Examination of the skin barrier repair/wound healing process using a living skin equivalent model and matrix-assisted laser desorption-ionization-mass spectrometry imaging.

OBJECTIVE: Examination of the skin barrier repair/wound healing process using a living skin equivalent (LSE) model and matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to identify lipids directly involved as potential biomarkers. These biomarkers may be used to determine whether an in vivo wound is going to heal for example if infected. METHODS: An in vitro LSE model was wounded with a scalpel blade and assessed at day 4 post-wounding by histology and MALDI-MSI. Samples were sectioned at wound site and were either formalin-fixed paraffin-embedded (FFPE) for histology or snapped frozen (FF) for MSI analysis. RESULTS: The combination of using an in vitro wounded skin model with MSI allowed the identification of lipids involved in the skin barrier repair/wound healing process. The technique was able to highlight lipids directly in the wound site and distinguish differences in lipid distribution between the epidermis and wound site. CONCLUSION: This novel method of coupling an in vitro LSE with MSI allowed in-depth molecular analysis of the skin barrier repair/wound healing process. The technique allowed the identification of lipids directly involved in the skin barrier repair/wound healing process, indicating these biomarkers may be potentially be used within the clinic. These biomarkers will help to determine, which stage of the skin barrier repair/wound healing process the wound is in to provide the best treatment.

European earwig (Forficula auricularia) as a novel host for the entomopathogenic nematode Steinernema carpocapsae.

The natural history of many entomopathogenic nematode species remains unknown, despite their wide commercial availability as biological control agents. The ambushing entomopathogenic nematode, Steinernema carpocapsae, and the introduced European earwig, Forficula auricularia, forage on the soil surface. Since they likely encounter one another in nature, we hypothesized that earwigs are susceptible to nematode infection. In the laboratory, the LC(50) for F. auricularia was 226 S. carpocapsae/earwig and the reproductive potential was 123.5 infective juvenile nematodes/mg tissue. This susceptibility depended on host body size with significantly higher mortality rates seen in larger earwigs. In a study of host recognition behavior, S. carpocapsae infective juveniles responded to earwig cuticle as strongly as they did to Galleria mellonella cuticle. We also found that earwigs exposed to S. carpocapsae cleaned and scratched their front, middle and back legs significantly more than controls. Coupled with previous field data, these findings lead us to suggest that F. auricularia may be a potential host for S. carpocapsae.

Effect of insect cadaver desiccation and soil water potential during rehydration on entomopathogenic nematode (Rhabditida: Steinernematidae and Heterorhabditidae) production and virulence.

We examined the influence of insect cadaver desiccation on the virulence and production of entomopathogenic nematodes (EPNs), common natural enemies of many soil-dwelling insects. EPNs are often used in biological control, and we investigated the feasibility of applying EPNs within desiccated insect cadavers. Desiccation studies were conducted using the factitious host, Galleria mellonella (Lepidoptera: Pyralidae, wax moth larvae) and three EPN species (Heterorhabditis bacteriophora 'HB1', Steinernema carpocapsae 'All', and Steinernema riobrave). Weights of individual insect cadavers were tracked daily during the desiccation process, and cohorts were placed into emergence traps when average mass losses reached 50%, 60%, and 70% levels. We tracked the proportion of insect cadavers producing infective juveniles (IJs), the number and virulence of IJs produced from desiccated insect cadavers, and the influence of soil water potentials on IJ production of desiccated insect cadavers. We observed apparent differences in the desiccation rate of the insect cadavers among the three species, as well as apparent differences among the three species in both the proportion of insect cadavers producing IJs and IJ production per insect cadaver. Exposure of desiccated insect cadavers to water potentials greater than -2.75 kPa stimulated IJ emergence. Among the nematode species examined, H. bacteriophora exhibited lower proportions of desiccated insect cadavers producing IJs than the other two species. Desiccation significantly reduced the number of IJs produced from insect cadavers. At the 60% mass loss level, however, desiccated insect cadavers from each of the three species successfully produced IJs when exposed to moist sand, suggesting that insect cadaver desiccation may be a useful approach for biological control of soil insect pests.

Effects of prasterone (dehydroepiandrosterone) on markers of cardiovascular risk and bone turnover in premenopausal women with systemic lupus erythematosus: a pilot study.

DHEA (dehydroepiandrosterone) is a weak androgen with proposed efficacy in the treatment of mild to moderate lupus, and possible beneficial effects on cardiovascular risk and bone mineral density. We hypothesized that treatment with 200 mg a day of Prasterone (DHEA) would improve pre-clinical measures of atherosclerosis: flow-mediated dilatation (FMD), nitroglycerin-mediated dilatation (NMD), and circulating apoptotic endothelial cells (CD 146(AnnV +)), as well markers of bone metabolism. Thirteen premenopausal female patients with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)

European corn borer (Lepidoptera: Crambidae) stalk tunneling on root-knot nematode (Tylenchida: Heteroderidae) fitness on corn.

Greenhouse experiments were conducted in 2004-2006 to examine the reciprocal effects of aboveground herbivory by European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), and belowground herbivory by root-knot nematode, Meloidogyne incognita Chitwood (Tylenchida: Heteroderidae), on one another at three corn, Zea mays L., growth stages. Two experiments were conducted to study the effect of aboveground herbivory by O. nubilalis on the number of M. incognita juvenile penetration/root system and eggs/root system. In the first experiment, the O. nubilalis infestation level by plant growth stage main effect interaction was not significant for either M. incognita juvenile penetration or eggs. The overall effect of stalk tunneling by O. nubilalis resulted in 48.9% fewer juvenile penetration and 40.0% fewer eggs than in the respective controls. In the second experiment, the main effects interaction was significant for juvenile penetration (P = 0.0422) and eggs (P = 0.0134). At the eight- and 10-leaf growth stages, the combined effect of one and three O. nubilalis larvae per plant resulted in 41.2 and 44.7% significantly fewer juvenile penetration than in the respective controls. Similarly, the combined effect of stalk tunneling (with the exception of one larvae per plant at the 10-leaf growth stage) at the six-, eight-, and 10-leaf growth stages resulted in 46.3, 53.3, and 55.2% fewer eggs than in the respective controls. In all instances, M. incognita juvenile penetration and eggs were significantly negatively correlated with O. nubilalis tunnel length. In a reciprocal experiment conducted two times, no significant (P > 0.05) effect of M. incognita inoculation level on stalk tunneling was found in either experiment.

Responses of the entomopathogenic nematode, Steinernema riobrave to its insect hosts, Galleria mellonella and Tenebrio molitor.

Potential hosts for infective juveniles of entomopathogenic nematodes can vary considerably in quality based on the characteristics of the host species/stage, physiological status (e.g. stress, feeding on toxins), and infection status (heterospecific or conspecific infection). In this study, we investigated responses of the entomopathogenic nematode Steinernema riobrave to hosts (Galleria mellonella or Tenebrio molitor) that were previously parasitized with conspecifics or injected with the nematode-symbiotic bacterium, Xenorhabdus sp., to determine if there is a preference for previously parasitized/injected hosts and when this preference might occur. In no-choice bioassays, the number of juveniles infecting both host species decreased with increasing time post-infection. However, infective juveniles continued to infect previously parasitized hosts up to 72 h. Significant preference was exhibited by S. riobrave for 24 h post-infection G. mellonella larvae over uninfected, and by 24 h post-injection G. mellonella larvae over 48 h post-injection larvae. No significant preference was exhibited by S. riobrave for T. molitor hosts previously parasitized with conspecifics or those injected with bacteria in any treatment combination. Such preference for, or continued infection of parasitized insects, has the potential to impact nematode efficacy.

Attraction behaviour of three entomopathogenic nematode species towards infected and uninfected hosts.

Entomopathogenic nematode infective juveniles are likely to encounter both uninfected and infected insects and host quality depends on the stage of the infection. We hypothesized that nematode response to infected hosts will change over the course of an infection. Here, we tested this hypothesis by focusing on the influence of host infection status on long-range attraction to host volatile cues. The attraction response of 3 nematode species (Steinernema carpocapsae, S. glaseri and S. riobrave) with different foraging strategies to infected and uninfected insects (Galleria mellonella and Tenebrio molitor) was tested at 24 h intervals from start of infection to emergence of infective juveniles from depleted host. As expected, based on their foraging strategies, S. carpocapsae was not very responsive to hosts, S. glaseri was highly responsive and S. riobrave was intermediate. Generally, the level of attraction did not change with time after infection and was similar between infected and uninfected hosts. An exception was S. glaseri infected T. molitor, which tended to be less attractive to S. glaseri than uninfected hosts. These results suggest that any influence of host infection status on infection behaviour is occurring at subsequent steps in the host-infection process than host attraction, or involves non-volatile cues.

Dispersal, Infectivity and Sex Ratio of Early- or Late-Emerging Infective Juveniles of the Entomopathogenic Nematode Steinernema carpocapsae.

Differences in activity between infective juveniles (IJ) of the entomopathogenic nematode Steinernema carpocapsae that emerged directly from cadavers onto either a sand or agar substrate compared with those emerging from a cadaver into water and then being placed on the same substrate are known to occur. Differences between S. carpocapsae IJ that emerged directly from a cadaver vs. those that emerged from a cadaver and held in water were further elucidated. Dispersed and non-dispersed IJ from a cadaver were compared with those held in water between two time periods designated as early- (first two days) or late-emerging IJ (seventh day). A significantly greater proportion of early-emerging IJ from the cadaver treatment dispersed, compared with late-emerging IJ from a cadaver or either group of emerging IJ held in aqueous suspension. Moreover, IJ from cadavers were more infectious than those from the aqueous suspensions, and IJ that dispersed were less infectious than those that did not disperse. IJ that emerged early were mostly males, whereas those that emerged late were mostly females. For the non-dispersed IJ, most that emerged early were males, and those that emerged later were females, but among dispersing IJ, there was no difference in sex ratio between early- and late-emerging nematodes.

Effect of Entomopathogenic Nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) on Meloidogyne mayaguensis Rammah and Hirschmann (Tylenchida: Meloidoginidae) Infection in Tomato Plants.

Some studies suggest that entomopathogenic nematodes (EPN) affect plant-parasitic nematode populations. Here, the effects of live and dead IJ of Heterorhabditis bacteriophora JPM4, H. baujardi LPP7, Steinernema feltiae SN and S. carpocapsae All were evaluated against eggs and J2 of the plant-parasitic nematode Meloidogyne mayaguensis. According to treatment, 100 IJ were applied with 350 eggs, 350 J2 or 175 eggs + 175 J2 to tomato plants. Bioassays were conducted in March to May and repeated in September to November 2005. Both experiments lasted 9 weeks, and the variable evaluated was number of galls per plant. When eggs were used for infections in the first trial, plants exhibited lower gall number compared to control when live and dead H. baujardi IJ and live S. feltiae IJ were added (9.7, 4.5, 7.3 and 85.7 galls, respectively). In the second trial, live S. feltiae and S. carpocapasae IJ influenced gall formation compared to control (14.33, 14.57 and 168.02 galls, respectively). When J2 were used for infections, plants with live H. baujardi IJ presented less galls when compared to control in both trials (38.3 and 355.7 galls in the first trial and 145.2 and 326.2 in the second one, respectively). Infection with a mixture of J2 and eggs resulted in fewer galls than when live S. feltiae IJ were present in both trials, compared to control (38.3 and 44.2 galls vs. 275.3 and 192.2 galls, respectively). We conclude that H. baujardi and S. feltiae apparently may be inhibiting egg hatching and J2 infection.

The ecology and biodemography of Caenorhabditis elegans.

The nematode Caenorhabditis elegans is a well-known model organism for research on aging and life span, but very little is known about its ecology and natural history. The strain N2 is the standard wild-type C. elegans and arose from the progeny of a single hermaphrodite. Since N2 has passed through laboratory culture, the influence of inadvertent selection and genetic drift on C. elegans strains kept in culture is unclear. Because it seems that other wild-type strains have also been subject to lengthy laboratory culture, the life span and biodemography of wild-caught C. elegans is of interest. We recovered C. elegans from snails (Helix aspersa) in ca. 50% of the California locations where we made collections. In experiments with one of the wild-caught isolates, it differed in important demographic properties, mortality, fertility, fitness, and activity patterns, from the standard N2 strain, when both strains were evaluated in a common laboratory environment. The differences were not only statistically significant; they were also large enough to be biologically important. The differences are consistent with the hypothesis that N2 has adapted to laboratory conditions.

Revising the standard wisdom of C. elegans natural history: ecology of longevity.

Here, we consider that most of the research concerning Caenorhabditis elegans has been laboratory focused and that only limited research has directly considered the worm's biology relative to its natural history in the wild. We describe that, although the worm has traditionally been considered a soil nematode, we could not find it in soil but frequently recovered it from snails. Finally, we discuss how a better understanding of the natural history of C. elegans may enhance its usefulness as a model organism for studying aging and other phenomena.

Effect of Entomopathogenic Nematodes on Mesocriconema xenoplax Populations in Peach and Pecan.

The effect of Steinernema riobrave and Heterorhabditis bacteriophora on population density of Mesocriconema xenoplax in peach was studied in the greenhouse. Twenty-one days after adding 112 M. xenoplax adults and juveniles/1,500 cm(3) soil to the soil surface of each pot, 50 infective juveniles/cm(2) soil surface of either S. riobrave or H. bacteriophora were applied. Another entomopathogenic nematode application of the same density was administered 3 months later. The experiment was repeated once. Mesocriconema xenoplax populations were not suppressed (P

Effect of application method on fitness of entomopathogenic nematodes emerging at different times.

The entomopathogenic nematode species Steinernema feltiae and Heterorhabditis bacteriophora were compared for survival and infectivity of infective juveniles (IJ) collected with a standard White trap (i.e., emerging from hosts and accumulating in water) and later applied to sand (treatment A) to IJ allowed to emerge from hosts into sand (treatment C). Percentage IJ survival and infectivity was compared between treatments for S. feltiae IJ that emerged between days 1 to 3 and days 4 to 6. For H. bacteriophora, percentage IJ survival and infectivity was compared between treatments only for infective juveniles that emerged between days 4 to 6. For S. feltiae IJ percentage survival and infectivity decreased with time (P

Evolution of host search strategies in entomopathogenic nematodes.

There is interspecific variation in infective juvenile behavior within the entomopathogenic nematode genus Steinernema. This variation is consistent with use of different foraging strategies along a continuum between ambush and cruise foraging. To address questions about the evolution of foraging strategy, behavioral and morphological characters were mapped onto a phylogeny of Steinernema. Three species, all in the same clade, were classified as ambushers based on standing bout duration and host-finding ability. One clade of six species were all cruisers based on both host-finding and lack of standing behavior. All species in the ambusher clade had a high rate of jumping, all species in the cruiser clade had no jumping, and most intermediate foragers exhibited some level of jumping. Response to volatile and contact host cues was variable, even within a foraging strategy. Infective juveniles in the ambusher clade were all in the smallest size category, species in the cruiser clade were in the largest size categories, and intermediate foragers tended to be more intermediate in size. We hypothesize that the ancestral Steinernema species was an intermediate forager and that ambush and cruise foraging both evolved at least once in the genus.

Formulation of entomopathogenic nematode-infected cadavers.

Entomopathogenic nematodes are commercially applied in aqueous suspension. These biocontrol agents may also be applied in nematode-infected insect cadavers, but this approach may entail problems in storage and ease of handling. We determined the feasibility of formulating nematode-infected insect cadavers to overcome these hindrances. All experiments were conducted with Heterorhabditis bacteriophora Poinar and Galleria mellonella (L.). Nonformulated cadavers were used as controls. Of 19 formulations tested (including combinations of starches, flours, clays, etc.) 1 (starch-clay combination) was found to adhere to the cadaver and to have no significant deleterious effects on nematode reproduction and infectivity; other formulations exhibited poor adhesion or reduced nematode reproduction. Two formulations enabled cadavers to be partially desiccated without affecting reproduction; other formulations and nonformulated cadavers exhibited reduced reproduction upon desiccation. Four-day-old cadavers were more amenable to desiccation than 8-day-old cadavers. Formulated cadavers were more resistant to rupturing and sticking together during agitation than nonformulated cadavers.

Nitrogen partitioning in Heterorhabditis bacteriophora-infected hosts and the effects of nitrogen on attraction/repulsion.

Entomopathogenic nematode behavior is affected by the condition of their infected hosts. We hypothesized that nitrogen compounds released from infected hosts may be one factor affecting entomopathogenic nematode host-finding and infection behaviors. Our objectives were to (1) investigate the partitioning of nitrogen in Galleria mellonella (L.) infected by Heterorhabditis bacteriophora Poinar and (2) determine attraction and repulsion of H. bacteriophora to various quantities of nitrogen (ammonia). Volatile (ammonia), organic, and inorganic nitrogen forms were monitored during the course of infection. Approximately 0.052 mg of nitrogen was released from a single infected host as volatile ammonia. Most of the ammonia release was detected within the first 3 days postinoculation. Organic nitrogen increased during the course of infection, whereas inorganic nitrogen decreased. The net nitrogen change in the infected host consisted of a loss of approximately 47 mg, most of which was lost within the 1st week of infection. Accelerated loss of nitrogen early in the infection process was likely correlated with activity and growth of bacterial symbionts. Increased organic nitrogen was likely associated with nematode reproduction within the host. Attraction or repulsion of H. bacteriophora to nitrogen (ammonium hydroxide) was measured on agar quadrant plates. Nematodes were attracted to 16 and 160 microg of nitrogen and repelled by concentrations of 1600 and 8000 microg. Our data indicate that nitrogen released from H. bacteriophora-infected hosts attracts nematodes at lower levels (early in the infection) and repels them at higher concentrations (later in the infection process).

Differential susceptibility of Dysmicoccus vaccinii (Homoptera: Pseudococcidae) to entomopathogenic nematodes (Rhabditida: Heterorhabditidae and Steinernematidae).

The susceptibility of the mealy bug, Dysmicoccus vaccinii Miller & Polavarapu, to infection by various species and strains of entomopathogenic nematodes was investigated in laboratory sand-dish and sand-column assays. Steinernemo carpocapsae (Weiser) (All strain), S. feltiae (Filipjev) (AB [Australia] strain), and S. glaseri (Steiner) (NC strain) were ineffective against individual mealybugs in sand-dish assays conducted in small petri dishes (1 cm high by 3.5 cm diameter) at 25 degrees C with doses of infective juvenile nematodes ranging up to 500 or 1,000 infective juveniles and exposure periods up to 5 d. However, Heterorhabditis bacteriophora Poinar (HP88 strain and 2 New Jersey isolates). H. hawaiiensis Gardner, Stock & Kaya (MG-13 strain), and H. indicus Poinar, Karunakar & David (EMS-13 strain) induced significant mortality (65.0-90.0%) at doses as low as 100 infective juveniles and an exposure of 5 d. H. bacteriophora (HBNJ strain) was effective at doses of 500 and 1,000 infective juveniles but, together with H. zealandica Poinar (V16 strain) and 4 other H. bacteriophora isolates from New Jersey, was ineffective at doses of 100 infective juveniles. Removal of the waxy coating from the mealybugs did not influence susceptibility to H. bacteriophora (HP88 strain). In the sand-column assay (5.5 cm high by 5 cm diameter, 5-d exposure, 25 degrees C), which more closely resembles host-finding in the field, H. bacteriophora (HP88 strain) induced no significant mortality against individual mealybugs at doses of 100 infective juveniles but produced 93.8% mortality at 500 infective juveniles, whereas H. indicus (EMS-13 strain) induced 56.3 and 100% mortality at 100 and 500 infective juveniles, respectively. H. bacteriophora (HP88 strain and some New Jersey isolates), H. hawaiiensis (MG-13 strain), and H. indicus (EMS-13 strain) successfully reproduced in and emerged from mealybug cadavers. This study demonstrates strong variability in the susceptibility of D. vaccinii to different species and strains of entomopathogenic nematodes, and implicates certain heterorhabditids as promising candidates for the biological control of this insect.

Host recognition behaviour predicts host suitability in the entomopathogenic nematode Steinernema carpocapsae (Rhabditida:Steinernematidae).

Steinernema carpocapsae (Rhabditida: Steinernematidae) host recognition behaviour was assessed and compared with 2 measures of host suitability. Previous research showed that S. carpocapsae infective juveniles respond to host cues in a hierarchical order, with attraction to Galleria mellonella volatiles being stimulated by contact with G. mellonella cuticle. We measured host recognition behaviour by calculating the percentage response of S. carpocapsae infective juveniles to volatiles produced by G. mellonella last instars after the nematodes were exposed to the cuticle of 11 candidate arthropod hosts and 2 control surfaces. Host suitability was measured by nematode-induced mortality to candidate hosts at 2 nematode doses and the level of reproduction supported by each host. The highest recognition response was scored for Agrotis ipsilon (Lepidoptera: Noctuidae). This insect also incurred nearly 100% mortality due to nematode infection and supported the highest level of reproduction. Non-insect arthropods tested (Chilopoda and Isopoda) stimulated no behavioural response and were not susceptible to nematode infection. Other insect species elicited intermediate levels of the recognition response. There were significant correlations between behavioural response and nematode-induced mortality at the lower dose. The level of reproduction supported by the candidate hosts was also correlated with S. carpocapsae behavioural response.

Location and behavioral effects of lectin binding on entomopathogenic nematodes with different foraging strategies.

Lectins have been used to locate and ascribe a function to sensory organs of nematodes. We had two objectives: (1) to determine the location of carbohydrate exudates on the surface of three species of entomopathogenic nematodes with different foraging strategies and (2) to evaluate the importance of carbohydrates in host finding and recognition.Steinernema carpocapsae finds hosts by ambushing, and most individuals respond poorly to host cues. No specific areas of lectin binding were recorded for this species. Wheat germ agglutinin bound specifically to the anterior region ofHeterorhabditis bacteriophora and to the anterior and posterior ofS. glaseri. These two nematode species are cruise foragers and are highly responsive to host cues. The behavioral effects of lectins varied. None of the species' responses to volatile host cues was affected.S. carpocapsae's rate of attachment during ambushing was increased by treatment with WGA, and this effect was negated by treatment with a competing sugar (N-acetylglucosamine). The infectivity ofS. glaseri in an assay that required movement through 5 cm of sand to find the host was significantly decreased by treatment with WGA.

Percutaneous transluminal angioplasty. Results and surgical implications.

This retrospective study of 91 percutaneous transluminal angioplasties in 80 patients showed an overall patency rate by life-table analysis of 46 percent with a follow-up period of 36 months. Success rates were significantly reduced by predilatation ankle-brachial ratios less than 0.45, by diabetes, by pain at rest or necrosis, and somewhat, by older age. The overall success rate for iliac dilatation was significantly better (58 percent) than that in the femopopliteal segments (18 percent). Although percutaneous angioplasty was performed on many patients thought to be high surgical risks, the complication rate was low, and complications that required surgical intervention were rare (4 percent). Angioplasty was used as an adjunct to vascular surgery in several ways. An attempt to dilate a Dacron graft-femoral artery anastomosis was unsuccessful, and one of two dilatations distal to a femoropopliteal bypass was successful. However, the combination of an iliac angioplasty with outflow femoropopliteal or femorofemoral bypass produced a long-term patency rate of over 85 percent, significantly better than that achieved with iliac dilatation alone. Percutaneous transluminal angioplasty is both an alternative and an adjunct which should be considered by vascular surgeons for their patients.