Simultaneous display of multiple foreign peptides in the FliD capping and FliC filament proteins of the Escherichia coli flagellum

The bacterial flagellum is composed of more than 20 different proteins. The filament, which constitutes the major extracellular part of the flagellum, is built up of approximately 20,000 FliC molecules that assemble at the growing distal end of the filament. A capping structure composed of five FliD molecules located at the tip of the filament promotes polymerization of FliC. Lack of FliD leads to release of the subunits into the growth medium. We show here that FliD can be successfully used in bacterial surface display. We tested various insertion sites in the capping protein, and the optimal region for display was at the variable region in FliD. Deletion and/or insertion at other sites resulted in decreased formation of flagella. We further developed the technique into a multihybrid display system in which three foreign peptides are simultaneously expressed within the same flagellum, i.e., D repeats of FnBPA from Staphylococcus aureus at the tip and fragments of YadA from Yersinia enterocolitica as well as SlpA from Lactobacillus crispatus along the filament. This technology can have biotechnological applications, e.g., in simultaneous delivery of several effector molecules.     

The impact of thermal seasonality on terrestrial endotherm food web dynamics: a revision of the Exploitation Ecosystem Hypothesis

Many terrestrial endotherm food webs constitute three trophic level cascades. Others have two trophic level dynamics (food limited herbivores; plants adapted to tackle intense herbivory) or one trophic level dynamic (herbivorous endotherms absent, thus plants compete for the few places where they can survive and grow). According to the Exploitation Ecosystems Hypothesis (EEH), these contrasting dynamics are consequences of differences in primary productivity. The productivity thresholds for changing food web dynamics were assumed to be global constants. We challenged this assumption and found that several model parameters are sensitive to the contrast between persistently warm and seasonally cold climates. In persistently warm environments, three trophic level dynamics can be expected to prevail almost everywhere, save the most extreme deserts. We revised EEH accordingly and tested it by compiling direct evidence of three and two trophic level dynamics and by studying the global distribution of felids. In seasonally cold environments, we found evidence for three trophic level dynamics only in productive ecosystems, while evidence for two trophic level dynamics appeared in ecosystems with low primary productivity. In persistently warm environments, we found evidence for three trophic level dynamics in all types of ecosystems. The distribution of felids corroborated these results. The empirical evidence thus indicates that two trophic level dynamics, as defined by EEH, are restricted to seasonally cold biomes with low primary productivity, such as the artic–alpine tundra and the temperate steppe.     

Acidification of the intimal fluid: the perfect storm for atherogenesis

Atherosclerotic lesions are often hypoxic and exhibit elevated lactate concentrations and local acidification of the extracellular fluids. The acidification may be a consequence of the abundant accumulation of lipid-scavenging macrophages in the lesions. Activated macrophages have a very high energy demand and they preferentially use glycolysis for ATP synthesis even under normoxic conditions, resulting in enhanced local generation and secretion of lactate and protons. In this review, we summarize our current understanding of the effects of acidic extracellular pH on three key players in atherogenesis: macrophages, apoB-containing lipoproteins, and HDL particles. Acidic extracellular pH enhances receptor-mediated phagocytosis and antigen presentation by macrophages and, importantly, triggers the secretion of proinflammatory cytokines from macrophages through activation of the inflammasome pathway. Acidity enhances the proteolytic, lipolytic, and oxidative modifications of LDL and other apoB-containing lipoproteins, and strongly increases their affinity for proteoglycans, and may thus have major effects on their retention and the ensuing cellular responses in the arterial intima. Finally, the decrease in the expression of ABCA1 at acidic pH may compromise cholesterol clearance from atherosclerotic lesions. Taken together, acidic extracellular pH amplifies the proatherogenic and proinflammatory processes involved in atherogenesis.     

Pre-targeting and direct immunotargeting of liposomal drug carriers to ovarian carcinoma

Background

Epidermal growth factor receptor (EGFR) is overexpressed in many solid tumor types, such as ovarian carcinoma. Immunoliposome based drug targeting has shown promising results in drug delivery to the tumors. However, the ratio of tumor-to-normal tissue concentrations should be increased to minimize the adverse effects of cytostatic drugs.

Methodology/Principal Findings

We studied the EGFR-targeted doxorubicin immunoliposomes using pre-targeting and local intraperitoneal (i.p.) administration of the liposomes. This approach was used to increase drug delivery to tumors as compared to direct intravenous (i.v.) administration of liposomes. EGFR antibodies were attached on the surface of PEG coated liposomes using biotin-neutravidin binding. Receptor mediated cellular uptake and cytotoxic efficacy of EGFR-targeted liposomes were investigated in human ovarian adenocarcinoma (SKOV-3 and SKOV3.ip1) cells. In vivo distribution of the liposomes in mice was explored using direct and pre-targeting approaches and SPECT/CT imaging. Targeted liposomes showed efficient and specific receptor-mediated binding to ovarian carcinoma cells in vitro, but the difference in cytotoxicity between targeted and non-targeted liposomes remained small. The relatively low cytotoxic efficacy is probably due to insufficient doxorubicin release from the liposomes rather than lack of target binding. Tumor uptake of targeted liposomes in vivo was comparable to that of non-targeted liposomes after both direct and pre-targeting administration. For both EGFR-targeted and non-targeted liposomes, the i.p. administration increased liposome accumulation to the tumors compared to i.v. injections.

Conclusions/Significance

Intraperitoneal administration of liposomes may be a beneficial approach to treat the tumors in the abdominal cavity. The i.p. pre-targeting method warrants further studies as a potential approach in cancer therapy.     

Transgenerational effects of parental larval diet on offspring development time, adult body size and pathogen resistance in Drosophila melanogaster

Environmental conditions experienced by parents are increasingly recognized to affect offspring performance. We set out to investigate the effect of parental larval diet on offspring development time, adult body size and adult resistance to the bacterium Serratia marcescens in Drosophila melanogaster. Flies for the parental generation were raised on either poor or standard diet and then mated in the four possible sex-by-parental diet crosses. Females that were raised on poor food produced larger offspring than females that were raised on standard food. Furthermore, male progeny sired by fathers that were raised on poor food were larger than male progeny sired by males raised on standard food. Development times were shortest for offspring whose one parent (mother or the father) was raised on standard and the other parent on poor food and longest for offspring whose parents both were raised on poor food. No evidence for transgenerational effects of parental diet on offspring disease resistance was found. Although paternal effects have been previously demonstrated in D. melanogaster, no earlier studies have investigated male-mediated transgenerational effects of diet in this species. The results highlight the importance of not only considering the relative contribution each parental sex has on progeny performance but also the combined effects that the two sexes may have on offspring performance.     

Targeted codon optimization improves translational fidelity for an Fc fusion protein

High levels of translational errors, both truncation and misincorporation in an Fc‐fusion protein were observed. Here, we demonstrate the impact of several commercially available codon optimization services, and compare to a targeted strategy. Using the targeted strategy, only codons known to have translational errors are modified. For an Fc‐fusion protein expressed in Escherichia coli, the targeted strategy, in combination with appropriate fermentation conditions, virtually eliminated misincorporation (proteins produced with a wrong amino acid sequence), and reduced the level of truncation. The use of full optimization using commercially available strategies reduced the initial errors, but introduced different misincorporations. However, truncation was higher using the targeted strategy than for most of the full optimization strategies. This targeted approach, along with monitoring of translation fidelity and careful attention to fermentation conditions is key to minimizing translational error and ensuring high‐quality expression. These findings should be useful for other biopharmaceutical products, as well as any other transgenic constructs where protein quality is important. Biotechnol. Bioeng. 2012; 109: 2770–2777. © 2012 Wiley Periodicals, Inc.     

Marfan syndrome: No evidence for heterogeneity in different populations, and more precise mapping of the gene

Marfan syndrome is a dominantly inherited connective tissue disorder with manifestations in the cardiovascular, ocular, and skeletal systems. The diagnosis is hampered by both high variability in the phenotypic expression and late manifestation of symptoms. The cause of Marfan syndrome remains unknown, but our group has recently reported the genetic linkage of Marfan syndrome to a polymorphic marker on chromosome 15. To analyze the possible heterogeneity behind Marfan syndrome, we have performed linkage analyses for four chromosome 15 markers in 17 families from five different populations: Scottish, English, Swiss, American, and Finnish. By combining the linkage data of all the studied families into a LINKMAP analysis we obtained a maximal LOD score of 11.2, which maps the Marfan syndrome locus between D15S25 and D15S45 on the long arm of chromosome 15. The data reveal no evidence for genetic heterogeneity behind Marfan syndrome and provide us with a more precise location of both the Marfan syndrome locus and flanking markers. This information will provide the basis for the DNA diagnostics of Marfan syndrome in the future.     

Cysteine protease cathepsin F is expressed in human atherosclerotic lesions, is secreted by cultured macrophages, and modifies low density lipoprotein particles in vitro

During atherogenesis, low density lipoprotein (LDL) particles in the arterial intima become modified and fuse to form extracellular lipid droplets. Proteolytic modification of apolipoprotein (apo) B-100 may be one mechanism of droplet formation from LDL. Here we studied whether the newly described acid protease cathepsin F can generate LDL-derived lipid droplets in vitro. Treatment of LDL particles with human recombinant cathepsin F led to extensive degradation of apoB-100, which, as determined by rate zonal flotation, electron microscopy, and NMR spectroscopy, triggered both aggregation and fusion of the LDL particles. Two other acid cysteine proteases, cathepsins S and K, which have been shown to be present in the arterial intima, were also capable of degrading apoB-100, albeit less efficiently. Cathepsin F treatment resulted also in enhanced retention of LDL to human arterial proteoglycans in vitro. Cultured monocyte-derived macrophages were found to secrete active cathepsin F. In addition, similarly with cathepsins S and K, cathepsin F was found to be localized mainly within the macrophage-rich areas of the human coronary atherosclerotic plaques. These results suggest that proteolytic modification of LDL by cathepsin F may be one mechanism leading to the extracellular accumulation of LDL-derived lipid droplets within the proteoglycan-rich extracellular matrix of the arterial intima during atherogenesis.     

Immune cell phenotype and functional defects in Netherton syndrome

Background

Netherton syndrome (NS) is a rare life-threatening syndrome caused by SPINK5 mutations leading to a skin barrier defect and a severe atopic diathesis. NS patients are prone to bacterial infections, but the understanding of the underlying immune deficiency is incomplete.

Results

We analyzed blood lymphocyte phenotypes and function in relation to clinical infections in 11 Finnish NS patients, aged 3 to 17?years, and healthy age-matched controls. The proportion of B cells (CD19⁺) and naïve B cells (CD27^?, IgD⁺) were high while memory B cells (CD27⁺) and switched memory B cells (CD27⁺IgM^?IgD^?), crucial for the secondary response to pathogens, was below or in the lowest quartile of the reference values in 8/11 (73%) and 9/11 (82%) patients, respectively. The proportion of activated non-differentiated B cells (CD21^low, CD38l^ow) was below or in the lowest quartile of the reference values in 10/11 (91%) patients. Despite normal T cell counts, the proportion of naïve CD4⁺ T cells was reduced significantly and the proportion of CD8⁺ T central memory significantly elevated. An increased proportion of CD57⁺ CD8⁺ T cells indicated increased differentiation potential of the T cells. The proportion of cytotoxic NK cells was elevated in NS patients in phenotypic analysis based on CD56DIM, CD16⁺ and CD27^? NK cells but in functional analysis, decreased expression of CD107a/b indicated impaired cytotoxicity.The T and NK cell phenotype seen in NS patients also significantly differed from that of age-matched atopic dermatitis (AD) patients, indicating a distinctive profile in NS. The frequency of skin infections correlated with the proportion of CD62L⁺ T cells, naïve CD4⁺ and CD27⁺ CD8⁺ T cells and with activated B cells. Clinically beneficial intravenous immunoglobulin therapy (IVIG) increased naïve T cells and terminal differentiated effector memory CD8⁺ cells and decreased the proportion of activated B cells and plasmablasts in three patients studied.

Conclusions

This study shows novel quantitative and functional aberrations in several lymphocyte subpopulations, which correlate with the frequency of infections in patients with Netherton syndrome. IVIG therapy normalized some dysbalancies and was clinically beneficial.