Complete genome sequencing of sixteen Francisella noatunensis subsp. orientalis isolates: A genomic approach for molecular characterization and spread dynamics of this clonal population

Francisella noatunensis subsp. orientalis (FNO) is an important emerging pathogen associated with disease outbreaks in farm-raised Nile tilapia. FNO genetic diversity using PCR-based typing, no intra-species discrimination was achieved among isolates/strains from different countries, thus demonstrating a clonal behaviour pattern. In this study, we aimed to evaluate the population structure of FNO isolates by comparing whole-genome sequencing data. The analysis of recombination showed that Brazilian isolates group formed a clonal population; whereas other lineages are also supported by this analysis for isolates from foreign countries. The whole-genome multilocus sequence typing (wgMLST) analysis showed varying numbers of dissimilar alleles, suggesting that the Brazilian clonal population are in expansion. Each Brazilian isolate could be identified as a single node by high-resolution gene-by-gene approach, presenting slight genetic differences associated to mutational events. The common ancestry node suggests a single entry into the country before 2012, and the rapid dissemination of this infectious agent may be linked to market sales of infected fingerlings.     

F508del disturbs the dynamics of the nucleotide binding domains of CFTR before and after ATP hydrolysis

The cystic fibrosis transmembrane conductance regulator (CFTR) channel is an ion channel responsible for chloride transport in epithelia and it belongs to the class of ABC transporters. The deletion of phenylalanine 508 (F508del) in CFTR is the most common mutation responsible for cystic fibrosis. Little is known about the effect of the mutation in the isolated nucleotide binding domains (NBDs), on dimer dynamics, ATP hydrolysis and even on nucleotide binding. Using molecular dynamics simulations of the human CFTR NBD dimer, we showed that F508del increases, in the prehydrolysis state, the inter-motif distance in both ATP binding sites (ABP) when ATP is bound. Additionally, a decrease in the number of catalytically competent conformations was observed in the presence of F508del. We used the subtraction technique to study the first 300 ps after ATP hydrolysis in the catalytic competent site and found that the F508del dimer evidences lower conformational changes than the wild type. Using longer simulation times, the magnitude of the conformational changes in both forms increases. Nonetheless, the F508del dimer shows lower C-α RMS values in comparison to the wild-type, on the F508del loop, on the residues surrounding the catalytic site and the portion of NBD2 adjacent to ABP1. These results provide evidence that F508del interferes with the NBD dynamics before and after ATP hydrolysis. These findings shed a new light on the effect of F508del on NBD dynamics and reveal a novel mechanism for the influence of F508del on CFTR.     

Identification and characterization of a new member of the prolactin family, placental lactogen-I variant

This report describes the identification and characterization of a new member of the placental prolactin (PRL) family, termed placental lactogen-I variant (PL-Iv). PL-Iv was isolated from medium conditioned by late gestation placental explants. Rat PL-Iv was found to be closely related to rat PL-I. Amino-terminal sequence analysis indicated that PL-Iv shared approximately 88% sequence identity with the amino terminus of PL-I. PL-Iv proteins cross-reacted with antiserum to recombinant mouse PL-I and PL-Iv mRNA hybridized with a PL-I cDNA. Multiple PL-I and PL-Iv species were present in placental cytosol. Despite the structural similarities between PL-I and PL-Iv, distinct differences were also evident. Antibodies generated to the amino-terminal 19 amino acids of PL-Iv specifically recognized PL-Iv, while failing to recognize PL-I. Secreted PL-Iv had an affinity for concanavalin A, whereas secreted PL-I lacked affinity for the lectin. PL-I was predominantly secreted as a 36-40-kDa species and PL-Iv was predominantly secreted as a 33-kDa species. Furthermore, PL-I and PL-Iv were synthesized at different times during gestation and by different cell types. PL-I was synthesized by trophoblast giant cells during the first half of gestation, while PL-Iv was predominantly synthesized by spongiotrophoblast cells during the later stages of gestation. PL-Iv was shown to stimulate the proliferation of rat Nb2 lymphoma cells, an in vitro measure of lactogenic activity. In summary, PL-Iv shares structural similarities with PL-I; however, it shows other structural differences in addition to unique cell- and temporal-specific patterns of expression in the rat chorioallantoic placenta.     

Fatty acid composition and biological activities of Isochrysis galbana T-ISO,Tetraselmis sp. and Scenedesmus sp.: possible application in the pharmaceutical and functional food industries

Organic and water extracts of Isochrysis galbana T-ISO (=Tisochrysis lutea), Tetraselmis sp. and Scenedesmus sp. were evaluated for their antioxidant activity, acetylcholinesterase (AChE) inhibition, cytotoxicity against tumour cell lines, and fatty acids and total phenolic content (TPC). I. galbana T-ISO had the highest TPC (3.18 mg GAE g^?1) and radical scavenging activity, with an IC₅₀ value of 1.9 mg mL^?1 on the acetone extract. The extracts exhibited a higher ability to chelate Fe²⁺ than Cu²⁺, and the maximum Fe²⁺ chelating capacity was observed in the hexane extract of Scenedesmus sp. (IC₅₀=0.73 mg mL^?1) and Scenedesmus sp. (IC₅₀?=?0.73 mg mL^?1). The highest ability to inhibit AChE was observed in the water and ether extracts of Scenedesmus sp., with IC₅₀ values of 0.11 and 0.15 mg mL^?1, respectively, and in the water extract of I. galbana (IC₅₀?=?0.16 mg mL^?1). The acetone extract of I. galbana T-ISO significantly reduced the viability of human hepatic carcinoma HepG2 cells (IC₅₀?=?81.3 μg mL^?1) as compared to the non-tumour murine stromal S17 cell line, and displayed a selectivity index of 3.1 at the highest concentration tested (125 μg mL^?1). All species presented a highly unsaturated fatty acids profile. Results suggest that these microalgae, particularly I. galbana T-ISO, could be a source of biomolecules for the pharmaceutical industry and the production of functional food ingredients and can be considered as an advantageous alternative to several currently produced microalgae.     

Plasma cell granuloma of the tongue. Report of a case

A case of plasma cell granuloma of the tongue in an otherwise symptomless 48-year-old caucasian female is reported. The polyclonal nature of the plasmocytes was revealed by immunostaining of kappa and lambda light chains. Electron microscopic observations showed typical mature plasmocytes. A parasitic etiology of this type of lesion is suggested.     

Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PP_i) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF‐hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PP_i) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP‐OE) resulted in a significant decrease in cytosolic PP_i, and short and long‐chain polyP levels. Additionally, the TcVSP‐OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PP_i and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.     

Growth and temperature‐related phenotypic plasticity in the cyanobacterium Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is an invasive and potentially toxic cyanobacterium, which has recently spread worldwide, mainly because of its tolerance to a wide range of climatic conditions. C. raciborskii is able to change several traits in response to environmental changes and its morphology is also affected by these changes (especially in nutrients). We also expected temperature to affect the morphology of this cyanobacterium. We examined the growth and morphology of C. raciborskii at different temperatures and compared laboratory results to the morphology of this cyanobacterium in situ. As expected, growth rates increased with temperature. In addition, a high carrying capacity at 32°C suggests that this cyanobacterium is able to form more dense blooms at high temperatures. Fragile trichomes and low growth rates were observed at 12°C. An increase in the growth rate related to temperature resulted in a decrease in trichome length, with shorter trichomes at 32°C. The same pattern was observed in wild populations of C. raciborskii in a tropical reservoir, where shorter trichomes were observed in warmer months, when biomass was highest. This species' high ability to adapt to different environmental conditions throughout the year (i.e., nutrients, temperature) may have provided it with an additional advantage to increase its perennial blooms, mainly in tropical regions.     

NMR determination of Electrophorus electricus acetylcholinesterase inhibition and reactivation by neutral oximes

Neurotoxic organophosphorus compounds (OPs), which are used as pesticides and chemical warfare agents lead to more than 700,000 intoxications worldwide every year. The main target of OPs is the inhibition of acetylcholinesterase (AChE), an enzyme necessary for the control of the neurotransmitter acetylcholine (ACh). The control of ACh function is performed by its hydrolysis with AChE, a process that can be completely interrupted by inhibition of the enzyme by phosphylation with OPs. Compounds used for reactivation of the phosphylated AChE are cationic oximes, which usually possess low membrane and hematoencephalic barrier permeation. Neutral oximes possess a better capacity for hematoencephalic barrier permeation.NMR spectroscopy is a very confident method for monitoring the inhibition and reactivation of enzymes, different from the Ellman test, which is the common method for evaluation of inhibition and reactivation of AChE. In this work ¹H NMR was used to test the effect of neutral oximes on inhibition of AChE and reactivation of AChE inhibited with ethyl-paraoxon. The results confirmed that NMR is a very efficient method for monitoring the action of AChE, showing that neutral oximes, which display a significant AChE inhibition activity, are potential drugs for Alzheimer disease. The NMR method showed that a neutral oxime, previously indicated by the Ellman test as better in vitro reactivator of AChE inhibited with paraoxon than pralidoxime (2-PAM), was much less efficient than 2-PAM, confirming that NMR is a better method than the Ellman test.     

Angiogenesis and Inflammation Signaling Are Targets of Beer Polyphenols on Vascular Cells

Emerging evidence indicates that chronic inflammation and oxidative stress cluster together with angiogenic imbalance in a wide range of pathologies. In general, natural polyphenols present health‐protective properties, which are likely attributed to their effect on oxidative stress and inflammation. Hops used in beer production are a source of polyphenols such as xanthohumol (XN), and its metabolites isoxanthohumol (IXN) and phytoestrogen 8‐prenylnaringenin (8PN). Our study aimed to evaluate XN, IXN, and 8PN effects on angiogenesis and inflammation processes. Opposite in vitro effects were observed between 8PN, stimulating endothelial and smooth muscle cell (SMC) growth, motility, invasion and capillary‐like structures formation, and XN and IXN, which inhibited them. Mouse matrigel plug and rat skin wound‐healing assays confirmed that XN and IXN treatments reduced vessel number as well as serum macrophage enzymatic activity, whereas 8PN increased blood vessels formation in both assays and enzyme activity in the wound‐healing assay. A similar profile was found for serum inflammatory interleukin‐1β quantification, in the wound‐healing assay. Our data indicate that whereas 8PN stimulates angiogenesis, XN and IXN manifested anti‐angiogenic and anti‐inflammatory effects in identical conditions. These findings suggest that the effects observed for individual compounds on vascular wall cells must be carefully taken into account, as these polyphenols are metabolized after in vivo administration. The modulation of SMC proliferation and migration is also of special relevance, given the role of these cells in many pathological conditions. Furthermore, these results may provide clues for developing useful therapeutic agents against inflammation‐ and angiogenesis‐associated pathologies. J. Cell. Biochem. 111: 1270–1279, 2010. © 2010 Wiley‐Liss, Inc.     

Separation of yeasts by addition of flocculent cells of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Separation of yeast cells using a co-flocculation process was investigated. Co-flocculation is a fast process (within few minutes), occurs in a broad pH range (3.0–8.0) and requires a small amount of calcium (0.1 mM). Agitation affects yeast aggregation; however, an agitation between 60 rev/min and 160 rev/min has only a little influence on the co-flocculation process. The ratio flocculent/non-flocculent cells that induced the settling of 50 and 90% of the cells of S. cerevisiae was 1:7 and 1:1, respectively. Separation of non-flocculent cells can be carried out at any time of the growth cycle. No difference in the efficiency of co-flocculation carried out in buffer (pH 4.0 with 10 mM calcium) or in 48 h-fermented broth was observed. Flocculent cells of Saccharomyces cerevisiae had the ability to sediment non-flocculent cells of S. cerevisiae and Kluyveromyces marxianus, which shows the suitability of the co-flocculation process for separation of different kinds of non-flocculent cells.