Stress homeostasis - the redox and auxin perspective

Under environmental stresses, plant development is adaptively modulated. This modulation is influenced by the steady-state balance (homeostasis) between reactive oxygen species (ROS) and phytohormones. Frequently observed symptoms in plant stress adaptation responses include growth retardation, reduced metabolism and photosynthesis, reallocation of metabolic resources and increased antioxidant activities to maximize plant survival under adverse environmental conditions. In view of stress-induced morphogenetic changes during adaptation, ROS and auxin are the main players in the regulatory networks because both are strongly affected by exposure to environmental cues. However, the mechanisms underlying the crosstalk between ROS and auxin are poorly understood. In this review, we aim at surveying how the integration of environmental stress-related signals is modulated by crosstalk between ROS and auxin regulatory networks.     

Enhanced leishmanicidal activity of cryptopeptide chimeras from the active N1 domain of bovine lactoferrin

Two antimicrobial cryptopeptides from the N1 domain of bovine lactoferrin, lactoferricin (LFcin17–30) and lactoferrampin (LFampin265–284), together with a hybrid version (LFchimera), were tested against the protozoan parasite Leishmania. All peptides were leishmanicidal against Leishmania donovani promastigotes, and LFchimera showed a significantly higher activity over its two composing moieties. Besides, it was the only peptide active on Leishmania pifanoi axenic amastigotes, already showing activity below 10?μM. To investigate their leishmanicidal mechanism, promastigote membrane permeabilization was assessed by decrease of free ATP levels in living parasites, entrance of the vital dye SYTOX Green (MW?=?600?Da) and confocal and transmission electron microscopy. The peptides induced plasma membrane permeabilization and bioenergetic collapse of the parasites. To further clarify the structural traits underlying the increased leishmanicidal activity of LFchimera, the activity of several analogues was assessed. Results revealed that the high activity of these hybrid peptides seems to be related to the order and sequence orientation of the two cryptopeptide moieties, rather than to their particular linkage through an additional lysine, as in the initial LFchimera. The incorporation of both antimicrobial cryptopeptide motifs into a single linear sequence facilitates chemical synthesis and should help in the potential clinical application of these optimized analogues.     

Negative responses of highland pines to anthropogenic activities in inland Spain: a palaeoecological perspective

Palaeoecological evidence indicates that highland pines were dominant in extensive areas of the mountains of Central and Northern Iberia during the first half of the Holocene. However, following several millennia of anthropogenic pressure, their natural ranges are now severely reduced. Although pines have been frequently viewed as first-stage successional species responding positively to human disturbance, some recent palaeobotanical work has proposed fire disturbance and human deforestation as the main drivers of this vegetation turnover. To assess the strength of the evidence for this hypothesis and to identify other possible explanations for this scenario, we review the available information on past vegetation change in the mountains of northern inland Iberia. We have chosen data from several sites that offer good chronological control, including palynological records with microscopic charcoal data and sites with plant macro- and megafossil occurrence. We conclude that although the available long-term data are still fragmentary and that new methods are needed for a better understanding of the ecological history of Iberia, fire events and human activities (probably modulated by climate) have triggered the pine demise at different locations and different temporal scales. In addition, all palaeoxylological, palynological and charcoal results obtained so far are fully compatible with a rapid human-induced ecological change that could have caused a range contraction of highland pines in western Iberia.     

Quantification of extraradical soil mycelium and ectomycorrhizas of Boletus edulis in a Scots pine forest with variable sporocarp productivity

The availability of most edible ectomycorrhizal mushrooms depends on their natural fructification. Sporocarp formation of these fungi is linked to habitat characteristics and climate conditions, but these data alone do not explain all the trends of fungal fruiting and dynamics. It could be hypothesized that the amount of soil mycelia could also be related to the production of carpophores. Soil samples (five cylinders of 250 cm³ per plot) were taken monthly, from September to November, in five fenced permanent plots (5 × 5 m) in Pinar Grande (Soria, Spain), a Pinus sylvestris stand situated in the north of the Sistema Ibérico mountain range. Plots were chosen to establish a gradient of Boletus edulis productivity from 0 to 38.5 kg/ha year, according to the mean fresh weight of sporocarps collected during the last 10 years. B. edulis ectomycorrhizal root tips were identified in each soil sample according to its morphology and counted. DNA extractions were performed with the PowerSoil^TM DNA Isolation Kit and quantification of extraradical soil mycelium by real-time polymerase chain reaction using specific primers and a TaqMan? probe. The concentration of soil mycelium of B. edulis (mg mycelium/g soil) did not differ significantly between plots (p = 0.1397), and sampling time (p = 0.7643) within the fructification period. The number of mycorrhizal short roots per soil volume showed significant differences between the plots (p = 0.0050) and the three sampling times (p < 0.0001). No significant correlation between the number of mycorrhizas and the productivity of the plot (kg of B. edulis/ha year) was detected (p = 0.615). A statistically significant positive correlation (p = 0.0481) was detected between the concentration of mycelia of B. edulis in the soil samples and the presence of short roots mycorrhizal with B. edulis in these samples. The productivity of the plots, in terms of sporocarps produced during the last 10 years, was not correlated either with the concentration of soil mycelium or with the presence or abundance of ectomycorrhizas.     

Hyaluronan receptors in the human ocular surface: a descriptive and comparative study of RHAMM and CD44 in tissues, cell lines and freshly collected samples

The purpose of this study was to demonstrate the presence of the receptor for hyaluronan-mediated motility (RHAMM) in human conjunctival epithelium and in two widely used cell lines from human corneal (HCE) and conjunctival (IOBA-NHC) epithelia. We compared the distribution of RHAMM proteins and mRNAs in human ocular surface tissues (corneal, limbal and conjunctival), HCE and IOBA-NHC cell lines, and corneal and conjunctival epithelia primary samples from healthy donors with the previously identified hyaluronan receptor CD44. We also aimed to determine if soluble CD44 (sCD44) was present in human tears, as it could have a role in the interaction of the tear fluid with hyaluronan. Protein expression was evaluated by Western blots and immunofluorescence microscopy. mRNA expression was evaluated by RT-PCR and Q-PCR. sCD44 was analyzed by ELISA in culture supernatants and in human tears. We describe the expression of RHAMM in human healthy conjunctiva and in HCE and IOBA-NHC cells at both protein and mRNA levels, and the presence of sCD44 in human tears. Furthermore, we detected CD44 and sCD44 expression variations in in vitro inflammatory conditions. This study also focused on the necessary caution with which the conclusions extracted from cell lines should be made, and in the great value of using primary samples as often as possible.     

Trade-offs in plankton species richness arising from drought: insights from long-term data of a National Park wetland (central Spain)

Wetlands are considered vulnerable ecosystems of both high species richness and socio-economic value. In semi-arid regions, these ecosystems often experience long drought periods that are usually aggravated by local water overexploitation. Drought leads to: (i) reduced flooding area, (ii) isolation of water bodies, (iii) increased areas of dry sediments and shoreline length, and (iv) increased ionic concentration. These processes affect aquatic populations in a species-specific way and can have antagonistic effects on taxon richness. Here, we highlight long-term (1997–2008) trade-off effects on plankton species richness linked to drought in a semi-arid wetland (Las Tablas de Daimiel National Park, central Spain). Annual average phytoplankton species richness increased from wet- (1997–1998) to dry years (2001–2002) and taxon richness diminished again when drought was more severe (2007–2008). Zooplankton changes were more complex depending on taxonomic groups and the body size of the organisms, total species loss being related to hydrological conditions. Half of the algal species recorded in 2007–2008 and one-tenth of total zooplankton taxa were new comers in the wetland, because salinization, eutrophication and submerged macrophytes occurring in different sites enhanced species turnover and mitigated homogenization of beta diversity. Maintenance of one water body with a constant water level and macrophytes was the key to preventing the collapse of plankton richness. Our study has demonstrated that plankton can be very useful for tracking environmental changes of wetlands, thus giving the environmental manager another tool to enhance the conservation of wetlands and their biota.     

Antibodies against the envelope glycoprotein promote infectivity of immature dengue virus serotype 2

Cross-reactive dengue virus (DENV) antibodies directed against the envelope (E) and precursor membrane (prM) proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM antibodies render essentially non-infectious immature DENV infectious in Fcγ-receptor-expressing cells. Immature DENV particles are abundantly present in standard (st) virus preparations due to inefficient processing of prM to M during virus maturation. Structural analysis has revealed that the E protein is exposed in immature particles and this prompted us to investigate whether antibodies to E render immature particles infectious. To this end, we analyzed the enhancing properties of 27 anti-E antibodies directed against distinct structural domains. Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner. The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV) mouse model. Remarkably, mice injected with immature WNV opsonized with anti-E mAbs or immune serum produced a lethal infection in a dose-dependent manner, whereas in the absence of antibody immature WNV virions caused no morbidity or mortality. Furthermore, enhancement infection studies with standard (st) DENV preparations opsonized with anti-E mAbs in the presence or absence of furin inhibitor revealed that prM-containing particles present within st virus preparations contribute to antibody-dependent enhancement of infection. Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles.     

New sources of resistance to lettuce aphids in Lactuca spp.

The use of resistant cultivars is one of the best ways to protect lettuce from aphid pests. At present, there are cultivars available with nearly complete resistance to Nasonovia ribisnigri biotype Nr:0 (based on the Nr gene) and partial resistance to Macrosiphum euphorbiae. Nevertheless, a new biotype of N. ribisnigri (Nr:1) able to overcome the resistance based on the Nr gene is expanding around Europe and has become a major threat of lettuce. In the present work, we report the presence of this new biotype in southeastern Spain, a major lettuce-producing region. Furthermore, a pool of 264 germplasm accessions belonging to Lactuca genus was tested in a greenhouse assay to search for new resistance sources to N. ribisnigri. The most promising accessions were retested in the laboratory to characterize the resistance by means of free-choice and antibiosis assays against biotypes Nr:0 and Nr:1 of N. ribisnigri and against a clone of M. euphorbiae. Three accessions of L. virosa showed resistance against the target aphid species and could be of interest for ongoing breeding programs. The accessions CGN16272 and CGN13361 were both partially resistant to the Nr:1 biotype of N. ribisnigri and to M. euphorbiae, and CGN13355, in spite of not being resistant to N. ribisnigri, showed a near complete resistance to M. euphorbiae. The study of the feeding behavior of N. ribisnigri biotypes showed that the Nr:1 biotype is able to maintain a similar phloem feeding ingestion pattern on genotypes bearing the Nr gene and on N. ribisnigri-susceptible lettuce genotypes. Moreover, as aphids rejected L. virosa as a feeding source due to superficial factors (high level of antixenosis), no differences in the level of antibiosis between such genotypes were detected. A second set of screening assays were conducted on 40 accessions of L. virosa in order to select for resistance against the Nr:1 biotype. The results showed three accessions with high levels of resistance (CGN05148, CGN21399 and CGN16274) against Nr:1 that could be of interest in lettuce breeding programs.     

Appraisal of a Leishmania major Strain Stably Expressing mCherry Fluorescent Protein for Both In Vitro and In Vivo Studies of Potential Drugs and Vaccine against Cutaneous Leishmaniasis

Background

Leishmania major cutaneous leishmaniasis is an infectious zoonotic disease. It is produced by a digenetic parasite, which resides in the phagolysosomal compartment of different mammalian macrophage populations. There is an urgent need to develop new therapies (drugs) against this neglected disease that hits developing countries. The main goal of this work is to establish an easier and cheaper tool of choice for real-time monitoring of the establishment and progression of this pathology either in BALB/c mice or in vitro assays. To validate this new technique we vaccinated mice with an attenuated Δhsp70-II strain of Leishmania to assess protection against this disease.

Methodology

We engineered a transgenic L. major strain expressing the mCherry red-fluorescent protein for real-time monitoring of the parasitic load. This is achieved via measurement of fluorescence emission, allowing a weekly record of the footpads over eight weeks after the inoculation of BALB/c mice.

Results

In vitro results show a linear correlation between the number of parasites and fluorescence emission over a range of four logs. The minimum number of parasites (amastigote isolated from lesion) detected by their fluorescent phenotype was 10,000. The effect of antileishmanial drugs against mCherry+L. major infecting peritoneal macrophages were evaluated by direct assay of fluorescence emission, with IC₅₀ values of 0.12, 0.56 and 9.20 µM for amphotericin B, miltefosine and paromomycin, respectively. An experimental vaccination trial based on the protection conferred by an attenuated Δhsp70-II mutant of Leishmania was used to validate the suitability of this technique in vivo.

Conclusions

A Leishmania major strain expressing mCherry red-fluorescent protein enables the monitoring of parasitic load via measurement of fluorescence emission. This approach allows a simpler, faster, non-invasive and cost-effective technique to assess the clinical progression of the infection after drug or vaccine therapy.     

Technical evaluation of nested PCR for the diagnosis of experimental sporotrichosis

BackgroundSporotrichosis caused by the dimorphic fungus Sporothrix schenckii can presents in a variety of clinical forms. Routine diagnosis is made by mycology and serology studies. Few investigations have been focused on the evaluation of the molecular diagnosis.AimTo determine the value of the nested PCR technique for the diagnosis of experimental sporotrichosis in organs of mice, and to compare the results with the established laboratory diagnostic procedures.MethodsBALB/c mice were inoculated with growing concentrations of the 2 morphological phases of the fungus. The infected animals were sacrificed one month later and specimens from liver, spleen, lung and testicle were obtained to perform wet mount, culture and molecular diagnosis by the nested PCR technique. Blood samples were obtained for determination of specific antibodies against S. schenckii by the double immunodiffusion procedure.ResultsThe pathogenicity observed with the different concentrations of the fungus inoculated and its isolation by culture, showed scarce differences in the study of specimens from organs infected with the 2 morphological phases of S. schenckii. Specimens from organs of mice inoculated with the mycelial phase when studied by wet mount and culture, showed a higher positivity (100 and 37.5%) than those from mice inoculated with the yeast phase (73 and 2%). However, diagnosis by the nested PCR molecular technique applied to the latter specimens showed a higher percentage of positivity (75%) and 43% of positive results coming from animals infected with the mycelial phase. Specific antibody detection was positive in 100% all groups of infected mice.ConclusionsIn the study of experimental sporotrichosis in mice, the culture, as well as the antibody detection, was an effective diagnostic procedure, while the nested PCR and microscopic studies had a lower diagnostic value.