucosal tissue and are likely to play a critical role in the long term survival of this pathogen in the urogenital tract. In this study, we used cell surface biotinylation and multidimensional protein identification technology to identify the surface proteome of six strains of Trichomonas vaginalis with differing adherence capacities to vaginal epithelial cells. A combined total of 411 proteins were identified, and of these, 11 were found to be more abundant in adherent strains relative to less adherent parasites. The mRNA levels of five differentially expressed proteins selected for quantitative RT-PCR analysis mirrored their observed protein levels, confirming their up-regulation in highly adherent strains. As proof of principle and to investigate a possible role in pathogenesis for differentially expressed proteins, gain of function experiments were performed using two novel proteins that were among the most highly expressed surface proteins in adherent strains. Overexpression of either of these proteins, TVAG_244130 or TVAG_166850, in a relatively non-adherent strain increased attachment of transfected parasites to vaginal epithelial cells ∼2.2-fold. These data support a role in adhesion for these abundant surface proteins. Our analyses demonstrate that comprehensive profiling of the cell surface proteome of different parasite strains is an effective approach to identify potential new adhesion factors as well as other surface molecules that may participate in establishing and maintaining infection by this extracellular pathogen.The flagellated protozoan parasite Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted infection worldwide with an estimated 174 million new cases annually (1). Although asymptomatic infection by T. vaginalis is common, multiple symptoms and pathologies can arise in both men and women, including vaginitis, urethritis, prostatitis, low birth weight infants and preterm delivery, premature rupture of membranes, and infertility (2–5). T. vaginalis has also emerged as an important cofactor in amplifying human immunodeficiency virus spread (6) as individuals infected with T. vaginalis have a significantly increased incidence of human immunodeficiency virus transmission (7, 8). T. vaginalis infection likewise increases the risk of cervical and aggressive prostate cancers (9–11).Despite the serious consequences that can arise from trichomoniasis, the underlying biochemical processes that lead to T. vaginalis pathogenesis are not well defined. Because T. vaginalis is an obligate extracellular pathogen, adherence to epithelial cells is critical for parasite survival within the human host (12). Several in vitro studies indicate that adhesion of the parasite to target mucosal epithelial cells is essential for the maintenance of infection and for cytopathogenicity (13, 14). T. vaginalis adherence to host cells is mediated, in part, by a lipophosphoglycan (LPG)¹ that coats the surface of the parasite, and altering the sugar content of this LPG reduces both adherence and cytotoxicity (15). Moreover, the mammalian protein galectin-1 binds to T. vaginalis in a carbohydrate-dependent manner via a direct interaction with parasite LPG (16). Knockdown of galectin-1 in mammalian cells, however, reduces parasite binding only by ∼17% (16). Although galectin-1-mediated interactions between T. vaginalis LPG and host cell glycoconjugates may be central in establishing infection, it is clear that parasite adhesion factors in addition to LPG are likely to be involved in host-parasite interaction. Surface proteins are likely to play important roles in the initial adherence to mucosal tissue as well as the long term survival of the pathogen on mucosal surfaces.The outcome of infection with T. vaginalis is highly variable. Possible explanations for this phenomenon include host immunity, host nutritional status, and the vaginal microbiota. Additionally, genetic differences between T. vaginalis isolates leading to differences in adherence and cytotoxicity capacities are likely to result in differences in disease progression. Recently, geographically diverse T. vaginalis strains that are significantly more cytotoxic to host cells than laboratory-adapted strains have become available (17, 18), paving the way toward comparative studies aimed at identifying proteins that correlate with virulent phenotypes.Despite the importance of T. vaginalis surface proteins as a critical interface for pathogen-host interactions, there has been no systematic investigation of the surface proteins of this parasite. The T. vaginalis genome is large and encodes a massive proteome with a considerable and diverse repertoire of candidate surface proteins (19). For example, sequence analysis programs that predict transmembrane protein topology identified over 5100 T. vaginalis proteins with one or more transmembrane domains (20). Furthermore, over 300 annotated proteins with predicted transmembrane domains also contain protein motifs common to surface proteins from other pathogens known to contribute to mucosal colonization and other pathogenic processes (20). The vast number and diversity of possible surface proteins necessitates a multitiered approach using complementary genomics and proteomics analyses to identify candidates for focused functional studies.Biotinylation of proteins at the cell surface with an impermeable reagent followed by specific purification of these proteins using streptavidin has successfully been used for the enrichment and identification of surface proteins (21–24). The high avidity binding of biotin to streptavidin greatly enhances membrane protein purification, a challenging feat because of the low abundance of membrane proteins in total cellular extracts. Here, we used this approach to profile the surface plasma membrane proteome of T. vaginalis and to identify proteins that are differentially expressed in adherent relative to less adherent strains of the parasite. To the best of our knowledge, this is the first study to systematically identify and characterize proteins at the surface of Trichomonas parasites. Defining the parasite cell surface proteome is a critical step toward understanding the relative abundance of surface proteins in strains with varying virulence properties. This information will be critical for defining the role surface proteins play in mediating contact between the parasite and host cells as well as the resulting intracellular and extracellular signals that contribute to establishing and maintaining infection. Additionally, conserved surface molecules unique to T. vaginalis that might serve as specific vaccine candidates can be revealed using this approach. The prevalence of trichomoniasis among women of reproductive age (25) and its correlation with AIDS transmission and cervical and prostate cancers (6, 8–11) provide strong arguments for the need to develop vaccines against this human pathogen.     

Fat-induced inflammation unchecked

Increased dietary saturated fat intake can lead to detrimental effects on human health. In this issue of Cell Metabolism, Lichtenstein et?al. (2010) show that by inhibiting lipoprotein lipase (LPL) activity in mesenteric lymph nodes, Angiopoietin-like protein 4 (Angptl4) protects resident macrophages from dietary saturated fatty acid (SFA)-induced inflammation.