Method for Spiking Soil Samples with Organic Compounds

We examined the harmful side effects on indigenous soil microorganisms of two organic solvents, acetone and dichloromethane, that are normally used for spiking of soil with polycyclic aromatic hydrocarbons for experimental purposes. The solvents were applied in two contamination protocols to either the whole soil sample or 25% of the soil volume, which was subsequently mixed with 75% untreated soil. For dichloromethane, we included a third protocol, which involved application to 80% of the soil volume with or without phenanthrene and introduction of Pseudomonas fluorescens VKI171 SJ132 genetically tagged with luxAB::Tn5. For both solvents, application to the whole sample resulted in severe side effects on both indigenous protozoa and bacteria. Application of dichloromethane to the whole soil volume immediately reduced the number of protozoa to below the detection limit. In one of the soils, the protozoan population was able to recover to the initial level within 2 weeks, in terms of numbers of protozoa; protozoan diversity, however, remained low. In soil spiked with dichloromethane with or without phenanthrene, the introduced P. fluorescens VKI171 SJ132 was able to grow to a density 1,000-fold higher than in control soil, probably due mainly to release of predation from indigenous protozoa. In order to minimize solvent effects on indigenous soil microorganisms when spiking native soil samples with compounds having a low water solubility, we propose a common protocol in which the contaminant dissolved in acetone is added to 25% of the soil sample, followed by evaporation of the solvent and mixing with the remaining 75% of the soil sample.     

A functional genomics approach identifies candidate effectors from the aphid species Myzus persicae (green peach aphid)

Aphids are amongst the most devastating sap-feeding insects of plants. Like most plant parasites, aphids require intimate associations with their host plants to gain access to nutrients. Aphid feeding induces responses such as clogging of phloem sieve elements and callose formation, which are suppressed by unknown molecules, probably proteins, in aphid saliva. Therefore, it is likely that aphids, like plant pathogens, deliver proteins (effectors) inside their hosts to modulate host cell processes, suppress plant defenses, and promote infestation. We exploited publicly available aphid salivary gland expressed sequence tags (ESTs) to apply a functional genomics approach for identification of candidate effectors from Myzus persicae (green peach aphid), based on common features of plant pathogen effectors. A total of 48 effector candidates were identified, cloned, and subjected to transient overexpression in Nicotiana benthamiana to assay for elicitation of a phenotype, suppression of the Pathogen-Associated Molecular Pattern (PAMP)-mediated oxidative burst, and effects on aphid reproductive performance. We identified one candidate effector, Mp10, which specifically induced chlorosis and local cell death in N. benthamiana and conferred avirulence to recombinant Potato virus X (PVX) expressing Mp10, PVX-Mp10, in N. tabacum, indicating that this protein may trigger plant defenses. The ubiquitin-ligase associated protein SGT1 was required for the Mp10-mediated chlorosis response in N. benthamiana. Mp10 also suppressed the oxidative burst induced by flg22, but not by chitin. Aphid fecundity assays revealed that in planta overexpression of Mp10 and Mp42 reduced aphid fecundity, whereas another effector candidate, MpC002, enhanced aphid fecundity. Thus, these results suggest that, although Mp10 suppresses flg22-triggered immunity, it triggers a defense response, resulting in an overall decrease in aphid performance in the fecundity assays. Overall, we identified aphid salivary proteins that share features with plant pathogen effectors and therefore may function as aphid effectors by perturbing host cellular processes.     

Extensive introgressive hybridization within the northern oriole group (Genus Icterus) revealed by three‐species isolation with migration analysis

Until recently, studies of divergence and gene flow among closely‐related taxa were generally limited to pairs of sister taxa. However, organisms frequently exchange genes with other non‐sister taxa. The “northern oriole” group within genus Icterus exemplifies this problem. This group involves the extensively studied hybrid zone between Baltimore oriole (Icterus galbula) and Bullock's oriole (I. bullockii), an alleged hybrid zone between I. bullockii and black‐backed oriole (I. abeillei), and likely mtDNA introgression between I. galbula and I. abeillei. Here, we examine the divergence population genetics of the entire northern oriole group using a multipopulation Isolation‐with‐Migration (IM) model. In accordance with Haldane's rule, nuclear loci introgress extensively beyond the I. galbula–I. bullockii hybrid zone, while mtDNA does not. We found no evidence of introgression between I. bullockii and I. abeillei or between I. galbula and I. abeillei when all three species were analyzed together in a three‐population model. However, traditional pairwise analysis suggested some nuclear introgression from I. abeillei into I. galbula, probably reflecting genetic contributions from I. bullockii unaccounted for in a two‐population model. Thus, only by including all members of this group in the analysis was it possible to rigorously estimate the level of gene flow among these three closely related species.     

Embryonic cell lineage of the marine nematode Pellioditis marina

We describe the complete embryonic cell lineage of the marine nematode Pellioditis marina (Rhabditidae) up to somatic muscle contraction, resulting in the formation of 638 cells, of which 67 undergo programmed cell death. In comparison with Caenorhabditis elegans, the overall lineage homology is 95.5%; fate homology, however, is only 76.4%. The majority of the differences in fate homology concern nervous, epidermal, and pharyngeal tissues. Gut and, remarkably, somatic muscle is highly conserved in number and position. Partial lineage data from the slower developing Halicephalobus sp. (Panagrolaimidae) reveal a lineage largely, but not exclusively, built up of monoclonal sublineage blocs with identical fates, unlike the polyclonal fate distribution in C. elegans and P. marina. The fate distribution pattern in a cell lineage could be a compromise between minimizing the number of specification events by monoclonal specification and minimizing the need for migrations by forming the cells close at their final position. The latter could contribute to a faster embryonic development. These results reveal that there is more than one way to build a nematode.     

Polymer coated liposomes for use in the oral cavity – a study of the in vitro toxicity,effect on cell permeability and interaction with mucin

In this study we investigated the in vitro toxicity, impact on cell permeability and mucoadhesive potential of polymer-coated liposomes intended for use in the oral cavity. A TR146 cell line was used as a model. The overall aim was to end up with a selection of safe polymer coated liposomes with promising mucoadhesive properties for drug delivery to the oral cavity. The following polymers were tested: chitosan, low-methoxylated pectin (LM-pectin), high-methoxylated pectin (HM-pectin), amidated pectin (AM-pectin), Eudragit, poly(N-isopropylacrylamide-co-methacrylic acid) (p(NIPAAM-co-MAA)), hydrophobically modified hydroxyethyl cellulose (HM-HEC), and hydrophobically modified ethyl hydroxyethyl cellulose (HM-EHEC). With chitosan as an exception, all the systems exhibited no significant effect on cell viability and permeability at the considered concentrations. Additionally, all the formulations showed to a varying degree an interaction with mucin (BSM type I-S); the positively charged formulations exhibited the strongest interaction, while the negatively and neutrally charged formulations displayed a moderate or low interaction. The ability to interact with mucin makes all the liposomal formulations promising for oromucosal administration. Although the chitosan-coated liposomes affected the cell viability, this formulation also influenced the cell permeability, which makes it an interesting candidate for systemic drug delivery from the oral cavity.     

The prodomain of a secreted hydrophobic mini-protein facilitates its export from the endoplasmic reticulum by hitchhiking on sorting receptors

Misfolded secretory proteins are retained in the endoplasmic reticulum (ER) by quality control mechanisms targeted to exposed hydrophobic surfaces. Paradoxically, certain conotoxins expose extensive hydrophobic surfaces upon folding to their bioactive structures. How then can such secreted mini-proteins traverse the secretory pathway? Here we show that secretion of the hydrophobic conotoxin-TxVI is strongly dependent on its propeptide domain, which enhances TxVI export from the ER. The propeptide domain interacts with sorting receptors from the sortilin Vps10p domain family. The sortilin-TxVI interaction occurs in the ER, and sortilin facilitates export of TxVI from the ER to the Golgi. Thus, the prodomain in a secreted hydrophobic protein acts as a tag that can facilitate its ER export by a hitchhiking mechanism.     

Bacillus thuringiensis in Fecal Samples from Greenhouse Workers after Exposure to B. thuringiensis-Based Pesticides

In a study of occupational exposure to Bacillus thuringiensis, 20 exposed greenhouse workers were examined for Bacillus cereus-like bacteria in fecal samples and on biomonitoring filters. Bacteria with the following characteristics were isolated from eight individuals: intracellular crystalline inclusions characteristic of B. thuringiensis, genes for and production of B. cereus enterotoxins, and positivity for cry11 as determined by PCR. DNA fingerprints of the fecal isolates were identical to those of strains isolated from the commercial products used. Work processes (i.e., spraying) correlated with the presence of B. thuringiensis in the fecal samples (10² to 10³ CFU/g of feces). However, no gastrointestinal symptoms correlated with the presence of B. thuringiensis in the fecal samples.     

Transfer of the Pheromone-Inducible Plasmid pCF10 among Enterococcus faecalis Microorganisms Colonizing the Intestine of Mini-Pigs

A new animal model, the streptomycin-treated mini-pig, was developed in order to allow colonization of defined strains of Enterococcus faecalis in numbers sufficient to study plasmid transfer. Transfer of the pheromone-inducible pCF10 plasmid between streptomycin-resistant strains of E. faecalis OG1 was investigated in the model. The plasmid encodes resistance to tetracycline. Numbers of recipient, donor, and transconjugant bacteria were monitored by selective plating of fecal samples, and transconjugants were subsequently verified by PCR. After being ingested by the mini-pigs, the recipient strain persisted in the intestine at levels between 10⁶ and 10⁷ CFU per g of feces throughout the experiment. The donor strain, which carried different resistance markers but was otherwise chromosomally isogenic to the recipient strain, was given to the pigs 3 weeks after the recipient strain. The donor cells were initially present in high numbers (10⁶ CFU per g) in feces, but they did not persist in the intestine at detectable levels. Immediately after introduction of the donor bacteria, transconjugant cells appeared and persisted in fecal samples at levels between 10³ and 10⁴ CFU per g until the end of the experiment. These observations showed that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from ingested E. faecalis cells to other E. faecalis organisms already present in the intestinal environment and that the plasmid subsequently persisted in the intestine.     

Specific binding of alpha-macroglobulin to complement-type repeat CR4 of the low-density lipoprotein receptor-related protein

The low-density lipoprotein receptor-related protein (LRP) is a large surface receptor that mediates binding and internalization of a large number of structurally and functionally unrelated ligands. The ligand binding sites are located in clusters of complement-type repeats (CR), where the general absence of mutual binding competition suggests that different ligands map to distinct sites. Binding of alpha(2)-macroglobulin-protease complexes to the LRP is mediated by the receptor binding domain (RBD) of alpha(2)-macroglobulin (alpha(2)M). To determine the major binding epitope(s) in the LRP, we generated a complete set of tandem CR proteins spanning the second cluster of CR domains, and identified a binding site for alpha(2)M in the N-terminal part of the cluster comprising CR3-CR6, using ligand blotting and surface plasmon resonance (SPR) analysis. The specific site involved in alpha(2)M recognition resides in the fourth CR domain, CR4, whereas another site is identified in CR5. An acidic epitope in CR4 is identified as important for binding alpha(2)M by mutagenesis and SPR analysis. The formation of the complex between the rat alpha(1)-macroglobulin RBD and CR domain pairs is characterized by analytical size-exclusion chromatography, which demonstrates a sufficiently strong interaction between the alpha(1)M RBD and CR34 or CR45 for the isolation of a complex.