Papillomavirus capsid binding and uptake by cells from different tissues and species.

The inability of papillomaviruses (PV) to replicate in tissue culture cells has hampered the study of the PV life cycle. We investigated virus-cell interactions by the following two methods: (i) using purified bovine PV virions or human PV type 11 (HPV type 11) virus-like particles (VLP) to test the binding to eukaryotic cells and (ii) using different VLP-reporter plasmid complexes of HPV6b, HPV11 L1 or HPV11 L1/L2, and HPV16 L1 or HPV16 L1/L2 to study uptake of particles into different cell lines. Our studies showed that PV capsids bind to a broad range of cells in culture in a dose-dependent manner. Binding of PV capsids to cells can be blocked by pretreating the cells with the protease trypsin. Penetration of PV into cells was monitored by using complexes in which the purified PV capsids were physically linked to DNA containing the gene for beta-galactosidase driven by the human cytomegalovirus promoter. Expression of beta-galactosidase occurred in < 1% of the cells, and the efficiency of PV receptor-mediated gene delivery was greatly enhanced (up to 10 to 20% positive cells) by the use of a replication-defective adenovirus which promotes endosomal lysis. The data generated by this approach further confirmed the results obtained from the binding assays, showing that PV enter a wide range of cells and that these cells have all functions required for the uptake of PV. Binding and uptake of PV particles can be blocked by PV-specific antisera, and different PV particles compete for particle uptake. Our results suggest that the PV receptor is a conserved cell surface molecule(s) used by different PV and that the tropism of infection by different PV is controlled by events downstream of the initial binding and uptake.     

Regulation by estrogen through the 5'-flanking region of the transforming growth factor alpha gene.

Expression of transforming growth factor alpha (TGF alpha) mRNA and protein can be stimulated by estrogens such as 17 beta-estradiol (E2) in estrogen-responsive rodent and human breast cancer cells. To ascertain if E2 can directly regulate TGF alpha expression through the 5'-flanking region of the human TGF alpha gene, E2-responsive MCF-7 or ZR-75-1 human breast cancer cells or E2-nonresponsive MDA-MB-231 breast cancer cells were transiently transfected with a plasmid containing an 1140-base pair (bp) Sac-I fragment of the TGF alpha 5'-flanking region ligated to the chloramphenicol acetyltransferase (CAT) gene. Cells that were transfected and subsequently treated with physiological concentrations of E2 (10(-11)-10(-8) M) for 24 h exhibited a 2- to 10-fold increase in CAT activity. The E2 stimulation of CAT activity was dose-dependent with an increase first found at 10(-10) M E2. The increase in CAT activity could be detected within 24-36 h after the addition of E2. There was no significant change in CAT activity in transiently transfected MDA-MB-231 cells as mediated through the TGF alpha 5'-flanking region after E2 treatment. MCF-7 cells were also transiently transfected with different fragments of the TGF alpha 5'-flanking region ligated to the luciferase gene. In the absence of E2 treatment, no detectable luciferase activity was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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.     

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 effects of ecolabels on environmentally- and health-friendly cars: an online survey and two experimental studies

The International Journal of Life Cycle Assessment - Given the increasing importance of political decision-making to reduce emission targets, the main purpose of the current paper is to identify...     

Aerobic training prevents cardiometabolic changes triggered by myocardial infarction in ovariectomized rats

This study aimed to evaluate the impact of aerobic training (AT) on autonomic, cardiometabolic, ubiquitin‐proteasome activity, and inflammatory changes evoked by myocardial infarction (MI) in ovariectomized rats. Female Wistar rats were ovariectomized and divided into four groups: sedentary + sham (SS), sedentary + MI (SI), AT + sham surgery (TS), AT + MI (TI). AT was performed on a treadmill for 8 weeks before MI. Infarcted rats previously subjected to AT presented improved physical capacity, increased interleukin‐10, and decreased pro‐inflammatory cytokines. Metabolomic analysis identified and quantified 62 metabolites, 9 were considered significant by the Vip Score. SS, SI, and TS groups presented distinct metabolic profiles; however, TI could not be distinguished from the SS group. MI dramatically increased levels of dimethylamine, and AT prevented this response. Our findings suggest that AT may be useful in preventing the negative changes in functional, inflammatory, and metabolic parameters related to MI in ovariectomized rats.     

The citrus plant pathogen Xanthomonas citri has a dual polyamine-binding protein

ATP-Binding Cassette transporters (ABC transporters) are protein complexes involved in the import and export of different molecules, including ions, sugars, peptides, drugs, and others. Due to the diversity of substrates, they have large relevance in physiological processes such as virulence, pathogenesis, and antimicrobial resistance. In Xanthomonas citri subsp. citri, the phytopathogen responsible for the citrus canker disease, 20% of ABC transporters components are expressed under infection conditions, including the putative putrescine/polyamine ABC transporter, PotFGHI. Polyamines are ubiquitous molecules that mediate cell growth and proliferation and play important role in bacterial infections. In this work, we characterized the X. citri periplasmic-binding protein PotF (XAC2476) using bioinformatics, biophysical and structural methods. PotF is highly conserved in Xanthomonas sp. genus, and we showed it is part of a set of proteins related to the import and assimilation of polyamines in X. citri. The interaction of PotF with putrescine and spermidine was direct and indirectly shown through fluorescence spectroscopy analyses, and experiments of circular dichroism (CD) and small-angle X-ray scattering (SAXS), respectively. The protein showed higher affinity for spermidine than putrescine, but both ligands induced structural changes that coincided with the closing of the domains and increasing of thermal stability.