Lipoproteins of Listeria monocytogenes are critical for virulence and TLR2-mediated immune activation

Numerous cell surface components of Listeria influence and regulate innate immune recognition and virulence. Here, we demonstrate that lipidation of prelipoproteins in Listeria monocytogenes is required to promote NF-kappaB activation via TLR2. In HeLa cells transiently expressing TLR2, L. monocytogenes and Listeria innocua mutants lacking the prolipoprotein diacylglyceryl transferase (lgt) gene are unable to induce TLR2-dependent activation of NF-kappaB, a property intrinsic to their isogenic parental strains. TLR2-dependent immune recognition is directed to secreted, soluble lipoproteins as evidenced by the sensitivity of the response to lipoprotein lipase. Studies of bone marrow-derived macrophages of C57BL/6 wild-type and TLR2-deficient mice infected with wild-type and lgt mutant strains indicate that the absence of host TLR2 receptor signaling has consequences similar to those of the absence of the bacterial TLR2 ligand, i.e., a delay in cellular immune responses directed toward the bacterium. Infection studies with the wild-type and TLR2(-/-) mice indicated attenuation of the lgt deletion mutant in both mouse strains, implying multiple roles of lipoproteins during infection. Further characterization of the Delta lgt mutant indicated that it is impaired for both invasion and intracellular survival and exhibits increased susceptibility to cationic peptides. Our studies identify lipoproteins as the immunologically active ligand of TLR2 and assign a critical role for this receptor in the recognition of these bacteria during infection, but they also reveal the overall importance of the lipoproteins for the pathogenicity of Listeria.     

Microwave-enhanced enzyme reaction for protein mapping by mass spectrometry: a new approach to protein digestion in minutes

Accelerated proteolytic cleavage of proteins under controlled microwave irradiation has been achieved. Selective peptide fragmentation by endoproteases trypsin or lysine C led to smaller peptides that were analyzed by matrix-assisted laser desorption ionization (MALDI) or liquid chromatography-electrospray ionization (LC-ESI) techniques. The efficacy of this technique for protein mapping was demonstrated by the mass spectral analyses of the peptide fragmentation of several biologically active proteins, including cytochrome c, ubiquitin, lysozyme, myoglobin, and interferon alpha-2b. Most important, using this novel approach digestion of proteins occurs in minutes, in contrast to the hours required by conventional methods.     

Algorithms for the evaluation of radiation induced chromosome aberration yields per cell from flow karyotypes

CHO-K1 cells were irradiated in G0/G1-phase with 150 kV X-rays. Single chromosomes isolated from metaphase cells and stained with DNA intercalating dye DAPI were analyzed in the ICP 22 with a modified flow chamber. In order to study dose-dependent changes in the flow karyotypes, they were split into peak- and background-portions by an iterative fit algorithm. As in a first approach, estimates of the frequencies of chromosome lesions were derived from an evaluation of the dose-dependent reduction in peak contents. The number of radiation-induced lesions per chromosome was found to be proportional to its length. As a second approach, the number of fluorescence events in the histogram background was corrected for non-chromosomal debris and evaluated interms of chromosome aberration frequency per cell, which was consistent with the yields of dicentric chromosomes and acentric fragments observed in microscopic investigations. As a third approach, lesion frequencies were calculated from the corrected background light sum in the karyotypes, utilizing a Monte Carlo model to simulate the effect of aberration formation on the flow histogram. The results indicate that the number of chromosome lesions observed by flow cytometry can be quantitatively related to the yield of structural chromosome aberrations detected by microscopic analysis. Dose-effect relations and split-dose kinetics are given as examples demonstrating the usefulness of this technique in radiobiology. Time saving compared to microscopic analysis was of the order of 90%.     

Ozone-induced gene expression occurs via ethylene-dependent and -independent signalling

Recent studies suggest that ethylene is involved in signalling ozone-induced gene expression. We show here that application of ozone increased glucuronidase (GUS) expression of chimeric reporter genes regulated by the promoters of the tobacco class I -1,3-glucanases (GLB and Gln2) and the grapevine resveratrol synthase (Vst1) genes in transgenic tobacco leaves. 5-deletion analysis of the class I -1,3-glucanase promoter revealed that ozone-induced gene regulation is mainly mediated by the distal enhancer region containing the positively acting ethylene-responsive element (ERE). In addition, application of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, blocked ozone-induced class I -1,3-glucanase promoter activity. Enhancer activity and ethylene-responsiveness depended on the integrity of the GCC boxes, cis-acting elements present in the ERE of the class I -1,3-glucanase and the basic-type pathogenesis-related PR-1 protein (PRB-1b) gene promoters. The minimal PRB-1b promoter containing only the ERE with intact GCC boxes, was sufficient to confer 10-fold ozone inducibility to a GUS-reporter gene, while a substitution mutation in the GCC box abolished ozone responsiveness. The ERE region of the class I -1,3-glucanase promoter containing two intact GCC boxes confered strong ozone inducibility to a minimal cauliflower mosaic virus (CaMV) 35S RNA promoter, whereas two single-base substitution in the GCC boxes resulted in a complete loss of ozone inducibility. Taken together, these data strongly suggest that ethylene is signalling ozone-induced expression of class I -1,3-glucanase and PRB-1b genes. Promoter analysis of the stilbene synthase Vst1 gene unravelled different regions for ozone and ethylene-responsiveness. Application of 1-MCP blocked ethylene-induced Vst1 induction, but ozone induction was not affected. This shows that ozone-induced gene expression occurs via at least two different signalling mechanisms and suggests an additional ethylene independent signalling pathway for ozone-induced expression of genes involved in phytoalexin biosynthesis.     

Rapid Identification and Typing of Listeria Species by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Listeria monocytogenes is a food-borne pathogen that is the causative agent of human listeriosis, an opportunistic infection that primarily infects pregnant women and immunologically compromised individuals. Rapid, accurate discrimination between Listeria strains is essential for appropriate therapeutic management and timely intervention for infection control. A rapid method involving matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) that shows promise for identification of Listeria species and typing and even allows for differentiation at the level of clonal lineages among pathogenic strains of L. monocytogenes is presented. A total of 146 strains of different Listeria species and serotypes as well as clinical isolates were analyzed. The method was compared with the pulsed-field gel electrophoresis analysis of 48 Listeria strains comprising L. monocytogenes strains isolated from food-borne epidemics and sporadic cases, isolates representing different serotypes, and a number of Listeria strains whose genomes have been completely sequenced. Following a short inactivation/extraction procedure, cell material from a bacterial colony was deposited on a sample target, dried, overlaid with a matrix necessary for the MALDI process, and analyzed by MALDI-TOF MS. This technique examines the chemistry of major proteins, yielding profile spectra consisting of a series of peaks, a characteristic “fingerprint” mainly derived from ribosomal proteins. Specimens can be prepared in a few minutes from plate or liquid cultures, and a spectrum can be obtained within 1 minute. Mass spectra derived from Listeria isolates showed characteristic peaks, conserved at both the species and lineage levels. MALDI-TOF MS fingerprinting may have potential for Listeria identification and subtyping and may improve infection control measures.     

Evidence and impact of neutrophil extracellular traps in malignant melanoma

Ulceration of melanoma is associated with neutrophil infiltrates and lower survival rates opposite to non‐ulcerated melanoma. Neutrophils release neutrophil extracellular traps (NETs) that are chromatin structures loaded with antimicrobial proteins. Since NETs have been correlated with tumor progression, we investigated whether NETs appear in melanoma and affect melanoma cells. Indeed, human primary melanoma biopsies revealed neutrophils releasing NETs in all of 27 ulcerated melanomas, whereas NETs were absent in all of 7 non‐ulcerated melanomas. However, the quantity of intratumoral NETs did not correlate with tumor progression of melanoma. Interestingly, in vitro assays showed that melanoma cells attach to NETs via integrin‐mediated adhesion and that NETs inhibit tumor cell migration. Moreover, co‐culturing of NETs and melanoma cells had a cytotoxic effect on melanoma cells resulting in necrosis. Hence, we discovered in vitro an antineoplastic role of NETs in melanoma.     

Swim Speed,Behavior, and Movement of North Atlantic Right Whales (Eubalaena glacialis) in Coastal Waters of Northeastern Florida,USA

In a portion of the coastal waters of northeastern Florida, North Atlantic right whales (Eubalaena glacialis) occur close to shore from December through March. These waters are included within the designated critical habitat for right whales. Data on swim speed, behavior, and direction of movement – with photo-identification of individual whales – were gathered by a volunteer sighting network working alongside experienced scientists and supplemented by aerial observations. In seven years (2001–2007), 109 tracking periods or “follows” were conducted on right whales during 600 hours of observation from shore-based observers. The whales were categorized as mother-calf pairs, singles and non-mother-calf pairs, and groups of 3 or more individuals. Sample size and amount of information obtained was largest for mother-calf pairs. Swim speeds varied within and across observation periods, individuals, and categories. One category, singles and non mother-calf pairs, was significantly different from the other two – and had the largest variability and the fastest swim speeds. Median swim speed for all categories was 1.3 km/h (0.7 kn), with examples that suggest swim speeds differ between within-habitat movement and migration-mode travel. Within-habitat right whales often travel back-and-forth in a north-south, along-coast, direction, which may cause an individual to pass by a given point on several occasions, potentially increasing anthropogenic risk exposure (e.g., vessel collision, fishing gear entanglement, harassment). At times, mothers and calves engaged in lengthy stationary periods (up to 7.5 h) that included rest, nursing, and play. These mother-calf interactions have implications for communication, learning, and survival. Overall, these behaviors are relevant to population status, distribution, calving success, correlation to environmental parameters, survey efficacy, and human-impacts mitigation. These observations contribute important parameters to conservation biology, predictive modeling, and management. However, while we often search for predictions, patterns, and means, the message here is also about variability and the behavioral characteristics of individual whales.     

Evaluation of biological and biorational control tactics for suppression of Nantucket pine tip moth damage in Virginia pine Christmas trees

A study was performed to evaluate the potential of the egg parasitoid Trichogramma exiguum Pinto and Platner (Hymenoptera: Trichogrammatidae), the newly registered insect growth regulator (IGR) tebufenozide (Confirm), and a modified spray technique (top whorl only pesticide application) for suppression of Nantucket pine tip moth, Rhyacionia frustrana (Comstock) (Lepidoptera: Tortricidae), damage in Virginia pine, Pinus virginiana Mill., Christmas trees. Augmentative releases of T. exiguum failed to increase parasitism levels in release plots compared with controls, and significant reduction in tip moth damage did not occur. High predation levels on released T. exiguum may have contributed to the failure of parasitoid augmentations. Whole-tree and top whorl tebufenozide treatments provided significantly greater damage control than corresponding applications of acephate (Orthene), a commonly used pesticide in Christmas trees. Damage to trees receiving whole-tree chemical applications did not differ significantly from trees receiving top whorl treatments for most measurements of damage. Top whorl chemical treatments resulted in a 67% reduction in time required for application and a 70% reduction in pesticide used.