Expression and immunolocalization of endothelin peptides and its receptors, ETA and ETB, in the carotid body exposed to chronic intermittent hypoxia.

Increased levels of endothelin-1 (ET-1) in the carotid body (CB) contribute to the enhancement of chemosensory responses to acute hypoxia in cats exposed to chronic intermittent hypoxia (CIH). However, it is not known if the ET receptor types A (ETA-R) and B (ETB-R) are upregulated. Thus, we studied the expression and localization of ETA-R and ETB-R using Western blot and immunohistochemistry (IHC) in CBs from cats exposed to cyclic hypoxic episodes, repeated during 8 hr for 4 days. In addition, we determined if ET-1 is expressed in the chemoreceptor cells using double immunofluorescence for ET-1 and tyrosine hydroxylase (TH). We found that ET-1 expression was ubiquitous in the blood vessels and CB parenchyma, although double ET-1 and TH-positive chemoreceptor cells were mostly found in the parenchyma. ETAR was expressed in most chemoreceptor cells and blood vessels of the CB vascular pole. ETB-R was expressed in chemoreceptor cells, parenchymal capillaries, and blood vessels of the vascular pole. CIH upregulated ETB-R expression by approximately 2.1 (Western blot) and 1.6-fold (IHC) but did not change ETA-R expression. Present results suggest that ET-1,ETA-R, and ETB-R are involved in the enhanced CB chemosensory responses to acute hypoxia induced by CIH.     

The Saccharomyces cerevisiae ADP/ATP carrier-iso 1 cytochrome c fusion protein: one-step purification and functional analysis in vitro

Genetic expression versus plasmidic overexpression of a functional recombinant fusion protein combining the yeast Saccharomyces cerevisiae mitochondrial ADP/ATP carrier (Anc2p) and the iso-1-cytochrome c (Cyc1p) has been investigated, with the main aim of increasing the polar surface of the carrier to improve its crystallization properties. The gene encoding the his6-tagged fusion protein was expressed in yeast under the control of the regulatory sequences of ScANC2 or under the control of the strong yeast PMA1 promoter. In both cases, the chimeric carrier, Anc2-Cyc1(His6)p, was able to restore growth on a non-fermentable carbon source of a yeast strain devoid of functional ADP/ATP carrier, demonstrating its transport activity. Nevertheless, when the expression vector was used, the level of expression of Anc2-Cyc1(His6)p was no greater than that of the chimeric carrier obtained in yeast mitochondria after homologous recombination. Optimal conditions to extract and to purify Anc2-Cyc1(His6)p were determined. A series of detergents was screened for their ability to extract and to preserve in vitro the chimeric carrier. A rapid, single step purification of Anc2-Cyc1(His6)p was developed, using n-dodecyl-beta-d-maltoside (DoDM) as the best detergent to solubilize the chimeric protein. Carboxyatractyloside- (CATR-) and nucleotide-binding sites were preserved in the purified protein. Moreover, the Cyc1p moiety of Anc2-Cyc1(His6)p-CATR complex solubilized in DoDM was still able to interact in vitro with the cytochrome c oxidase (COX), with the same affinity as yeast Cyc1p. Improved production and purification of Anc2-Cyc1(His6)p-CATR complex opens up new possibilities for the use of this protein in crystallographic approaches to the yeast ADP/ATP carrier. Furthermore, Anc2-Cyc1(His6)p may be an useful molecular tool to investigate in vivo interactions between components of the respiratory chain complexes such as COX and the proteins implicated in ATP biogenesis, such as the ATP/ADP carrier.     

Detection of Gambierdiscus and Fukuyoa single cells using recombinase polymerase amplification combined with a sandwich hybridization assay

Dinoflagellates of the genera Gambierdiscus and Fukuyoa are known to produce several bioactive compounds including the potent neurotoxic ciguatoxins (CTXs) which are able to accumulate in fish and through the food web. When humans ingest fish contaminated with CTXs, it can result in an intoxication named ciguatera. Although not all the currently recognized species are able to produce toxins, G. australes and G. excentricus have been highlighted to be the most abundant and toxic among the species present in the Atlantic. Even though the genera Gambierdiscus and Fukuyoa are endemic to tropical areas, recently their presence was recorded in subtropical and temperate regions. In this work, the development of three molecular assays for the detection of the Gambierdiscus and Fukuyoa genera and for G. australes and G. excentricus species, based on the combination of recombinase polymerase amplification with detection via hybridization, is successfully described. Furthermore, a remarkable limit of detection of a single cell was achieved. Additionally, six different species have been used to check the ability of each primer set to give an amplified product, even in presence of potentially interfering non-target DNAs. Therefore, these developments provide a rapid and cost-effective strategy for detection of both genera and two of the most toxic species, which will undoubtedly contribute to reliable screening of samples and ciguatera risk assessment, guaranteeing seafood safety and protection of human health.

     

Differential effects of local habitat and landscape characteristics on bird communities in Mediterranean afforestations motivated by the EU Common Agrarian Policy

We investigated the effects of local habitat structure and surrounding landscape characteristics (proportion of land use types and connectedness) on species density and composition of bird communities inhabiting the interior of young tree plantations on former cropland in central Spain, which were motivated by the Common Agrarian Policy. Variation of species density (number of species/0.78 ha) among tree plantations showed different environmental associations across seasons: local habitat was more important than landscape characteristics during winter, whereas they were similarly important during spring. Species density increased with the development of the tree layer in winter and with the presence of urban areas around tree plantations and cover of the herbaceous layer within them in the breeding season. We identified 15 species that exhibit high relative abundance in woodland habitats within the Mesomediterranean region of Central Spain that were absent in both seasons in the studied tree plantations, which were an attractive habitat for urban exploiter species but an unfavorable habitat for the regional forest species pool except for forest generalist species. Composition of bird assemblages was more related to local habitat structure than to landscape characteristics around tree plantations and was rather similar in winter and spring seasons. The very different effects of local habitat and landscape characteristics on bird communities make difficult suggesting management practices with positive effects for all avifauna species during the entire year. We conclude that the small size and low maturity of the studied tree plantations do not contribute to enhancing the bird diversity value of current CAP aids to afforest former cropland with pines in the Mediterranean region.     

Species-specific population trends detected for penguins,gulls and cormorants over 20 years in sub-Antarctic Fuegian Archipelago

Understanding the dynamics and causes of population trends are essential for seabird conservation. Long-term studies of seabirds at high-latitude (Antarctic, sub-Antarctic and Arctic) regions have shown contrasting species-specific trends in population size in response to climate change and anthropogenic pressures. We have studied for the last 20 years (1992–2012) the population trends of seven seabird species that breed in the Beagle Channel, south-eastern Tierra del Fuego and at Staten Island, a sub-Antarctic region in southern Argentina. The numbers of Magellanic and Gentoo Penguins increased significantly since 1992 (by >15 % year^?1). In comparison, the populations of Imperial Cormorants, Dolphin Gulls and Kelp Gulls increased at slower rates (<5 % year^?1), while the Rock Cormorant population even decreased by 1.3 % year^?1. At Staten Island, the numbers of Rockhopper Penguins decreased by 24 % between the censuses of 1998 and 2010, whereas the population of Magellanic Penguins increased by 227 % during the same period. Over the study period, air and sea-surface temperatures remained stable in our study area, suggesting that the detected population changes are not driven by the climate. This finding contrasts with the detected links between increasing temperature trends and seabird population changes reported from Antarctic and Arctic regions. The level of tourism and size of the permanent human population has increased in the Beagle Channel area during the last 20 years and could be responsible for the increase of gull populations. The seabird species that received the highest number of visitors (Imperial Cormorants and penguin species) seem to be adapted or at least indifferent to pressures exerted by tourism, as their populations increased during the study period. In addition, increasing numbers of seabirds in the area may generally be leading to higher abundances of scavenging species (e.g. gulls).     

Bradyrhizobium valentinum sp. nov., isolated from effective nodules of Lupinus mariae-josephae, a lupine endemic of basic-lime soils in Eastern Spain

Bacterial strains isolated from nitrogen-fixing nodules of Lupinus mariae-josephae have been characterized following genetic, phenotypic and symbiotic approaches. Analysis of 16S rRNA genes placed them in a group together with Bradyrhizobium elkanii USDA 76^T, B. pachyrhizi PAC48^T, B. jicamae PAC68^T, ‘B. retamae’ Ro19^T and B. lablabi CCBAU 23086^T with over 99.0% identity. Phylogenetic analysis of concatenated housekeeping genes, recA, atpD and glnII, suggested that L. mariae-josephae strains represent a new Bradyrhizobium species, closely related to B. lablabi CCBAU 23086^T, B. jicamae PAC68^T and ‘B. retamae’ Ro19^T with 92.1, 91.9 and 90.8% identity, respectively. These results are consistent with overall genomic identities calculated as Average Nucleotide Identity (ANIm) using draft genomic sequences obtained for relevant strains. While L. mariae-josephae strains LmjM3^T/LmjM6 exhibited a 99.2% ANIm value, they were significantly distant (<93% ANIm) from type strains of their closest species (‘B. retamae’ Ro19^T, B. lablabi CCBAU 23086^T and B. jicamae PAC68^T). Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (WC-MALDI-TOF-MS) analysis of proteomic patterns of the same strains was consistent with these results. The symbiosis-related genes nodC, nodA and nifH genes from strains nodulating L. mariae-josephae were phylogenetically related to those from ‘B. retamae’ Ro19^T, but divergent from those of strains that nodulate other lupine species. Based on genetic, genomic, proteomic and phenotypic data presented in this study, L. mariae-josephae nodulating strains LmjM3^T, LmjM6 and LmjM2 should be grouped within a new species for which the name Bradyrhizobium valentinum sp. nov. is proposed (type strain LmjM3^T = CECT 8364^T, LMG 2761^T)     

Lactate and the Lactate-to-Pyruvate Molar Ratio Cannot Be Used as Independent Biomarkers for Monitoring Brain Energetic Metabolism: A Microdialysis Study in Patients with Traumatic Brain Injuries

Background

For decades, lactate has been considered an excellent biomarker for oxygen limitation and therefore of organ ischemia. The aim of the present study was to evaluate the frequency of increased brain lactate levels and the LP ratio (LPR) in a cohort of patients with severe or moderate traumatic brain injury (TBI) subjected to brain microdialysis monitoring to analyze the agreement between these two biomarkers and to indicate brain energy metabolism dysfunction.

Methods

Forty-six patients with an admission Glasgow coma scale score of ≤13 after resuscitation admitted to a dedicated 10-bed Neurotraumatology Intensive Care Unit were included, and 5305 verified samples of good microdialysis data were analyzed.

Results

Lactate levels were above 2.5 mmol/L in 56.9% of the samples. The relationships between lactate and the LPR could not be adequately modeled by any linear or non-linear model. Neither Cohen’s kappa nor Gwet’s statistic showed an acceptable agreement between both biomarkers to classify the samples in regard to normal or abnormal metabolism. The dataset was divided into four patterns defined by the lactate concentrations and the LPR. A potential interpretation for these patterns is suggested and discussed. Pattern 4 (low pyruvate levels) was found in 10.7% of the samples and was characterized by a significantly low concentration of brain glucose compared with the other groups.

Conclusions

Our study shows that metabolic abnormalities are frequent in the macroscopically normal brain in patients with traumatic brain injuries and a very poor agreement between lactate and the LPR when classifying metabolism. The concentration of lactate in the dialysates must be interpreted while taking into consideration the LPR to distinguish between anaerobic metabolism and aerobic hyperglycolysis.