Karyology,pollen and seed morphology,and distribution of eight endangered species in the Veneto region (Northern Italy)

ABSTRACT

Eight endangered species of the Veneto region were examined from the karyological and micromorphological points of view. Their geographical distribution, the exsiccata of Herbarium Venetum (HV-PAD) and conservation problems were also considered. These species are: Cortusa matthioli L., a taxon sporadically distributed in Veneto with 2n=24 chromosomes; Calianthemum kernerianum Freyn ex Kerner, only known on Mt. Baldo (Verona) and with 2n=4x=32; Scrophularia vernalis L. with 2n=28; Kosteletzkya pentacarpos (L.) Ledeb., an entity with no regional conservation regulations and with 2n=34; Athamanta vestina A.Kerner, endemic to Italy and with 2n=22; Hottonia palustris L. with 2n=20 and in danger because of the destruction of its habitat; Sagittaria sagttifolia L. with 2n=22; Trapa natans L., a protected species in Veneto with 2n=36 and 2n=48.     

Immunization with extracellular vesicles excreted by Toxoplasma gondii confers protection in murine infection,activating cellular and humoral responses

The study aim was to analyze whether microvesicles and exosomes, named extracellular vesicles (EVs), purified from Toxoplasma gondii are able to stimulate the protective immunity of experimental mice when administered, as challenge, a highly virulent strain. EVs excreted from T. gondii tachyzoites (RH strain) were purified by chromatography and used for immunization assays in inbred mouse groups (EV-IM). Chronic infected (CHR) and naive (NI) mice were used as control groups, since the immune response is well known. After immunizations, experimental groups were challenged with 100 tachyzoites. Next, parasitemias were determined by real-time PCR (qPCR), and survival levels were evaluated daily. The humoral response was analyzed by detection of IgM, IgG, IgG1 and IgG2a, and opsonization experiments. The cellular response was evaluated in situ by immunohistochemistry on IFN-γ, IL-10, TNF-α and IL-17 expression in cells of five organs (brain, heart, liver, spleen and skeletal muscles). EV immunization reduced parasitemia and increased the survival index in two mouse lineages (A/Sn and BALB/c) infected with a lethal T. gondii strain. EV-IM mice had higher IgG1 levels than IgM or IgG2a. IgGs purified from sera of EV-IM mice were able to opsonize tachyzoites (RH strain), and mice that received these parasites had lower parasitemias, and mortality was delayed 48 h, compared with the same results from those receiving parasites opsonized with IgG purified from NI mice. Brain and spleen cells from EV-IM mice more highly expressed IFN-γ, IL-10 and TNF-α. In conclusion, EV-immunization was capable of inducing immune protection, eliciting high production of IgG1, IFN-γ, IL-10 and TNF-α.     

Absolute quantitation of endogenous proteins with precision and accuracy using a capillary Western system

Precise and accurate quantification of protein expression levels in a complex biological setting is challenging. Here, we describe a method for absolute quantitation of endogenous proteins in cell lysates using an automated capillary immunoassay system, the size-based Simple Western system (recently developed by ProteinSimple). The method was able to accurately measure the absolute amounts of target proteins at picogram or sub-picogram levels per nanogram of cell lysates. The measurements were independent of the cell matrix or the cell lysis buffer and were not affected by different antibody affinities for their specific epitopes. We then applied this method to quantitate absolute levels of expression of protein kinase C (PKC) isoforms in LNCaP and U937 cells, two cell lines used extensively for probing the downstream biological responses to PKC targeted ligands. Our absolute quantitation confirmed the predominance of PKCδ in both cells, supporting the important functional role of this PKC isoform in these cell lines. The method described here provides an approach to accurately quantitate levels of protein expression and correlate protein level with function. In addition to enhanced accuracy relative to conventional Western analysis, it circumvents the distortions inherent in comparison with signal intensities from different antibodies with different affinities.     

In Vitro Susceptibility of Environmental Isolates of Exophiala dermatitidis to Five Antifungal Drugs

Several dematiaceous fungi frequently isolated from nature are involved in cases of superficial lesions to lethal cerebral infections. Antifungal susceptibility data on environmental and clinical isolates are still sparse despite the advances in testing methods. The objective of this study was to examine the activities of 5-flucytosine, amphotericin B, itraconazole, voriconazole and terbinafine against environmental isolates of Exophiala strains by minimum inhibition concentration (MIC) determination. The strains were obtained from hydrocarbon-contaminated soil, ant cuticle and fungal pellets from the infrabuccal pocket of attine gynes. Broth microdilution assay using M38-A2 reference methodology for the five antifungal drugs and DNA sequencing for fungal identification were applied. Terbinafine was the most active drug against the tested strains. It was observed that amphotericin B was less effective, notably against Exophiala spinifera, also studied. High MICs of 5-flucytosine against Exophiala dermatitidis occurred. This finding highlights the relevance of studies on the antifungal resistance of these potential opportunistic species. Our results also contribute to a future improvement of the standard methods to access the drug efficacy currently applied to black fungi.     

Stocking efficiency and the effects of diet preconditioning on the post-release adaptation of hatchery-reared juveniles of Atlantic salmon (Salmo salar L.) in an Atlantic temperate stream

This study explores the stocking efficiency of Atlantic salmon juveniles in a 4th order stream located at the southern limit of its Eastern Atlantic distribution. Moreover, with the idea of implementing new protocols and methods to improve ??traditional?? hatchery practices and rear a more ??wild?? like fish, the study examines the response of this species to natural diet preconditioning prior to release. The field study indicated that most juveniles disappeared from the stocked reaches within two months of their release, resulting in a recapture rate after a year of 0.0?C1.1?%. There were no differences in stocked juvenile??s density along the stream during the first months following stocking. The preconditioning experiments showed that juveniles of Atlantic salmon suffered a significant decrease in their condition factor when diet was changed from artificial pellets to live preys, either if the change occurred before (i.e., in a manipulative experiment) or after their release in the stream. Although weight, length, condition factor and specific growth rates reached by juveniles at the end of the rearing period were higher in individuals fed on pellets than in those fed on macroinvertebrates, differences disappeared only a week after their release in the stream. These results may have implications regarding the stocking success when releasing hatchery-reared Atlantic salmons in suboptimal environments. In particular, the study indicates that stocked salmon adaptation in the selected stream may not be improved by short conditioning periods to natural diet prior to their release, while we discuss potential explanations for the observed results.     

Reactive-Oxygen-Species-Mediated P. aeruginosa Killing Is Functional in Human Cystic Fibrosis Macrophages

Pseudomonas aeruginosa is the most common pathogen for chronic lung infection in cystic fibrosis (CF) patients. About 80% of adult CF patients have chronic P. aeruginosa infection, which accounts for much of the morbidity and most of the mortality. Both bacterial genetic adaptations and defective innate immune responses contribute to the bacteria persistence. It is well accepted that CF transmembrane conductance regulator (CFTR) dysfunction impairs the airways-epithelium-mediated lung defence; however, other innate immune cells also appear to be affected, such as neutrophils and macrophages, which thus contribute to this infectious pathology in the CF lung. In macrophages, the absence of CFTR has been linked to defective P. aeruginosa killing, increased pro-inflammatory cytokine secretion, and reduced reactive oxygen species (ROS) production. To learn more about macrophage dysfunction in CF patients, we investigated the generation of the oxidative burst and its impact on bacterial killing in CF macrophages isolated from peripheral blood or lung parenchyma of CF patients, after P. aeruginosa infection. Our data demonstrate that CF macrophages show an oxidative response of similar intensity to that of non-CF macrophages. Intracellular ROS are recognized as one of the earliest microbicidal mechanisms against engulfed pathogens that are activated by macrophages. Accordingly, NADPH inhibition resulted in a significant increase in the intracellular bacteria survival in CF and non-CF macrophages, both as monocyte-derived macrophages and as lung macrophages. These data strongly suggest that the contribution of ROS to P. aeruginosa killing is not affected by CFTR mutations.     

Malvidin,a red wine polyphenol,modulates mammalian myocardial and coronary performance and protects the heart against ischemia/reperfusion injury

A moderate red wine consumption and a colored fruit-rich diet protect the cardiovascular system, thanks to the presence of several polyphenols. Malvidin-3-0-glucoside (malvidin), an anthocyanidine belonging to polyphenols, is highly present in red grape skin and red wine. Its biological activity is poorly characterized, although a role in tumor cell inhibition has been found. To analyze whether and to which extent, like other food-derived polyphenols, malvidin affects the cardiovascular function, in this study, we have performed a quantitative analysis by high-performance liquid chromatography of polyphenolic content of red grape skins extract, showing that it contains a high malvidin amount (63.93±12.50 mg/g of fresh grape skin). By using the isolated and Langendorff perfused rat heart, we found that the increasing doses (1–1000 ng/ml) of the extract induced positive inotropic and negative lusitropic effects associated with coronary dilation. On the same cardiac preparations, we observed that malvidin (10^?10–10^?6 mol/L) elicited negative inotropism and lusitropism and coronary dilation. Analysis of mechanism of action revealed that malvidin-dependent cardiac effects require the activation of the phosphatidylinositol 3-kinase (PI3K)/nitric oxide (NO)/cGMP/PKG pathway and are associated with increased intracellular cGMP and the phosphorylation of endothelial NO synthase (eNOS), PI3K–AKT, ERK1/2, and GSK-3β. AKT and eNOS phosphorylation was confirmed in human umbilical vein endothelial cell. We also found that malvidin act as a postconditioning agent, being able to elicit cardioprotection against ischemia/reperfusion damages. Our results show the cardioactivity of polyphenols-rich red grape extracts and indicate malvidin as a new cardioprotective principle. This is of relevance not only for a better clarification of the beneficial cardiovascular effects of food-derived polyphenols but also for nutraceutical research.     

Somatic meiosis in the life history of Bonnemaisonia asparagoides and Bonnemaisonia clavata (Bonnemaisoniales,Rhodophyta) from the Iberian peninsula

Carpospores isolated from Bonnemaisonia asparagoides and Bonnemaisonia clavata (Bonnemaisoniaceae, Rhodophyta) and grown in culture developed into their respective ‘Hymenoclonium’ prostrate phases. In both species, young gametophytes were initiated directly on the prostrate phase from tetrasporangia-like protuberances. Comparison of the relative fluorescence area (rfa) of nuclear DNA over the sequence of life-history stages indicated that the lowest ploidy levels (1–2C) occurred in the gametophytes, whereas the lowest ploidy levels in the prostrate phases were 2–4C. Rfa data demonstrated that meiosis occurred in the tetrasporangia-like protuberances where 1C values were recorded. The present observations establish that B. asparagoides and B. clavata have a heteromorphic diplohaplontic life history, which involves a haploid gametophyte produced directly on a diploid prostate phase after somatic meiosis. We conclude that the life history of these taxa corresponds to the Lemanea-type. This indicates that the life history of several Bonnemaisoniales with a ‘Hymenoclonium’ phase but lacking tetrasporangia requires re-investigation.     

Polymer based biosensor for rapid electrochemical detection of virus infection of human cells

The demand in the field of medical diagnostics for simple, cost efficient and disposable devices is growing. Here, we present a label free, all-polymer electrochemical biosensor for detection of acute viral disease. The dynamics of a viral infection in human cell culture was investigated in a micro fluidic system on conductive polymer PEDOT:TsO microelectrodes by electrochemical impedance spectroscopy and video time lapse microscopy. Employing this sensitive, real time electrochemical technique, we could measure the immediate cell response to cytomegalovirus, and detect an infection within 3h, which is several hours before the cytopathic effect is apparent with conventional imaging techniques. Atomic force microscopy and scanning ion conductance microscopy imaging consolidate the electrochemical measurements by demonstrating early virus induced changes in cell morphology of apparent programmed cell death.     

New positive allosteric modulators of the metabotropic glutamate receptor 2 (mGluR2): identification and synthesis of N-propyl-8-chloro-6-substituted isoquinolones

A series of N-propyl-8-chloro-6-substituted isoquinolones was identified as positive allosteric modulators of metabotropic glutamate receptor 2 (mGluR2 PAM) via high throughput screening (HTS). The subsequent synthesis and initial SAR exploration that led to the identification of compound 28 is described.