Investigation of photosensitizing dyes for pathogen reduction in red cell suspensions

Despite recent advances in blood safety by careful donor selection and implementation of infectious disease testing, transmission of viruses, bacteria and parasites by transfusion can still rarely occur. One approach to reduce the residual risk from currently tested pathogens and to protect against the emergence of new ones is to investigate methods for pathogen inactivation. The use of photosensitizing dyes for pathogen inactivation has been studied in both red cell and platelet blood components. Optimal properties of sensitizing dyes for use in red cell suspensions include selection of dyes that traverse cell and viral membranes, bind to nucleic acids, absorb light in the red region of the spectrum, inactivate a wide range of pathogens, produce little red cell photodamage from dye not bound to nucleic acid and do not hemolyze red cells in the dark. Early research at the American Red Cross focused on the use of a class of dyes with rigid structures, such as the phenothiazine dyes, beginning with the prototypical sensitizer methylene blue. Results revealed that methylene blue phototreatment could inactivate extracellular virus, but resulted in undesirable defects in the red cell membrane that resulted in enhanced hemolysis that became evident during extended refrigerated blood storage. In addition, methylene blue phototreatment could neither inactivate intracellular viruses nor appreciably inactivate bacteria under conditions of extracellualar viral killing. Attempts to improve intracellular viral inactivation led to the investigations of more hydrophobic phenothiazines, such as methylene violet or dimethylmethylene blue. Although these dyes could inactivate intracellular virus, problems with increased red cell membrane damage and hemolysis persisted or increased. Further studies using red cell additive storage solutions containing high levels of the impermeable ion, citrate, to protect against colloidal osmotic hemolysis as well as competitive inhibitors to limit sensitizer binding to red cell membranes revealed that photoinduced hemolysis stemmed from dye bound to the red cell membrane as well as dye free in solution. Use of red cell additive solutions to prevent colloidal-osmotic hemolysis and use of novel flexible dyes that only act as sensitizers when bound to their targets are two techniques that currently are under investigation for reducing red cell damage. Ultimately, the decision to implement a photodynamic method for pathogen reduction will be determined by weighing the risks of unintended adverse consequences of the procedure itself, such as the potential for genotoxicity and allergic reactions, against the cost and benefits of its implementation.     

Apparent competition between two species of aphid via the fungal pathogen Erynia neoaphidis and its interaction with the aphid parasitoid Aphidius ervi

Abstract 1. Motivated by a community study on aphids and their fungal pathogens, three hypotheses were tested experimentally to investigate the influence of the fungal pathogen, Erynia neoaphidis Remaudière and Hennebert, on aphid population and community ecology.
2. Field experiments were performed in 2 years to test whether two susceptible aphid species on different host plants might interact through the shared fungal pathogen. No strong pathogen-mediated indirect interactions (apparent competition) between populations of pea aphid Acyrthosiphon pisum Harris and nettle aphid Microlophium carnosum Buckton were detected.
3. In the first of the field experiments, pea aphids exposed to the fungus showed a weak tendency to produce more winged dispersal morphs than control populations not exposed to the fungus. In a laboratory test, however, no support was found for the hypothesis that the presence of volatiles from fungus-infected cadavers promotes production of winged offspring.
4. The response of the pea aphid parasitoid Aphidius ervi Halliday to colonies containing hosts infected 1 and 3 days previously was assessed. Wasps initiated fewer attacks on 1-day-old infected colonies than on healthy colonies, with the numbers on 3-day-old fungus-infected colonies intermediate.     

The evolutionary significance of parasitism: do parasite‐driven genetic dynamics occur ex silico?

It has long been recognized that reciprocal antagonism might lock host and parasite populations into a process of constant change, adapting and reacting in open‐ended coevolution. A significant body of theory supports this intuition: dynamic genetic polymorphisms are a common outcome of computer simulations of host–parasite coevolution. These in silico experiments have also shown that dynamical interactions could be responsible for high levels of genetic diversity in host populations, and even be the principle determinant of rates of genetic recombination and sexuality. The evolutionary significance of parasitism depends on the strength and prevalence of parasite‐mediated selection in nature. Here I appraise whether parasitism is a pervasive agent of evolutionary change by detailing empirical evidence for selection. Although there is considerable evidence of genetic variation for resistance, and hence the potential for selection, direct observation of parasite‐driven genetic change is lacking.     

Species delimitation in downy mildews: the case of Hyaloperonospora in the light of nuclear ribosomal ITS and LSU sequences

Species definitions for plant pathogens have considerable practical impact for measures such as plant protection or biological control, and are also important for comparative studies involving model organisms. However, in many groups, the delimitation of species is a notoriously difficult taxonomic problem. This is particularly evident in the obligate biotrophic downy mildew genera (Peronosporaceae, Peronosporales, Oomycetes), which display a considerable diversity with respect to genetic distances and host plants, but are, for the most part, morphologically rather uniform. The recently established genus Hyaloperonospora is of particular biological interest because it shows an impressive radiation on virtually a single host family, Brassicaceae, and it contains the downy mildew parasite, Arabidopsis thaliana, of importance as a model organism. Based on the most comprehensive molecular sampling of specimens from a downy mildew genus to date, including various collections from different host species and geographic locations, we investigate the phylogenetic relationships of Hyaloperonospora by molecular analysis of the nuclear ribosomal ITS and LSU sequences. Phylogenetic trees were inferred with ML and MP from the combined dataset; partitioned Bremer support (PBrS) was used to assess potential conflict between data partitions. As in other downy mildew groups, the molecular data clearly corroborate earlier results that supported the use of narrow species delimitations and host ranges as taxonomic markers. With few exceptions, suggested species boundaries are supported without conflict between different data partitions. The results indicate that a combination of molecular and host features is a reliable means to discriminate downy mildew species for which morphological differences are unknown.     

丹东市熟肉制品中食源性致病菌污染状况的调查研究

为了解丹东市熟肉制品中沙门菌、金黄色葡萄球菌和单核细胞增生性李斯特菌的污染及菌相分布状况,为丹东市熟肉制品中致病菌污染提供本地资料,以及熟肉制品卫生监督提供科学依据。依据国标方法以及采用全自动酶联免疫荧光快速筛选仪进行筛选和BBL Crystal细菌鉴定仪鉴定,对130份样品分别进行上述致病菌分离、血清学和生化鉴定。结果共检出致病菌15株,总检出率为11．5％,酱卤类检出率为14．0％、烧烤类检出率为7．5％、白切类检出率为12．5％,其中沙门菌4株、单核细胞增生性李斯特菌4株和金黄色葡萄球菌7株。说明丹东市熟肉制品中存在食源性致病菌污染,其中金黄色葡萄球菌污染最严重。     

Solution structure of the phytotoxic protein PcF: The first characterized member of the Phytophthora PcF toxin family

The PcF protein from Phytophthora cactorum is the first member of the “PcF toxin family” from the plant pathogens Phytophthora spp. It is able to induce withering in tomato and strawberry leaves. The lack of sequence similarity with other proteins hampers the identification of the molecular mechanisms responsible for its toxicity. Here, we show that the six cysteines form a disulphide pattern that is exclusive for PcF and essential for the protein withering activity. The NMR solution structure identifies a novel fold among protein effectors: a helix‐loop‐helix motif. The presence of a negatively charged surface suggests that it might act as a site of electrostatic interaction. Interestingly, a good fold match with Ole e 6, a plant protein with allergenic activity, highlighted the spatial superimposition of a stretch of identical residues. This finding suggests a possible biological activity based on molecular mimicry.     

Microsatellite markers for the diploid basidiomycete fungus Armillaria mellea

We isolated and characterized 12 microsatellite markers for two North American populations (California, Pennsylvania) of Armillaria mellea, a fungal pathogen responsible for Armillaria root disease of numerous woody plants. Allele frequency ranged from two to nine alleles per locus, and gene diversity ranged from 0.05 to 0.86. Of the 12 loci, eight loci were polymorphic in the California and Pennsylvania populations, and showed no evidence of heterozygote deficiencies or severe linkage disequilibrium. Our results suggest that we have isolated and characterized variable loci to estimate genotypic diversity, gene flow and migration, and to determine population structure of North American A. mellea.     

Emerging trends in plasma-free manufacturing of recombinant protein therapeutics expressed in mammalian cells

Mammalian cells are the expression system of choice for therapeutic proteins, especially those requiring complex post-translational modifications. Traditionally, these cells are grown in medium supplemented with serum and other animal- or human-derived components to support viability and productivity. Such proteins are also typically added as excipients and stabilizers in the final drug formulation. However, the transmission of hepatitis B in the 1970s and of hepatitis C and HIV in the 1980s through plasma-derived factor VIII concentrates had catastrophic consequences for hemophilia patients. Thus, due to regulatory concerns about the inherent potential for transmission of infectious agents as well as the heterogeneity and lack of reliability of the serum supply, a trend has emerged to eliminate the use of plasma-derived additives in the production and formulation of recombinant protein therapeutics. This practice began with products used in the treatment of hemophilia and is progressively expanding throughout the entire industry. The plasma-free method of producing recombinant therapeutics is accomplished by the use of both cell culture media and final product formulations that do not contain animal- or human-derived additives. A number of recombinant therapeutic proteins for the treatment of several different diseases have been produced by plasma-free processes, with the objective of improving safety by eliminating blood-borne pathogens or by reducing immunogenicity. This review describes the factors that drove the development of plasma-free protein therapeutics and provides examples of advances in manufacturing that have made possible the removal of human and animal-derived products from all steps of recombinant protein production.