Leishmanicidal activity of amphotericin B encapsulated in PLGA–DMSA nanoparticles to treat cutaneous leishmaniasis in C57BL/6 mice

The major goal of this work was to design a new nanoparticle drug delivery system for desoxycholate amphotericin B (D-AMB), based on controlled particle size, looking for the most successful release of the active agents in order to achieve the best site-specific action of the drug at the therapeutically optimal rate and dose regimen. For this, AMB nanoencapsulated in poly(lactic-co-glycolic acid) (PLGA) and dimercaptosuccinic acid (DMSA) nanoparticles (Nano-D-AMB) has been developed, and its efficacy was evaluated in the treatment of experimental cutaneous leishmaniasis in C57BL/6 mice, to test if our nano-drug delivery system could favor the reduction of the dose frequency required to achieve the same therapeutic level of free D-AMB, and so, an extended dosing interval. Magnetic citrate-coated maghemite nanoparticles were added to this nanosystem (Nano-D-AMB-MG) aiming to increase controlled release of AMB by magnetohyperthermia. Female mice (N = 6/group) were infected intradermally in the right footpad with promastigotes of Leishmania amazonensis in the metacyclic phase, receiving the following intraperitoneal treatments: 1% PBS for 10 consecutive days; D-AMB at 2 mg/kg/day for 10 days (totalizing 20 mg/kg/animal); Nano-D-AMB and Nano-D-AMB-MG at 6 mg/kg on the 1st, 4th and 7th days and at 2 mg/kg on the 10th day, also totalizing 20 mg/kg/animal by treatment end. The Nano-D-AMB-MG group was submitted to an AC magnetic field, allowing the induction of magnetohyperthermia. The evaluations were through paw diameter measurements; parasite number and cell viability were investigated by limiting dilution assay. D-AMB-coated PLGA–DMSA nanoparticles showed the same efficacy as free D-AMB to reduce paw diameter; however, the Nano-D-AMB treatment also promoted a significantly greater reduction in parasite number and cell viability compared with free D-AMB. The nano-drug AMB delivery system appeared more effective than free D-AMB therapy to reduce the dose frequency required to achieve the same therapeutic level. It thus favors a longer interval between doses, as expected with development of a new nano drug delivery system, and may be useful in the treatment of many different pathologies, from cancer to neurodegenerative diseases.     

The Predation Strategy of the Recluse Spider <Emphasis Type="Italic">Loxosceles rufipes</Emphasis> (Lucas, 1834) against four Prey Species

Generalist predators have to deal with prey with sometimes very different morphologies and defensive behaviors. Therefore, such predators are expected to express plasticity in their predation strategy. Here we investigated the predatory behavior of the recluse spider Loxosceles rufipes (Araneae, Sicariidae) when attacking prey with different morphologies and defensive mechanisms. We expected L. rufipes to show different prey capture strategies and variable acceptance towards each prey type. Potential prey species were collected directly from the web or in the surroundings of the web-building site of L. rufipes. We collected and used the following in our experiments: termite workers (Nasutitermes sp.), lepidopteran larvae (Eurema salome), ants (Camponotus sp.) and isopods (Tylidae). We paired these prey with L. rufipes and recorded their behavior in captivity, quantifying acceptance rate, immobilization time and the sequence of behaviors by the predator. The acceptance rate was lower for isopods but not different among other prey. The immobilization time was higher for isopods than for termites and similar for the other pairwise comparisons. The behavioral sequence was similar for all prey except for isopods, which were also bit more often. Our combined results show plasticity in the behavior of L. rufipes and also show it subdues a potentially dangerous prey (ant) and an armored prey (isopod).     

Interaction between endophytic bacteria from citrus plants and the phytopathogenic bacteria Xylella fastidiosa, causal agent of citrus-variegated chlorosis

AIMS: To isolate endophytic bacteria and Xylella fastidiosa and also to evaluate whether the bacterial endophyte community contributes to citrus-variegated chlorosis (CVC) status in sweet orange (Citrus sinensis [L.] Osbeck cv. Pera). METHODS AND RESULTS: The presence of Xylella fastidiosa and the population diversity of culturable endophytic bacteria in the leaves and branches of healthy, CVC-asymptomatic and CVC-symptomatic sweet orange plants and in tangerine (Citrus reticulata cv. Blanco) plants were assessed, and the in vitro interaction between endophytic bacteria and X. fastidiosa was investigated. There were significant differences in endophyte incidence between leaves and branches, and among healthy, CVC-asymptomatic and CVC-symptomatic plants. Bacteria identified as belonging to the genus Methylobacterium were isolated only from branches, mainly from those sampled from healthy and diseased plants, from which were also isolated X. fastidiosa. CONCLUSIONS: The in vitro interaction experiments indicated that the growth of X. fastidiosa was stimulated by endophytic Methylobacterium extorquens and inhibited by endophytic Curtobacterium flaccumfaciens. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides the first evidence of an interaction between citrus endophytic bacteria and X. fastidiosa and suggests a promising approach that can be used to better understand CVC disease.     

RAPD profile and antibiotic susceptibility of Xylella fastidiosa, causal agent of citrus variegated chlorosis

AIMS: The aim of this study was to evaluate the diversity of Xylella fastidiosa isolated from citrus trees affected by Citrus Variegated Chlorosis (CVC). METHODS AND RESULTS: The antibiotic susceptibility by agar disc diffusion and minimum inhibitory concentration (MIC) methods was observed for all drug evaluated, except for penicillin-G. Genetic diversity by RAPD analysis revealed three major groups (citrus, coffee and grapevine), being the citrus group more similar with the coffee group than with the grapevine group. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the possibility to use these antibiotics susceptibility as markers in the development of a cloning vector and penicillin-G could be used as a selective marker for the isolation of X. fastidiosa from citrus plants.     

Early behavioral changes and quantitative analysis of neuropathological features in murine prion disease: Stereological analysis in the albino Swiss mice model

Behavioral and neuropathological changes have been widely investigated in murine prion disease but stereological based unbiased estimates of key neuropathological features have not been carried out. After injections of ME7 infected (ME7) or normal brain homogenates (NBH) into dorsal CA1 of albino Swiss mice and C57BL6, we assessed behavioral changes on hippocampal-dependent tasks. We also estimated by optical fractionator at 15 and 18 weeks post-injections (w.p.i.) the total number of neurons, reactive astrocytes, activated microglia and perineuronal nets (PN) in the polymorphic layer of dentate gyrus (PolDG), CA1 and septum in albino Swiss mice. On average, early behavioral changes in albino Swiss mice start four weeks later than in C57BL6. Cluster and discriminant analysis of behavioral data in albino Swiss mice revealed that four of nine subjects start to change their behavior at 12 w.p.i. and reach terminal stage at 22 w.p.i and the remaining subjects start at 22 w.p.i. and reach terminal stage at 26 w.p.i. Biotinylated dextran-amine BDA-tracer experiments in mossy fiber pathway confirmed axonal degeneration and stereological data showed that early astrocytosis, microgliosis and reduction in the perineuronal nets are independent of a change in the number of neuronal cell bodies. Statistical analysis revealed that the septal region had greater levels of neuroinflammation and extracellular matrix damage than CA1. This stereological and multivariate analysis at early stages of disease in an outbred model of prion disease provided new insights connecting behavioral changes and neuroinflammation and seems to be important to understand the mechanisms of prion disease progression.Key words: prion disease, optical fractionator, neuropathology, behavioral changes, albino Swiss mice     

Metformin (dimethyl-biguanide) induced DNA damage in mammalian cells

Metformin (dimethyl-biguanide) is an insulin-sensitizing agent that lowers fasting plasma-insulin concentration, wherefore it's wide use for patients with a variety of insulin-resistant and prediabetic states, including impaired glucose tolerance. During pregnancy it is a further resource for reducing first-trimester pregnancy loss in women with the polycystic ovary syndrome. We tested metformin genotoxicity in cells of Chinese hamster ovary, CHO-K1 (chromosome aberrations; comet assays) and in mice (micronucleus assays). Concentrations of 114.4 μg/mL and 572 μg/mL were used in in vitro tests, and 95.4 mg/kg, 190.8 mg/kg and 333.9 mg/kg in assaying. Although the in vitro tests revealed no chromosome aberrations in metaphase cells, DNA damage was detected by comet assaying after 24 h of incubation at both concentrations. The frequency of DNA damage was higher at concentrations of 114.4 μg/mL. Furthermore, although mortality was not observed in in vitro tests, the highest dose of metformin suppressed bone marrow cells. However, no statistically significant differences were noted in micronuclei frequencies between treatments. In vitro results indicate that chronic metformin exposure may be potentially genotoxic. Thus, pregnant woman undergoing treatment with metformin should be properly evaluated beforehand, as regards vulnerability to DNA damage.     

Social Environment Affects Acquisition and Color of Structural Nuptial Plumage in a Sexually Dimorphic Tropical Passerine

Structural colors result from the physical interaction of light with organic materials of differing refractive indexes organized at nanoscale dimensions to produce significant interference effects. Because color properties emerge from these finely organized nanostructures, the production of structural coloration could respond to environmental factors and be developmentally more plastic than expected, functioning as an indicator of individual quality. However, there are many unknown factors concerning the function and mechanisms regulating structural coloration, especially relative to social environment. We hypothesized that social environment, in the form of competitive settings, can influence the developmental pathways involving production of feather structural coloration. We experimentally assessed the impact of social environment upon body condition, molt and spectral properties of two types of structural color that compose the nuptial plumage in blue-black grassquits: black iridescent plumage and white underwing patches. We manipulated male social environment during nine months by keeping individuals in three treatments: (1) pairs; (2) all-male groups; and (3) male-female mixed groups. All morphological characters and spectral plumage measures varied significantly through time, but only acquisition of nuptial plumage coverage and nuptial plumage color were influenced by social environment. Compared with males in the paired treatment, those in treatments with multiple males molted into nuptial plumage faster and earlier, and their plumage was more UV-purple-shifted. Our results provide experimental evidence that social context strongly influences development and expression of structural plumage. These results emphasize the importance of long-term experimental studies to identify the phenotypic consequences of social dynamics relative to ornament expression.     

Web building and silk properties functionally covary among species of wolf spider

Although phylogenetic studies have shown covariation between the properties of spider major ampullate (MA) silk and web building, both spider webs and silks are highly plastic so we cannot be sure whether these traits functionally covary or just vary across environments that the spiders occupy. As MaSp2‐like proteins provide MA silk with greater extensibility, their presence is considered necessary for spider webs to effectively capture prey. Wolf spiders (Lycosidae) are predominantly non‐web building, but a select few species build webs. We accordingly collected MA silk from two web‐building and six non‐web‐building species found in semirural ecosystems in Uruguay to test whether the presence of MaSp2‐like proteins (indicated by amino acid composition, silk mechanical properties and silk nanostructures) was associated with web building across the group. The web‐building and non‐web‐building species were from disparate subfamilies so we estimated a genetic phylogeny to perform appropriate comparisons. For all of the properties measured, we found differences between web‐building and non‐web‐building species. A phylogenetic regression model confirmed that web building and not phylogenetic inertia influences silk properties. Our study definitively showed an ecological influence over spider silk properties. We expect that the presence of the MaSp2‐like proteins and the subsequent nanostructures improves the mechanical performance of silks within the webs. Our study furthers our understanding of spider web and silk co‐evolution and the ecological implications of spider silk properties.