Cyclodextrin-Based Nanosponges for Delivery of Resveratrol: <Emphasis Type="Italic">In Vitro</Emphasis> Characterisation,Stability, Cytotoxicity and Permeation Study

The aim of this work was to increase the solubility, stability and permeation of resveratrol by complexation with cyclodextrin-based nanosponges (NS). Nanosponges are recently developed hyper-cross-linked cyclodextrin polymers nanostructured to form three-dimensional networks; they are obtained by reacting cyclodextrin with a cross-linker such as carbonyldiimidazole. They have been used to increase the solubility and stability of poorly soluble actives. This study aimed at formulating complexes of resveratrol with β-cyclodextrin nanosponges in different weight ratios. DSC, FTIR and X-ray powder diffraction (XRPD) studies confirmed the interaction of resveratrol with NS. XRPD showed that the crystallinity of resveratrol decrease after encapsulation. The particle sizes of resveratrol-loaded NS are in between 400 to 500 nm with low polydispersity indices. Zeta potential is sufficiently high to obtain a stable colloidal nanosuspension. TEM measurement also revealed a particle size around 400 nm for NS complexes. The in vitro release and stability of resveratrol complex were increased compared with plain drug. Cytotoxic studies on HCPC-I cell showed that resveratrol formulations were more cytotoxic than plain resveratrol. The permeation study indicates that the resveratrol NS formulation showed good permeation in pigskin. The accumulation study in rabbit mucosa showed better accumulation of resveratrol NS formulation than plain drug. These results signify that resveratrol NS formulation can be used for buccal delivery and topical application.     

H ferritin gene silencing in a human metastatic melanoma cell line: a proteomic analysis

Ferritin, the major intracellular iron-storage protein, is made of 24 subunits of two types, H and L. Besides regulating intracellular iron homeostasis, it has been found that ferritin, in particular the H subunit (FHC), is involved in different biological events such as cell differentiation and pathologic states (i.e., neurodegeneration and cancer). This study is aimed at investigating the whole-cell proteome of FHC-expressing and sh-RNA-silenced human metastatic melanoma cells (MM07(m)) in the attempt to identify and classify the highest number of proteins directly or indirectly controlled by the FHC. We identified about 200 differentially expressed proteins and classified them in clusters on the basis of their functions, as proteins involved in metabolic processes, cell adhesion, migration, and proliferation processes. Some of them have captured our attention because of their involvement in metabolic pathways related to tumor progression and metastasis. In vitro assays confirmed that the FHC-silenced MM07(m) cells are characterized by a decreased growth activity, a reduced invasiveness, and a reduced cell adhesion capability. Moreover, nude mice (CD1 nu/nu), subcutaneously injected with FHC-silenced MM07(m) cells, showed a remarkable 4-fold reduction of their tumor growth capacity compared to those who received the FHC-unsilenced MM07(m) counterpart. In conclusion, these data indicate that gene silencing technology, coupled to proteomic analysis, is a powerful tool for a better understanding of H ferritin signaling pathways and lend support to the hypothesis that specific targeting of this gene might be an attractive and potentially effective strategy for the management of metastatic melanoma.     

Exposure to tebuconazol in rice field and laboratory conditions induces oxidative stress in carp (Cyprinus carpio)

Pesticides can have an effect on the biochemical and physiological functions of living organisms. The changes seen in fish and their response to pesticides can be used as an example for vertebrate toxicity. In this study, carp fish (Cyprinus carpio) were exposed to different concentrations of tebuconazol fungicide, by rice field (31.95 μg/L) and laboratory (33.47 and 36.23 μg/L) conditional testing, during a 7 day period. Parameters such thiobarbituric acid-reactive substance levels (TBARS), protein carbonyl, catalase, glutathione S-transferase and acetylcholinesterase activities were studied, using the liver, brain and white muscle of the fish. The field experiment showed that the TBARS levels were increased in all the analyzed tissues. Similarly, the protein carbonyl of the liver and the brain AChE activity increased after 7 days. The laboratory experiment demonstrated that the TBARS levels in the liver were increased in both of the concentration tests. TBARS levels in the muscle increased only by the lowest test concentration. On the other hand, the protein carbonyl was increased only by the highest concentration. The results indicate that the tebuconazol exposure from the field and laboratory conditions directly affected the health of the fish, showing the occurrence of oxidative stress.     

Identification of proteins associated with ligand-activated estrogen receptor α in human breast cancer cell nuclei by tandem affinity purification and nano LC-MS/MS

Estrogen receptor α (ER-α) is a key mediator of estrogen actions in breast cancer (BC) cells. Understanding the effects of ligand-activated ER-α in target cells requires identification of the molecular partners acting in concert with this nuclear receptor to transduce the hormonal signal. We applied tandem affinity purification (TAP), glycerol gradient centrifugation and MS analysis to isolate and identify proteins interacting with ligand-activated ER-α in MCF-7 cell nuclei. This led to the identification of 264 ER-associated proteins, whose functions highlight the hinge role of ER-α in the coordination of multiple hormone-regulated nuclear processes in BC cells.     

Characterization of Salmonella occurring at high prevalence in a population of the land iguana Conolophus subcristatus in Galápagos Islands, Ecuador

The aim of the study was to elucidate the association between the zoonotic pathogen Salmonella and a population of land iguana, Colonophus subcristatus, endemic to Galápagos Islands in Ecuador. We assessed the presence of Salmonella subspecies and serovars and estimated the prevalence of the pathogen in that population. Additionally, we investigated the genetic relatedness among isolates and serovars utilising pulsed field gel electrophoresis (PFGE) on XbaI-digested DNA and determined the antimicrobial susceptibility to a panel of antimicrobials. The study was carried out by sampling cloacal swabs from animals (n = 63) in their natural environment on in the island of Santa Cruz. A high prevalence (62/63, 98.4%) was observed with heterogeneity of Salmonella subspecies and serovars, all known to be associated with reptiles and with reptile-associated salomonellosis in humans. Serotyping revealed 14 different serovars among four Salmonella enterica subspecies: S. enterica subsp. enterica (n = 48), S. enterica subsp. salamae (n = 2), S. enterica subsp. diarizonae (n = 1), and S. enterica subsp. houtenae (n = 7). Four serovars were predominant: S. Poona (n = 18), S. Pomona (n = 10), S. Abaetetuba (n = 8), and S.Newport (n = 5). The S. Poona isolates revealed nine unique XbaI PFGE patterns, with 15 isolates showing a similarity of 70%. Nine S. Pomona isolates had a similarity of 84%. One main cluster with seven (88%) indistinguishable isolates of S. Abaetetuba was observed. All the Salmonella isolates were pan-susceptible to antimicrobials representative of the most relevant therapeutic classes. The high prevalence and absence of clinical signs suggest a natural interaction of the different Salmonella serovars with the host species. The interaction may have been established before any possible exposure of the iguanas and the biocenosis to direct or indirect environmental factors influenced by the use of antimicrobials in agriculture, in human medicine or in veterinary medicine.     

PKCδ sensitizes neuroblastoma cells to L-buthionine-sulfoximine and etoposide inducing reactive oxygen species overproduction and DNA damage

Neuroblastoma is a type of pediatric cancer. The sensitivity of neuroblastoma (NB) cancer cells to chemotherapy and radiation is inhibited by the presence of antioxidants, such as glutathione (GSH), which is crucial in counteracting the endogenous production of reactive oxygen species (ROS). We have previously demonstrated that cells depleted of GSH undergo apoptosis via oxidative stress and Protein kinase C (PKC) δ activation. In the present study, we transfected PKCδ in NB cells resistant to oxidative death induced by L-buthionine-S,R-sulfoximine (BSO), a GSH-depleting agent. Cell responses, in terms of ROS production, apoptosis and DNA damage were evaluated. Moreover, PKCδ activation was monitored by analyzing the phosphorylation status of threonine 505 residue, carrying out PKC activity assay and investigating the subcellular localization of the kinase. The cell responses obtained in BSO-resistant cells were also compared with those obtained in BSO-sensitive cells subjected to the same experimental protocol. Our results demonstrate, for the first time, that PKCδ induces DNA oxidation and ROS overproduction leading to apoptosis of BSO-resistant NB cells and potentiates the cytotoxic effects induced by BSO in sensitive cells. Moreover, PKCδ overexpression enhances the sensitivity of NB cells to etoposide, a well-characterised drug, commonly used in neuroblastoma therapy. Altogether our data provide evidence of a pro-oxidant role of PKCδ that might be exploited to design new therapeutic strategies aimed at selective killing of cancer cells and overcoming drug resistance. However, it becomes evident that a more detailed understanding of ROS-mediated signaling in cancer cells is necessary for the development of redox-modulated therapeutic approaches.     

IPHEN—a real-time network for phenological monitoring and modelling in Italy

This paper aims to describe the Italian PHEnology Network (IPHEN), a cooperative project started in 2006 with the aim of producing nationwide maps of analysis and forecast of plants phenological stages mainly used to satisfy the needs of agriculture, health and environmental care. Iphen is a data processing system composed of the following main segments (a) collection of atmospheric and phenological data, (b) processing of data with suitable phenological and geo-statistical models and (c) phenological maps of analysis and forecast. In more detail, IPHEN maps of analysis (featuring phenological stages reached at the date of processing) are produced with models based on a Normal Heat Hours approach which weighs hourly air temperature effectiveness for plant phenological progression applied to national grids of hourly temperature derived from the operational agro-meteorological network of CRA-CMA. A correction scheme based on phenological surveys provided by volunteer observers is applied to the first guess maps of analysis to obtain final maps. Forecast maps (prediction of the days of occurrence of relevant phenological stages) are produced on the basis of GFS model medium range forecasts and climatic data. Freeware IPHEN maps for grapevine, common and Arizona cypress, black elder, olive and locust trees are broadcasted weekly on the CRA-CMA website. The positive operational results of IPHEN are testified by 150 maps broadcasted during the 2011 season for the above-mentioned species. The system performances and reliability have been analysed focusing on the analysis of phenological simulation errors and on the sensitivity of phenological maps to anomalous atmospheric circulation patterns. The error analysis shows that phenological models are characterized by advances/delays that justify the adoption of an observation based correction scheme. The sensitivity analysis highlights that the system is responsive to the effects of circulation blocking systems leading to phenological advances and delays.     

Humpback whales washed ashore in southeastern Brazil from 1981 to 2011: stranding patterns and microbial pathogens survey

In the Southwest Atlantic Ocean the wintering breeding ground of Megaptera novaeangliae is located in northeastern coast of Brazil, mainly in the Abrolhos Bank (16°55′ S, 38°50′ W) and its surroundings. During migratory percussion, events of strandings are thought to occur. A total of 58 stranded humpback whales were recorded between 1981 and 2011 along the coast of the Rio de Janeiro state, Brazil. The number of strandings in 20 years (1992–2011) increased during the period of study, with a mean of 2.6 events per year. In 2010 a peak occurred with 13 records of strandings. Most of them occurred preferably in the southern half of the study area, or in the southeast area of Rio de Janeiro state. Three cases of entanglement were found, two of these involving calves with less than eight meters of body length. Stranding events were more frequent during winter and spring. No statistical differences were found between age categories, but 33% were classified as “dependent calf” (< 8 m length). Males stranded more often than females. Only one whale out of 16 specimens that had the stomach contents examined presented food remains, but comprising only two cephalopod beaks of the squid Doryteuthis sanpaulensis. Bacteriological survey of Vibrionaceae and Aeromonadaceae agents in three live stranded whales on the Brazilian coast indicated evidence of animal impairment that resulted or were associated with the cause of death and stranding event.     

Effect of ultraviolet-B radiation in laboratory on morphological and ultrastructural characteristics and physiological parameters of selected cultivar of Oryza sativa L.

Ultraviolet-B radiation (UVBR) affects plants in many important ways, including reduction of growth rate and primary productivity, and changes in ultrastructures. Rice (Oryza sativa) is one of the most cultivated cereals in the world, along with corn and wheat, representing over 50 % of agricultural production. In this study, we examined O. sativa plants exposed to ambient outdoor radiation and laboratory-controlled photosynthetically active radiation (PAR) and PAR + UVBR conditions for 2 h/day during 30 days of cultivation. The samples were studied for morphological and ultrastructural characteristics, and physiological parameters. PAR + UVBR caused changes in the ultrastructure of leaf of O. sativa and leaf morphology (leaf index, leaf area and specific leaf area, trichomes, and papillae), plant biomass (dry and fresh weight), photosynthetic pigments, phenolic compounds, and protein content. As a photoprotective acclimation strategy against PAR + UVBR damage, an increase of 66.24 % in phenolic compounds was observed. Furthermore, PAR + UVBR treatment altering the levels of chlorophylls a and b, and total chlorophyll. In addition, total carotenoid contents decreased after PAR + UVBR treatment. The results strongly suggested that PAR + UVBR negatively affects the ultrastructure, morphology, photosynthetic pigments, and growth rates of leaf of O. sativa and, in the long term, it could affect the viability of this economically important plant.