SERS‐based biosensor for Alzheimer disease evaluation through the fast analysis of human serum

Alzheimer disease (AD) is the most common form of dementia in the elderly, progressively affecting the cognitive functions with a complex diagnostic procedure that limits the time for a prompt intervention. In this study we optimized a reliable protocol for the analysis of AD patients and healthy subjects' serum using the Surface Enhanced Raman Spectroscopy (SERS), taking into consideration the effect of different variables on the final spectra, analyzed and compared through multivariate analysis and correlated with hippocampus volume. As results, we demonstrated a statistical difference between the spectra collected from the two investigated groups, with an accuracy, precision and specificity of respectively 83%, 86%, and 86%. The correlation of these data with those obtained from MRI, demonstrated a direct correlation between Raman spectra and hippocampus degeneration showing the Raman Spectroscopy (RS) as a potential tool for the monitoring of AD progression and rehabilitation treatments.     

Structural and ecophysiological adaptations to forest gaps

To survive new microclimatic conditions of a forest gap environment, plant species must physiologically and structurally adjust. A morpho-anatomical, ultrastructural and ecophysiological study was performed at three different times in a forest gap that was created by illegal selective logging. The study followed the early successional Actinostemon verticillatus and the late-successional Metrodorea brevifolia, to elucidate the adaptive strategies of acclimation to gaps. Additionally, Schinus terebinthifolius was included in the study in order to test the plasticity of a pioneer species that grows on forest edges, where this species had higher values of leaf thickness, leaf mass area and succulence. M. brevifolia had succulent leaves, high leaf area and a thin cuticle. A. verticillatus presented the densest leaves and was the only species to show leaf morpho-anatomical plasticity. Ultrastructural and physiological differences were observed only in A. verticillatus and M. brevifolia leaves from the gap: increase in the stroma volume, oil droplets, plastoglobuli, photochemical and non-photochemical quenching. Photosynthetic efficiency showed that the early stages of gap formation are the most critical. Acclimation strategies of A. verticillatus suggest this species invests in the efficiency of photosynthesis by increasing its leaf thickness, leaf mass area and in water content maintenance by increasing the density of its leaves, at the expense of gas exchange, was compensated by a high density of stomata. M. brevifolia compensates for the higher cost of leaves and lower leaf plasticity with ultrastructural changes that are used to adjust the photosynthetic process, which promotes a shorter leaf payback time.     

Use of nonradiative decays of extrinsic fluorophores as structural and dynamical probes in protein environments: Fluorescence quenching

In this work a combined pulsed-laser, time-resolved photoacoustic calorimetry (PAC) and fluorescence study is presented on two widely used covalent protein probes, fluorescein-5-isothiocyanate (FITC) and 6-acryloyl-2-dimethylaminonaphtalene (acrylodan). Three proteins that contain a single free thiol, namely carbonic anhydrase, bovine serum albumin (BSA) and papain, have been selectively labelled with FITC and acrylodan, and their fluorescence emission was quenched with KI. Nonradiative decays of the excited states of FITC are used to complement the information usually obtained by monitoring the quenching of fluorescence emssion. Data analysis evidences the dependence of the nonradiative quenching constants on the exposure of the dye to the solvent, and shows the involvement of a triplet state of FITC in the non radiative deexcitation. The shielding of the binding sites from the solvent is demonstrated also by the fluorescence emission of acrylodan and by the Stern-Volmer analysis of fluorescence quenching by KI. From photoacoustic data, an estimate of the fluorescent quantum yield of bound FITC is obtained. This work demonstrates the complete equivalence of quenching data obtained by fluorescence and photoacoustics measurements and shows that this combined approach allows a better control of the photophysics of the dyes involved in the quenching process.     

Molecular Epidemiology of Carbapenem Non-Susceptible Acinetobacter baumannii in France

Carbapenem-resistant Acinetobacter baumannii have emerged globally. The objective of this study was to investigate the epidemiology, clonal diversity and resistance mechanisms of imipenem non-susceptible A. baumannii isolates in France. Between December 2010 and August 2011, 132 notifications were collected, including 37 outbreaks corresponding to 242 cases (2 to 55 per cluster). Multilocus sequence typing, pulsed-field gel electrophoresis (PFGE) and characterisation of carbapenemase-encoding genes were performed on 110 non-repetitive isolates. Gene bla _OXA-23 was the most frequently detected (82%), followed by bla _OXA-24 (11%) and bla _OXA-58 (7%). Eleven sequence types (ST) were distinguished, among which sequence types ST1, ST2 (64%), ST20, ST25, ST85 and ST107. Isolates from epidemiological clusters had the same ST and resistance genes, indicating probable transmission within centres. In contrast, PFGE types of isolates differed among centres, arguing against transmission among centers. This study provides the first epidemiological snapshot of the population of A. baumannii with reduced susceptibility to carbapenems from France, and further underlines the predominance of international clones.     

Vicariance and endemism in a Neotropical savanna hotspot: distribution patterns of Cerrado squamate reptiles

Aim To test predictions of the vicariance model, to define basic biogeographical units for Cerrado squamates, and to discuss previous biogeographical hypotheses. Location Cerrado; South American savannas south of the Amazon, extending across central Brazil, with marginal areas in Bolivia and Paraguay and isolated relictual enclaves in adjacent regions. Methods We compiled species occurrence records via field sampling and revision of museum specimens and taxonomic literature. All species were mapped according to georeferenced locality records, and classified as (1) endemic or non‐endemic, (2) typical of plateaus or depressions, and (3) typical of open or forested habitats. We tested predictions of the vicariance model using biotic element analysis, searching for non‐random clusters of species ranges. Spatial congruence of biotic elements was compared with putative areas of endemism revealed by sympatric restricted‐range species. Effects of topographical and vegetational mosaics on distribution patterns were studied according to species composition in biotic elements and areas of endemism. Results We recorded 267 Cerrado squamates, of which 103 (39%) are endemics, including 20 amphisbaenians (61% endemism), 32 lizards (42%) and 51 snakes (32%). Distribution patterns corroborated predictions of the vicariance model, revealing groups of species with significantly clustered ranges. An analysis of endemic species recovered seven biotic elements, corroborating results including non‐endemics. Sympatric restricted‐range taxa delimited 10 putative areas of endemism, largely coincident with core areas of biotic elements detected with endemic taxa. Distribution patterns were associated with major topographical and vegetational divisions of the Cerrado. Endemism prevailed in open, elevated plateaus, whereas faunal interchange, mostly associated with forest habitats, was more common in peripheral depressions. Main conclusions Our results indicate that vicariant speciation has strongly shaped Cerrado squamate diversity, in contrast to earlier studies emphasizing faunal interchange and low endemism in the Cerrado vertebrate fauna. Levels of squamate endemism are higher than in any other Cerrado vertebrate group. The high number of recovered endemics revealed previously undetected areas of evolutionary relevance, indicating that biogeographical patterns in the Cerrado were poorly represented in previous analyses. Although still largely undocumented, effects of vicariant speciation may be prevalent in a large fraction of Cerrado and Neotropical biodiversity.     

Visual Evoked Cortical Potential (VECP) Elicited by Sinusoidal Gratings Controlled by Pseudo-Random Stimulation

The contributions of contrast detection mechanisms to the visual cortical evoked potential (VECP) have been investigated studying the contrast-response and spatial frequency-response functions. Previously, the use of m-sequences for stimulus control has been almost restricted to multifocal electrophysiology stimulation and, in some aspects, it substantially differs from conventional VECPs. Single stimulation with spatial contrast temporally controlled by m-sequences has not been extensively tested or compared to multifocal techniques. Our purpose was to evaluate the influence of spatial frequency and contrast of sinusoidal gratings on the VECP elicited by pseudo-random stimulation. Nine normal subjects were stimulated by achromatic sinusoidal gratings driven by pseudo random binary m-sequence at seven spatial frequencies (0.4–10 cpd) and three stimulus sizes (4°, 8°, and 16° of visual angle). At 8° subtence, six contrast levels were used (3.12–99%). The first order kernel (K1) did not provide a consistent measurable signal across spatial frequencies and contrasts that were tested–signal was very small or absent–while the second order kernel first (K2.1) and second (K2.2) slices exhibited reliable responses for the stimulus range. The main differences between results obtained with the K2.1 and K2.2 were in the contrast gain as measured in the amplitude versus contrast and amplitude versus spatial frequency functions. The results indicated that K2.1 was dominated by M-pathway, but for some stimulus condition some P-pathway contribution could be found, while the second slice reflected the P-pathway contribution. The present work extended previous findings of the visual pathways contribution to VECP elicited by pseudorandom stimulation for a wider range of spatial frequencies.     

First record of anomalous otoliths of Menticirrhus americanus in the South Atlantic

Sagitta otoliths are usually formed of calcium carbonate polymorphs as aragonite. The objective of this study was to verify which carbonate polymorph is predominant in the sagitta otolith of Menticirrhus americanus and check whether this pattern remains in otoliths with morphological alterations. Otoliths of M. americanus were obtained from five sites on the southeast‐south coast of Brazil (São Sebastião (SS) 23°45′S–45°24′O, n = 29; Cananéia‐Iguape Estuarine Complex (CI) 25°02′S–47°54′O, n = 30; Paranaguá Estuarine Complex (PEC) 25°28′S–48°20′O, n = 35; Itapoá (IT) 26°07′S–48°36′O, n = 31; Laguna (LA) 28°28′S–48°46′O, n = 13). The characterization of carbonate polymorphs of otoliths was performed through Raman spectroscopy, a photonic and non‐destructive technique that analyzes molecular vibrations induced by laser. We analyzed 138 pairs of M. americanus otoliths, of which eight otoliths from different pairs presented morphological alterations (SS n = 1, CEP n = 5, IT n = 1, LA n = 1). The Raman spectra show that normal otoliths, that is, without morphological alterations, presented only aragonite in their structure. Among the otoliths that presented morphological alterations, the Raman spectra allowed to identify in six otoliths the deposition of aragonite and in only two otoliths the deposition of vaterite (one specimen of the PEC and one of SS).     

The novel ruthenium—γ‐linolenic complex [Ru2(aGLA)4Cl] inhibits C6 rat glioma cell proliferation and induces changes in mitochondrial membrane potential,increased reactive oxygen species generation and apoptosis in vitro

The present study reports the synthesis of a novel compound with the formula [Ru₂(aGLA)4Cl] according to elemental analyses data, referred to as Ru₂GLA. The electronic spectra of Ru₂GLA is typical of a mixed valent diruthenium(II,III) carboxylate. Ru₂GLA was synthesized with the aim of combining and possibly improving the anti‐tumour properties of the two active components ruthenium and γ‐linolenic acid (GLA). The properties of Ru₂GLA were tested in C6 rat glioma cells by analysing cell number, viability, lipid droplet formation, apoptosis, cell cycle distribution, mitochondrial membrane potential and reactive oxygen species. Ru₂GLA inhibited cell proliferation in a time and concentration dependent manner. Nile Red staining suggested that Ru₂GLA enters the cells and ICP‐AES elemental analysis found an increase in ruthenium from <0.02 to 425 mg/Kg in treated cells. The sub‐G1 apoptotic cell population was increased by Ru₂GLA (22 ± 5.2%) when analysed by FACS and this was confirmed by Hoechst staining of nuclei. Mitochondrial membrane potential was decreased in the presence of Ru₂GLA (44 ± 2.3%). In contrast, the cells which maintained a high mitochondrial membrane potential had an increase (18 ± 1.5%) in reactive oxygen species generation. Both decreased mitochondrial membrane potential and increased reactive oxygen species generation may be involved in triggering apoptosis in Ru₂GLA exposed cells. The EC₅₀ for Ru₂GLA decreased with increasing time of exposure from 285 µM at 24 h, 211 µM at 48 h to 81 µM at 72 h. In conclusion, Ru₂GLA is a novel drug with antiproliferative properties in C6 glioma cells and is a potential candidate for novel therapies in gliomas. Copyright © 2009 John Wiley & Sons, Ltd.