Influence of drought on algal biofilms and meiofaunal assemblages of temperate reservoirs and rivers

The role of hydrological droughts in shaping meiofauna abundance through alterations in biofilm biomass and composition was investigated. In January 2005, continental Portugal was under a moderate to severe drought resulting from a 40% to 60% decrease in rainfall during the previous 12 months relative to the long-term average (1961–1990). Reservoir capacity was reduced by 30–50% relative to average values and the width of streams was reduced by 20–80% in the Zêzere River Basin (central Portugal). Algal biomass and algal class composition of biofilms was assessed through quantification of algal pigments in three reservoir and six river locations. During drought, habitat alterations are expected to be sharp in rivers while, in the absence of water quality deterioration, the habitat characteristics of reservoirs are expected to remain fairly unaffected. Chlorophylls and carotenoid pigments were extracted from biofilm samples and analysed using high performance liquid chromatography (HPLC). In the winter of 2003, during the period of average rainfall, biofilm biomass did not exceed 5 μg chlorophyll a cm⁻² at any location. River biofilm biomass was roughly half of that measured in the reservoirs. In the winter of 2005 (drought), biofilm biomass increased by more than 5-fold in river locations and remained low or decreased in the reservoirs. Algal biofilms were either dominated by Bacillariophyceae or by Chlorophyceae regardless of the existence of drought. The relative contribution of Bacillariophyceae to total biofilm biomass was higher during the drought than under average hydrological conditions. The abundance of harpacticoids, cladocerans and ostracods was favoured by the drought only in the reservoirs where an increase in diatom proportion in biofilms was observed. The increase in the abundance of cyclopoid copepods, turbellarians, nematodes and chironomids in rivers during the drought could be explained by algal class composition and biomass of biofilms and environmental variables (organic matter sediment content, phosphorus availability content and sediment granulometry). The hydrological drought appears to regulate meiofauna abundance only in river locations, possibly through the promotion of the growth of biofilms and the availability of organic matter deposited in rivers during the drought. Handling editor: D. Ryder     

Identification and preliminary characterization of temperature-sensitive mutations affecting HlyB, the translocator required for the secretion of haemolysin (HlyA) from Escherichia coli

We have carried out a genetic analysis of Escherichia coli HlyB using in vitro(hydroxylamine) mutagenesis and regionally directed mutagenesis. From random mutagenesis, three mutants, temperature sensitive (Ts) for secretion, were isolated and the DNA sequenced: Glyl0Arg close to the N-terminus, Gly408Asp in a highly conserved small periplasmic loop region PIV, and Pro624Leu in another highly conserved region, within the ATP-binding region. Despite the Ts character of the Gly10 substitution, a derivative of HlyB, in which the first 25 amino acids were replaced by 21 amino acids of the Cro protein, was still active in secretion of HlyA. This indicates that this region of HlyB is dispensable for function. Interestingly, the Gly408Asp substitution was toxic at high temperature and this is the first reported example of a conditional lethal mutation in HlyB. We have isolated 4 additional mutations in PIV by directed mutagenesis, giving a total of 5 out of 12 residues substituted in this region, with 4 mutations rendering HlyB defective in secretion. The Pro624 mutation, close to the Walker B-site for ATP binding in the cytoplasmic domain is identical to a mutation in HisP that leads to uncoupling of ATP hydrolysis from the transport of histidine. The expression of a fully functional haemolysin translocation system comprising HlyC,A,B and D increases the sensitivity of E. coli to vancomycin 2.5-fold, compared with cells expressing HlyB and HlyD alone. Thus, active translocation of HlyA renders the cells hyperpermeable to the drug. Mutations in hlyB affecting secretion could be assigned to two classes: those that restore the level of vancomycin resistance to that of E. coli not secreting HlyA and those that still confer hypersensitivity to the drug in the presence of HlyA. We propose that mutations that promote vancomycin resistance will include mutations affecting initial recognition of the secretion signal and therefore activation of a functional transport channel. Mutations that do not alter HlyA-dependent vancomycin sensitivity may, in contrast, affect later steps in the transport process.     

Assessing spatial patterns and density of a dolphin population through signature whistles

Some dolphin species produce signature whistles, which may allow the identification of individual dolphins using passive acoustic monitoring (PAM). Identifying individuals by their sounds may enhance the opportunities for monitoring and addressing biological and ecological questions about these species. Here, we explored the potential of signature whistles to investigate ecological aspects of a resident bottlenose dolphin population. Using a limited data set, with few individuals recognized by signature whistles, combined with spatial capture-recapture (SCR) methods, we investigated how effective such approach is describing spatial use patterns and estimating density for this population. The data were collected using 4–6 stationary bottom-moored recorders. Since only eight signature whistles were identified, our density estimate may represent a subset of the entire population. However, even with only a few signature whistles identified, our results confirmed the center of the core area used by these dolphins as the area with the highest encounter probability. In addition, our results provided evidence that these dolphins have the same spatial use pattern at night as during the day. This study shows that SCR analysis of signature whistle data can improve our ecological knowledge and understanding of dolphin populations.     

Chemical Composition,Antibacterial, Antibiofilm and Synergistic Properties of Essential Oils from Eucalyptus globulus Labill. and Seven Mediterranean Aromatic Plants

Essential oils (EOs) from Eucalyptus globulus Labill . ssp. globulus and from Mediterranean autochthonous aromatic plants – Thymus mastichina L., Mentha pulegium L., Rosmarinus officinalis L., Calamintha nepeta (L.) Savi ssp. nepeta, Cistus ladanifer L., Foeniculum vulgare L., Dittrichia viscosa (L.) Greuter ssp. viscosa – were extracted by hydrodistillation and characterized by GC‐FID and NMR spectroscopy. EOs were evaluated for antimicrobial properties against several bacterial strains, using diverse methods, namely, the agar disc‐diffusion method, the microdilution method, the crystal violet assay and the Live/Dead staining for assessment of biofilm formation. Potential synergy was assessed by a checkerboard method. EOs of R. officinalis and C. ladanifer showed a predominance in monoterpene hydrocarbons (> 60%); EOs of C. nepeta, M. pulegium, T. mastichina, E. globulus and F. vulgare were rich in oxygenated monoterpenes (62 – 96%) whereas EO of D. viscosa was mainly composed of oxygenated sesquiterpenes (54%). All EOs showed antimicrobial activity; M. pulegium and E. globulus generally had the strongest antimicrobial activity. EO of C. nepeta was the most promising in hampering the biofilm formation. The combinations D. viscosa/C. nepeta and E. globulus/T. mastichina were synergistic against Staphylococcus aureus. These results support the notion that EOs from the aromatic plants herein reported should be further explored as potential pharmaceuticals and/or food preservatives.     

alpha-cardiac actin (ACTC) binds to the band 3 (AE1) cardiac isoform

The band 3 protein is the major integral protein present in the erythrocyte membrane. Two tissue-specific isoforms are also expressed in kidney alpha intercalated cells and in cardiomyocytes. It has been suggested that the cardiac isoform predominantly mediates the anion exchange in cardiomyocytes, but the role of the cytoplasmic domain of the band 3 (CDB3) protein in the cardiac tissue is unknown. In order to characterize novel associations of the CDB3 in the cardiac tissue, we performed the two-hybrid assay, using a bait comprising the region from leu 258 to leu 311 of the erythrocyte band 3, which must also be present in the cardiac isoform. The assay revealed two clones containing the C-terminal region of the alpha-cardiac actin. Immunoprecipitation of whole rat heart using an anti-actin antibody, immunoblotted with anti-human band 3, showed that actin binds to band 3 which was confirmed in the reverse assay. The confocal microscopy showed band 3 in the intercalated discs. Thus, besides the in vivo physical interaction in the Saccharomyces cerevisiae cell, we demonstrated using immunopreciptation that there is a physical association of band 3 with alpha-cardiac actin in cardiomyocyte, and we suggest that the binding occur "in situ," in the intercalated disc, a site of cell-cell contact and attachment of the sarcomere to the plasma membrane.     

Solid-state NMR Study Reveals Collagen I Structural Modifications of Amino Acid Side Chains upon Fibrillogenesis

In vivo, collagen I, the major structural protein in human body, is found assembled into fibrils. In the present work, we study a high concentrated collagen sample in its soluble, fibrillar, and denatured states using one and two dimensional {¹H}-¹³C solid-state NMR spectroscopy. We interpret ¹³C chemical shift variations in terms of dihedral angle conformation changes. Our data show that fibrillogenesis increases the side chain and backbone structural complexity. Nevertheless, only three to five rotameric equilibria are found for each amino acid residue, indicating a relatively low structural heterogeneity of collagen upon fibrillogenesis. Using side chain statistical data, we calculate equilibrium constants for a great number of amino acid residues. Moreover, based on a ¹³C quantitative spectrum, we estimate the percentage of residues implicated in each equilibrium. Our data indicate that fibril formation greatly affects hydroxyproline and proline prolyl pucker ring conformation. Finally, we discuss the implication of these structural data and propose a model in which the attractive force of fibrillogenesis comes from a structural reorganization of 10 to 15% of the amino acids. These results allow us to further understand the self-assembling process and fibrillar structure of collagen.     

Leucine and HMB Differentially Modulate Proteasome System in Skeletal Muscle under Different Sarcopenic Conditions

In the present study we have compared the effects of leucine supplementation and its metabolite β-hydroxy-β-methyl butyrate (HMB) on the ubiquitin-proteasome system and the PI3K/Akt pathway during two distinct atrophic conditions, hindlimb immobilization and dexamethasone treatment. Leucine supplementation was able to minimize the reduction in rat soleus mass driven by immobilization. On the other hand, leucine supplementation was unable to provide protection against soleus mass loss in dexamethasone treated rats. Interestingly, HMB supplementation was unable to provide protection against mass loss in all treatments. While solely fiber type I cross sectional area (CSA) was protected in immobilized soleus of leucine-supplemented rats, none of the fiber types were protected by leucine supplementation in rats under dexamethasone treatment. In addition and in line with muscle mass results, HMB treatment did not attenuate CSA decrease in all fiber types against either immobilization or dexamethasone treatment. While leucine supplementation was able to minimize increased expression of both Mafbx/Atrogin and MuRF1 in immobilized rats, leucine was only able to minimize Mafbx/Atrogin in dexamethasone treated rats. In contrast, HMB was unable to restrain the increase in those atrogenes in immobilized rats, but in dexamethasone treated rats, HMB minimized increased expression of Mafbx/Atrogin. The amount of ubiquitinated proteins, as expected, was increased in immobilized and dexamethasone treated rats and only leucine was able to block this increase in immobilized rats but not in dexamethasone treated rats. Leucine supplementation maintained soleus tetanic peak force in immobilized rats at normal level. On the other hand, HMB treatment failed to maintain tetanic peak force regardless of treatment. The present data suggested that the anti-atrophic effects of leucine are not mediated by its metabolite HMB.     

Hypersomnolence and Accidents in Truck Drivers: A Cross‐Sectional Study

Truck drivers are more likely to suffer severe injury and death due to certain truck driving characteristics. Identifying and preventing factors associated with accidents in this population is important to minimize damage and improve road safety. Excessive daytime sleepiness is a major public health problem, leading to impaired cognitive function, reduced alertness, and increased risk of motor vehicle crashes. The aim of this cross‐sectional study was to determine the prevalence and predictors of hypersomnolence (defined as an Epworth Sleepiness Scale score greater than 10) among truck drivers. Three hundred male truck drivers were studied. Quality of sleep was assessed by the Pittsburgh Sleep Quality Index, and the association between demographic, clinical, and occupational data with excessive sleepiness was analyzed. The mean daily sleep duration was 5.6±1.3 h, and poor quality of sleep was found in 46.3% of the individuals. Hypersomnolence was found in 46% of the drivers and was associated with younger age, snoring, and working >10 h without rest. A positive correlation between hypersomnolence and previous accidents was detected (p=0.005). These results show that sleep deprivation and hypersomnolence are frequent among truck drivers. The treatment of sleep‐disordered breathing and the implementation of educational programs, particularly targeting younger drivers and promoting increased awareness of the deleterious effects of sleep loss and work overload, may help to reduce hypersomnolence and accidents among truck drivers.     

Metabolic pathway for propionate utilization by phosphorus-accumulating organisms in activated sludge: 13C labeling and in vivo nuclear magnetic resonance

In vivo 13C and 31P nuclear magnetic resonance techniques were used to study propionate metabolism by activated sludge in enhanced biological phosphorus removal systems. The fate of label supplied in [3-13C]propionate was monitored in living cells subjected to anaerobic/aerobic cycles. During the anaerobic phase, propionate was converted to polyhydroxyalkanoates (PHA) with the following monomer composition: hydroxyvalerate, 74.2%; hydroxymethylvalerate, 16.9%; hydroxymethylbutyrate, 8.6%; and hydroxybutyrate, 0.3%. The isotopic enrichment in the different carbon atoms of hydroxyvalerate (HV) produced during the first anaerobic stage was determined: HV5, 59%; HV4, 5.0%; HV3, 1.1%; HV2, 3.5%; and HV1, 2.8%. A large proportion of the supplied label ended up on carbon C-5 of HV, directly derived from the pool of propionyl-coenzyme A (CoA), which is primarily labeled on C-3; useful information on the nature of operating metabolic pathways was provided by the extent of labeling on C-1, C-2, and C-4. The labeling pattern on C-1 and C-2 was explained by the conversion of propionyl-CoA to acetyl-CoA via succinyl-CoA and the left branch of the tricarboxylic acid cycle, which involves scrambling of label between the inner carbons of succinate. This constitutes solid evidence for the operation of succinate dehydrogenase under anaerobic conditions. The labeling in HV4 is explained by backflux from succinate to propionyl-CoA. The involvement of glycogen in the metabolism of propionate was also demonstrated; moreover, it was shown that the acetyl moiety to the synthesis of PHA was derived preferentially from glycogen. According to the proposed metabolic scheme, the decarboxylation of pyruvate is coupled to the production of hydrogen, and the missing reducing equivalents should be derived from a source other than glycogen metabolism.