Proteophosphoglycan confers resistance of Leishmania major to midgut digestive enzymes induced by blood feeding in vector sand flies

Leishmania synthesize abundant phosphoglycan‐containing molecules made up of [Gal‐Man‐PO₄] repeating units, including the surface lipophosphoglycan (LPG), and the surface and secreted proteophosphoglycan (PPG). The vector competence of Phlebotomus duboscqi and Lutzomyia longipalpis sand flies was tested using L. major knockout mutants deficient in either total phosphoglycans (lpg2^‐ or lpg5A^‐/5B^‐) or LPG alone (lpg1^‐) along with their respective gene add‐back controls. Our results confirm that LPG, the major cell surface molecule of Leishmania promastigotes known to mediate attachment to the vector midgut, is necessary to prevent the loss of infection during excretion of the blood meal remnants from a natural vector, P. duboscqi, but not an unnatural vector, L. longipalpis. Midgut digestive enzymes induced by blood feeding pose another potential barrier to parasite survival. Our results show that 36–72 h after the infective feed, all parasites developed well except the lpg2^‐ and lpg5A^‐/5B^‐ mutants, which showed significantly reduced survival and growth. Protease inhibitors promoted the early survival and growth of lpg2^‐ in the blood meal. PPG was shown to be the key molecule conferring resistance to midgut digestive enzymes, as it prevented killing of lpg2^‐ promastigotes exposed to midgut lysates prepared from blood‐fed flies. The protection was not associated with inhibition of enzyme activities, but with cell surface acquisition of the PPG, which appears to function similar to mammalian mucins to protect the surface of developing promastigotes against proteolytic damage.     

Expression,regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases

Background

Inflammatory lung diseases are a major morbidity factor in children. Therefore, novel strategies for early detection of inflammatory lung diseases are of high interest. Bacterial lipopolysaccharide (LPS) is recognized via Toll-like receptors and CD14. CD14 exists as a soluble (sCD14) and membrane-associated (mCD14) protein, present on the surface of leukocytes. Previous studies suggest sCD14 as potential marker for inflammatory diseases, but their potential role in pediatric lung diseases remained elusive. Therefore, we examined the expression, regulation and significance of sCD14 and mCD14 in pediatric lung diseases.

Methods

sCD14 levels were quantified in serum and bronchoalveolar lavage fluid (BALF) of children with infective (pneumonia, cystic fibrosis, CF) and non-infective (asthma) inflammatory lung diseases and healthy control subjects by ELISA. Membrane CD14 expression levels on monocytes in peripheral blood and on alveolar macrophages in BALF were quantified by flow cytometry. In vitro studies were performed to investigate which factors regulate sCD14 release and mCD14 expression.

Results

sCD14 serum levels were specifically increased in serum of children with pneumonia compared to CF, asthma and control subjects. In vitro, CpG induced the release of sCD14 levels in a protease-independent manner, whereas LPS-mediated mCD14 shedding was prevented by serine protease inhibition.

Conclusions

This study demonstrates for the first time the expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases and suggests sCD14 as potential marker for pneumonia in children.     

Initial development and structure of biofilms on microbial fuel cell anodes

Background  

Microbial fuel cells (MFCs) rely on electrochemically active bacteria to capture the chemical energy contained in organics and convert it to electrical energy. Bacteria develop biofilms on the MFC electrodes, allowing considerable conversion capacity and opportunities for extracellular electron transfer (EET). The present knowledge on EET is centred around two Gram-negative models, i.e. Shewanella and Geobacter species, as it is believed that Gram-positives cannot perform EET by themselves as the Gram-negatives can. To understand how bacteria form biofilms within MFCs and how their development, structure and viability affects electron transfer, we performed pure and co-culture experiments.     

Relationship between external stink bug (Hemiptera: Pentatomidae) boll-feeding symptoms and internal boll damage with respect to cotton lint gin-out and fiber quality

Cotton, Gossypium hirsutum L., bolls from 17 field locations in northeastern North Carolina and southeastern Virginia, having 20% or greater internal boll damage, were studied to determine the relationship between external feeding symptoms and internal damage caused by stink bug (Hemiptera: Pentatomidae) feeding. In 2006 and 2007, two cohorts of 100 bolls each were sampled at all field locations. The first cohort was removed as bolls reached approximately quarter size in diameter (2.4 cm). External and internal symptoms of stink bug feeding were assessed and tabulated. Concurrent to when the first cohort was collected, a second cohort of quarter-size-diameter bolls was identified, tagged, examined in situ for external feeding symptoms (sunken lesions), and harvested at the black seed coat stage. Harvested bolls were assessed for internal damage and locks were categorized (undamaged, minor damage, or major damage), dried, and ginned. Lint samples from each damage category were submitted for high volume instrument and advanced fiber information system quality analyses. Significant, moderately strong Pearson correlation coefficients existed between number of external stink bug feeding lesions and internal damage. Pearson correlation of total external lesions with total internal damage was stronger than any correlation among the other single components compared. Predictability plots indicated a rapid increase in relationship strength when relating external stink bug lesions to internal damage as the number of external lesions increased. Approximately 90% predictability of internal damage was achieved with four (2006) or six (2007) external lesions per boll. Gin-turnout and fiber quality decreased with increasing intensity of internal stink bug damage.     

Selection for resistance to imidacloprid in the house fly (Diptera: Muscidae)

The house fly, Musca domestica L. (Diptera: Muscidae), continues to be a primary pest of livestock facilities worldwide. This pest also has shown a propensity for pesticide resistance development when under high selection pressures. In this study the house fly strain FDm was created by a 20% contribution from each of five colonies collected from dairies in Florida with known imidacloprid resistance. The FDm strain was used to evaluate the level ofimidacloprid resistance after five selections near the LC70 value of each selected generation. Overall, the mean selection mortality was 72.7, with males being considerably more susceptible than females. The unselected (F0) FDm strain showed considerable susceptibility to imidacloprid after its creation, compared with the five parental strains. Between 9500 and 14,000 virgin house flies were used in each selection. After the fifth and final selection, a 331-fold increase in imidacloprid resistance at the LC70 was observed over the parental FDm strain. In parallel studies, the FDm strain showed increasing tolerance of the commercial imidacloprid product QuickBayt. These results suggest that livestock producers should use caution when choosing pesticides and consider rotating fly baits, as is encouraged with other pesticide treatment regimes on farms.     

Identification and Biochemical Characterization of Molybdenum Cofactor-binding Proteins from Arabidopsis thaliana

The molybdenum cofactor (Moco) forms part of the catalytic center in all eukaryotic molybdenum enzymes and is synthesized in a highly conserved pathway. Among eukaryotes, very little is known about the processes taking place subsequent to Moco biosynthesis, i.e. Moco transfer, allocation, and insertion into molybdenum enzymes. In the model plant Arabidopsis thaliana, we identified a novel protein family consisting of nine members that after recombinant expression are able to bind Moco with K_D values in the low micromolar range and are therefore named Moco-binding proteins (MoBP). For two of the nine proteins atomic structures are available in the Protein Data Bank. Surprisingly, both crystal structures lack electron density for the C terminus, which may indicate a high flexibility of this part of the protein. C-terminal truncated MoBPs showed significantly decreased Moco binding stoichiometries. Experiments where the MoBP C termini were exchanged among MoBPs converted a weak Moco-binding MoBP into a strong binding MoBP, thus indicating that the MoBP C terminus, which is encoded by a separate exon, is involved in Moco binding. MoBPs were able to enhance Moco transfer to apo-nitrate reductase in the Moco-free Neurospora crassa mutant nit-1. Furthermore, we show that the MoBPs are localized in the cytosol and undergo protein-protein contact with both the Moco donor protein Cnx1 and the Moco acceptor protein nitrate reductase under in vivo conditions, thus indicating for the MoBPs a function in Arabidopsis cellular Moco distribution.     

Early detection of population fragmentation using linkage disequilibrium estimation of effective population size

Population subdivision due to habitat loss and modification, exploitation of wild populations and altered spatial population dynamics is of increasing concern in nature. Detecting population fragmentation is therefore crucial for conservation management. Using computer simulations, we show that a single sample estimator of N _e based on linkage disequilibrium is a highly sensitive and promising indicator of recent population fragmentation and bottlenecks, even with some continued gene flow. For example, fragmentation of a panmictic population of N _e = 1,000 into demes of N _e = 100 can be detected with high probability after a single generation when estimates from this method are compared to prefragmentation estimates, given data for ~20 microsatellite loci in samples of 50 individuals. We consider a range of loci (10–40) and individuals (25–100) typical of current studies of natural populations and show that increasing the number of loci gives nearly the same increase in precision as increasing the number of individuals sampled. We also evaluated effects of incomplete fragmentation and found this N _e-reduction signal is still apparent in the presence of considerable migration (m ~ 0.10–0.25). Single-sample genetic estimates of N _e thus show considerable promise for early detection of population fragmentation and decline.     

Synthesis of “Difficult” Fluorescence Quenched Substrates of Granzyme C

The synthesis of fluorescence quenched peptide substrates of granzyme C is presented. These peptides which incorporate some unusual amino acids and have “difficult sequence” elements, in some cases could not be prepared by standard Fmoc-based SPPS. Application of three different contemporary strategies, namely the use of pseudoproline dipeptides, PEG-based solid supports and the application of microwave heating were able to provide for successful synthesis of our desired substrate peptides.