Dihydrotanshinone,a Natural Diterpenoid,Preserves Blood-Retinal Barrier Integrity via P2X7 Receptor

Activation of P2X7 signaling, due to high glucose levels, leads to blood retinal barrier (BRB) breakdown, which is a hallmark of diabetic retinopathy (DR). Furthermore, several studies report that high glucose (HG) conditions and the related activation of the P2X7 receptor (P2X7R) lead to the over-expression of pro-inflammatory markers. In order to identify novel P2X7R antagonists, we carried out virtual screening on a focused compound dataset, including indole derivatives and natural compounds such as caffeic acid phenethyl ester derivatives, flavonoids, and diterpenoids. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) rescoring and structural fingerprint clustering of docking poses from virtual screening highlighted that the diterpenoid dihydrotanshinone (DHTS) clustered with the well-known P2X7R antagonist JNJ47965567. A human-based in vitro BRB model made of retinal pericytes, astrocytes, and endothelial cells was used to assess the potential protective effect of DHTS against HG and 2′(3′)-O-(4-Benzoylbenzoyl)adenosine-5′-triphosphate (BzATP), a P2X7R agonist, insult. We found that HG/BzATP exposure generated BRB breakdown by enhancing barrier permeability (trans-endothelial electrical resistance (TEER)) and reducing the levels of ZO-1 and VE-cadherin junction proteins as well as of the Cx-43 mRNA expression levels. Furthermore, HG levels and P2X7R agonist treatment led to increased expression of pro-inflammatory mediators (TLR-4, IL-1β, IL-6, TNF-α, and IL-8) and other molecular markers (P2X7R, VEGF-A, and ICAM-1), along with enhanced production of reactive oxygen species. Treatment with DHTS preserved the BRB integrity from HG/BzATP damage. The protective effects of DHTS were also compared to the validated P2X7R antagonist, JNJ47965567. In conclusion, we provided new findings pointing out the therapeutic potential of DHTS, which is an inhibitor of P2X7R, in terms of preventing and/or counteracting the BRB dysfunctions elicited by HG conditions.     

Topical laryngo-tracheal anesthesia and intraocular pressure in anesthesia for ophthalmic surgery

OBJECTIVE: The reflex response to orotracheal intubation provokes an increase of arterial pressure accompanied by an increase of chorioides volume and a consequent ocular hypertone. There are several methods to reduce the reflex response due to intubation. One of the most effective is topical anaesthesia of larynx and trachea. Experiments were directed to evaluate the efficacy of topical anaesthesia to reduce the intraocular hypertone due to orotracheal intubation. DESIGN: A prospective randomized mask study was conduct on patients undergoing ophthalmologic (anterior segment) surgery at the Eye Clinic of Florence University. METHODS: Intraocular pressure was measured by a Goldman tonometer at four times: T0 = basal, T1 = 2' minutes after induction of general anaesthesia, T2 = 2' minutes after laryngoscopy, T3 = 2' minutes after orotracheal intubation. At the same moments, systolic blood pressure, heart rate, rate pressure pro duct were measured. Patients were randomly divided in two groups: Group L (n = 10) in which was evaluated the efficacy of laryngotracheal topical spray of lidocaine 4% (2 ml) and Group F (n = 10) in which saline was used instead of anesthetic. The filling of the LTA kit (Abbott) was made by a person not involved in the experiments. DATA ANALYSIS: Student's t test for unpaired data. RESULTS: Topical anaesthesia reduces the increase of intraocular pressure, hypertension and rate pressure product due to intubation. The intraocular pressure reduces to 13% less than basal value in Group L and increase to 50% more than basal value in Group F. CONCLUSION: The topical anaesthesia of larynx and trachea is effective to reduce the intraocular hypertension due to the reflex response evoked by orotracheal intubation.     

Direct monitoring of the calcium concentration in the sarcoplasmic and endoplasmic reticulum of skeletal muscle myotubes

Direct monitoring of the free Ca2+ concentration in the sarcoplasmic reticulum (SR) was carried out in rat skeletal myotubes transfected with a specifically targeted aequorin chimera (srAEQ). Myotubes were also transfected with a chimeric aequorin (erAEQ) that we have demonstrated previously is retained in the endoplasmic reticulum (ER). Immunolocalization analysis showed that although both recombinant proteins are distributed in an endomembrane network identifiable with immature SR, the erAEQ protein was retained also in the perinuclear membrane. The difficulty of measuring [Ca2+] in 100-1000 microM range was overcome with the use of the synthetic coelenterazine analogue, coelenterazine n. We demonstrate that the steady state levels of [Ca2+] measured with srAEQ is around 300 microM, whereas that measured with erAEQ is significantly lower, i.e. around 200 microM. The effects of caffeine, high KCl, and nicotinic receptor stimulation, in the presence or absence of external calcium or after blockade of the Ca-ATPase, were investigated with both chimeras. The kinetics of [Ca2+] changes revealed by the erAEQ were similar, but not identical, neither quantitatively nor qualitatively, to those monitored with the srAEQ, indicating that at this stage of muscle development, differences exist between SR and ER in their mechanisms of Ca2+ handling. The functional implications of these findings are discussed.     

Evaluation of a dual-color flow cytometry immunophenotyping panel in a multicenter quality assurance program

A basic immunophenotyping panel that employed dual-color combinations of fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugated monoclonal antibodies (mAb; FITC-CD45/PE-CD14, FITC-IgG1/PE-IgG2, FITC-CD3/PE-CD8, FITC-CD3/PE-CD4, FITC-CD3/PE-CD16 + PE-CD56, and PE-CD19) was utilized in a quality assurance program to determine whether the 4 laboratories participating in a multicenter AIDS study obtained similar lymphocyte subset percentage values for T cells, B cells, NK cells, and CD4+ and CD8+ T cells. Over a 1 1/2 year period, 78 shared peripheral blood specimens were prepared and analyzed in each laboratory. The CD45bright CD14- percentage for each specimen was used to correct that individual's lymphocyte subset values. Interlaboratory coefficients of variation (CV) for the human immunodeficiency virus type I (HIV) seronegative (n = 38) and HIV-seropositive (n = 40) specimens using this panel were < 3% for total T cells; < 5% for CD4+ T cells and CD8+ T cells; < or = 17% for B and NK cells; and < 8% for CD4T/CD8T ratios. The 6-tube basic immunophenotyping panel has several notable features: a) for clinical studies, it permits comprehensive evaluation of an individual's major lymphocyte subsets, i.e., T, B, NK, and CD4+ and CD8+ T cells; b) for interlaboratory proficiency testing programs, it allows the detection of differences among laboratories in measurements of several functionally distinct cell populations; and c) for within-sample quality assurance, it provides several quality control checks, including the lymphosum, i.e., the sum of an individual's corrected T+B+NK values, a sum that was generally 100 +/- 5% on the HIV-seronegative specimens analyzed in this study.     

Neuroprotective Effects of Testosterone in the Hypothalamus of an Animal Model of Metabolic Syndrome

Metabolic syndrome (MetS) is known to be associated to inflammation and alteration in the hypothalamus, a brain region implicated in the control of several physiological functions, including energy homeostasis and reproduction. Previous studies demonstrated the beneficial effects of testosterone treatment (TTh) in counteracting some MetS symptoms in both animal models and clinical studies. This study investigated the effect of TTh (30 mg/kg/week for 12 weeks) on the hypothalamus in a high-fat diet (HFD)-induced animal model of MetS, utilizing quantitative RT-PCR and immunohistochemical analyses. The animal model recapitulates the human MetS features, including low testosterone/gonadotropin plasma levels. TTh significantly improved MetS-induced hypertension, visceral adipose tissue accumulation, and glucose homeostasis derangements. Within hypothalamus, TTh significantly counteracted HFD-induced inflammation, as detected in terms of expression of inflammatory markers and microglial activation. Moreover, TTh remarkably reverted the HFD-associated alterations in the expression of important regulators of energy status and reproduction, such as the melanocortin and the GnRH-controlling network. Our results suggest that TTh may exert neuroprotective effects on the HFD-related hypothalamic alterations, with positive outcomes on the circuits implicated in the control of energy metabolism and reproductive tasks, thus supporting a possible role of TTh in the clinical management of MetS.     

The AFLATOX® Project: Approaching the Development of New Generation,Natural-Based Compounds for the Containment of the Mycotoxigenic Phytopathogen Aspergillus flavus and Aflatoxin Contamination

The control of the fungal contamination on crops is considered a priority by the sanitary authorities of an increasing number of countries, and this is also due to the fact that the geographic areas interested in mycotoxin outbreaks are widening. Among the different pre- and post-harvest strategies that may be applied to prevent fungal and/or aflatoxin contamination, fungicides still play a prominent role; however, despite of countless efforts, to date the problem of food and feed contamination remains unsolved, since the essential factors that affect aflatoxins production are various and hardly to handle as a whole. In this scenario, the exploitation of bioactive natural sources to obtain new agents presenting novel mechanisms of action may represent a successful strategy to minimize, at the same time, aflatoxin contamination and the use of toxic pesticides. The Aflatox^® Project was aimed at the development of new-generation inhibitors of aflatoxigenic Aspergillus spp. proliferation and toxin production, through the modification of naturally occurring molecules: a panel of 177 compounds, belonging to the thiosemicarbazones class, have been synthesized and screened for their antifungal and anti-aflatoxigenic potential. The most effective compounds, selected as the best candidates as aflatoxin containment agents, were also evaluated in terms of cytotoxicity, genotoxicity and epi-genotoxicity to exclude potential harmful effect on the human health, the plants on which fungi grow and the whole ecosystem.     

Regional Climate Simulations on EU-INDIA Grid Infrastructures: Methodologies and Performance

In this paper, we present a possible approach to use different Grid infrastructures across Europe and India for regional climate simulations and discuss in details the advantages and limitations in using them. The application taken into consideration is the Regional Climate Model RegCM4, which has been recently re-written for making it more efficient and easier to be fully exploited on any kind of computational infrastructure. We describe here the methods applied to port this package on the Grid infrastructures made available by the EU-IndiaGrid project. We also discuss different approaches, the way to run the model on both European and Indian infrastructures and our promising approach to deal with data management issues. Use of RegCM on Grid infrastructure has further been compared with that on HPC resources. The domain worked upon for these tests is the South-Asia CORDEX domain, which is of great importance for the Indian Summer Monsoon. Our final conclusion is that, for certain class of experiments, RegCM4 model can be efficiently and easily integrated on Grid infrastructures, by means of the procedures described in this paper.     

Enzymatic hydrolysis of cotton wastes. Effect of γ-ray pretreatments

Textile cotton wastes were irradiated with γ-rays at different doses and solubilized with 10% NaOH solutions. The regenerated products underwent enzymatic hydrolysis and their degradation pattern was compared with that of the cotton celluloses untreated or treated only with γ-rays. The different samples were characterized by measurements of X-ray diffraction, degree of polymerization, moisture regain and water retention. The structural and morphological modifications induced by the pretreatments were correlated with the variations of kinetic parameters, particularly the V_max/K_m ratio, defined as specificity constant. Only the combination of irradiation with dissolution pretreatments gave a considerable increase of hydrolysis reaction effectiveness.     

On the human ability to discriminate audio ambiances from similar locations of an urban environment

When developing advanced location-based systems augmented with audio ambiances, it would be cost-effective to use a few representative samples from typical environments for describing a larger number of similar locations. The aim of this experiment was to study the human ability to discriminate audio ambiances recorded in similar locations of the same urban environment. A listening experiment consisting of material from three different environments and nine different locations was carried out with nineteen subjects to study the credibility of audio representations for certain environments which would diminish the need for collecting huge audio databases. The first goal was to study to what degree humans are able to recognize whether the recording has been made in an indicated location or in another similar location, when presented with the name of the place, location on a map, and the associated audio ambiance. The second goal was to study whether the ability to discriminate audio ambiances from different locations is affected by a visual cue, by presenting additional information in form of a photograph of the suggested location. The results indicate that audio ambiances from similar urban areas of the same city differ enough so that it is not acceptable to use a single recording as ambience to represent different yet similar locations. Including an image was found to increase the perceived credibility of all the audio samples in representing a certain location. The results suggest that developers of audio-augmented location-based systems should aim at using audio samples recorded on-site for each location in order to achieve a credible impression.     

A data fusion and spatial data analysis approach for the estimation of wheat grain nitrogen uptake from satellite data

The selection of the optimal band combination for the estimation of specific crop variables is a key aspect in order to obtain reliable estimation of in-field variability from multi- and hyperspectral remote-sensing data. The selection of the bands is strongly influenced by the phenological stage of the crop at the acquisition time. In this work, the influence of the growing stage on the combination of spectral bands related to grain nitrogen (N) uptake in wheat was evaluated using multispectral (Satellite Pour l’Observation de la Terre – SPOT) and hyperspectral (Compact High Resolution Imaging Spectrometer – CHRIS-PROBA) satellite images at different growth stages over two wheat growth seasons in central Italy. In order to identify the more appropriate covariates (spectral bands) for each phenological stage, stepwise regression with backward selection was combined with stepwise variance inflation factors (VIFs) analysis and linear mixed effect model (LMEM). The results obtained in this study suggest that the spectral region most related to N uptake varies over the growing season of the wheat crop. For SPOT data, near-infrared (NIR) region was selected at all the phenological stages in both growing seasons, except for the latest stage, with low chlorophyll content due to the onset of senescence, in which the red band was selected. At stem elongation, the shortwave infrared (SWIR) band of SPOT data was also selected. At this stage, the best N estimation accuracy was obtained using an LMEM (root mean square error, RMSE = 0.012 t ha^?1). The inclusion of a spatial component in the estimation model by means of LMEMs provided a more accurate estimation than ordinary least square (OLS) models at all growth stages. The test carried out with CHRIS-PROBA data at the fourth stage confirmed the importance of NIR and in particular of the red-edge region for N uptake prediction. A novel methodology is proposed, which involves two crucial aspects in the context of the use of remote-sensing data in precision agriculture: i) the standardization of the spatial resolution for in-field and satellite data by a geostatistical data technique (data fusion); and ii) the selection of the most appropriate spectral bands for each phenological stage, taking into account both correlation with the target variable and collinearity.