Ceramium Roth (Ceramiales, Rhodophyta) from Venice lagoon (Adriatic Sea, Italy): Comparative studies of Mediterranean and Atlantic taxa

The cosmopolitan genus Ceramium (Ceramiaceae, Rhodophyta) is a large and systematically complex group. The taxonomy of this genus remains in a chaotic state due to the high degree of morphological variation. Culture studies, suggesting a strong influence of environment on phenotype, and the use of molecular tools have recently questioned the validity of morphological features used in species recognition. Here we compare three Ceramium taxa from Venice lagoon with samples from northwest Europe using the plastid ribulose-1,5-bisphosphate carboxylase/oxygenase gene (rbcL) and the rbcL-rbcS intergenic spacer combined with morphological observations. A strongly banded species, previously identified as member of a poorly understood and misnamed group, the Ceramium diaphanum complex sensu Feldmann-Mazoyer, is probably conspecific with British samples of Ceramium diaphanum sensu Harvey, for which no valid name has been identified up to now. We show that Ceramium polyceras (Kutzing) Zanardini is a valid name for this species. A fully corticated Ceramium species morphologically resembling C. secundatum differs at the species level from Atlantic C. secundatum; a valid name for this entity is Ceramium derbesii Solier ex Kutzing, described from Mediterranean France. A third species characterized by cortical spines, previously listed as Ceramium ciliation var. robustum (J. Agardh) Mazoyer, is shown to be Ceramium nudiusculum (Kutzing) Rabenhorst, originally described from Venice.

Rescue of glandular dysmorphogenesis in PTEN-deficient colorectal cancer epithelium by PPARγ-targeted therapy

Disruption of glandular architecture associates with poor clinical outcome in high-grade colorectal cancer (CRC). Phosphatase and tensin homolog deleted on chromosome ten (PTEN) regulates morphogenic growth of benign MDCK (Madin Darby Canine Kidney) cells through effects on the Rho-like GTPase cdc42 (cell division cycle 42). This study investigates PTEN-dependent morphogenesis in a CRC model. Stable short hairpin RNA knockdown of PTEN in Caco-2 cells influenced expression or localization of cdc42 guanine nucleotide exchange factors and inhibited cdc42 activation. Parental Caco-2 cells formed regular hollow gland-like structures (glands) with a single central lumen, in three-dimensional (3D) cultures. Conversely, PTEN-deficient Caco-2 ShPTEN cells formed irregular glands with multiple abnormal lumens as well as intra- and/or intercellular vacuoles evocative of the high-grade CRC phenotype. Effects of targeted treatment were investigated. Phosphatidinylinositol 3-kinase (PI3K) modulating treatment did not affect gland morphogenesis but did influence gland number, gland size and/or cell size within glands. As PTEN may be regulated by the nuclear receptor peroxisome proliferator-activated receptor-? (PPAR?), cultures were treated with the PPAR? ligand rosiglitazone. This treatment enhanced PTEN expression, cdc42 activation and rescued dysmorphogenesis by restoring single lumen formation in Caco-2 ShPTEN glands. Rosiglitazone effects on cdc42 activation and Caco-2 ShPTEN gland development were attenuated by cotreatment with GW9662, a PPAR? antagonist. Taken together, these studies show PTEN-cdc42 regulation of lumen formation in a 3D model of human CRC glandular morphogenesis. Treatment by the PPAR? ligand rosiglitazone, but not PI3K modulators, rescued colorectal glandular dysmorphogenesis of PTEN deficiency.

Haloalkane degradation and assimilation by Rhodococcus rhodochrous NCIMB -13064

The bacterium Rhodococcus rhodochrous NCIMB 13064, isolated from an industrial site, could use a wide range of 1-haloalkanes as sole carbon source but apparently utilized several different mechanisms simultaneously for assimilation of substrate. Catabolism of 1-chlorobutane occurred mainly by attack at the C-1 atom by a hydrolytic dehalogenase with the formation of butanol which was metabolized via butyric acid. The detection of small amounts of gamma-butyrolactone in the medium suggested that some oxygenase attack at C-4 also occurred, leading to the formation of 4-chlorobutyric acid which subsequently lactonized chemically to gamma-butyrolactone. Although 1-chlorobutane-grown cells exhibited little dehalogenase activity on 1-chloroalkanes with chain lengths above C-10, the organism utilized such compounds as growth substrates with the release of chloride. Concomitantly, gamma-butyrolactone accumulated to 1 mM in the culture medium with 1-chlorohexadecane as substrate. Traces of 4-hydroxybutyric acid were also detected. It is suggested that attack on the long-chain chloroalkane is initiated by an oxygenase at the non-halogenated end of the molecule leading to the formation of an omega-chlorofatty acid. This is degraded by beta-oxidation to 4-chlorobutyric acid which is chemically lactonized to gamma-butyrolactone which is only slowly further catabolized via 4-hydroxybutyric acid and succinic acid. However, release of chloride into the medium during growth on long-chain chloroalkanes was insufficient to account for all the halogen present in the substrate. Analysis of the fatty acid composition of 1-chlorohexadecane-grown cells indicated that chlorofatty acids comprised 75% of the total fatty acid content with C-14:0, C-16:0, C-16:1, and C-18:1 acids predominating. Thus the incorporation of 16-chlorohexadecanoic acid, the product of oxygenase attack directly into cellular lipid represents a third route of chloroalkane assimilation. This pathway accounts at least in part for the incomplete mineralization of long-chain chloroalkane substrates. This is the first report of the coexistence of a dehalogenase and the ability to incorporate long-chain haloalkanes into the lipid fraction within a single organism and raises important questions regarding the biological treatment of haloalkane containing effluents.

Long-chain haloalkanes are incorporated into fatty-acids by Rhodococcus rhodochrous NCIMB-13064

The fatty acid composition of the cellular lipids of Rhodococcus rhodochrous NCIMB 13064 grown on various long-chain haloalkanes has been investigated and the influence of halogen substituents, carbon chain length and the position of halogen substitution in the growth substrate explored. Of the total fatty acids present in cells grown on 1-chloro-, 1-bromo- and 1-iodohexadecane, 75, 90 and 81%, respectively, were substituted in the omega-position by the corresponding halogen but only 1% of the fatty acids present after growth on 1-fluorotetradecane were fluorinated in this position. The extent of the halofatty acid incorporation with different halogen substituents in the growth substrate appears to reflect the degree to which oxygenase attack is restricted to the non-halogenated end of the haloalkane. Studies of the fatty acid composition of cells after growth on a series of 1-chloroalkanes containing an even number of carbon atoms between C-10 and C-18 indicated chlorofatty acid incorporation from C-12 to C-18 substrates at levels ranging from 21% with C-12 to 75% with C-16. The chlorofatty acids formed by initial oxidation of the chloroalkane were chain-lengthened or chain-shortened by from two to eight carbon atoms, with accompanying desaturation in some instances. Substantial quantities of a methyl-branched C-19:0 chlorofatty acid were also present with several chloroalkane substrates, When the fatty acid composition of cells after growth on 1-bromoalkanes containing an odd number of carbon atoms between C-11 and C-17 was examined, the incorporation of bromofatty acids was observed with C-13, C-15 and C-17 substrates; a maximum of 76% was recorded for the C-15 bromoalkane. As with even chain-length chloroalkanes, both chain-lengthening and -shortening occurred predominantly via two-carbon units so that most bromoacids present possessed an odd number of carbon atoms, When 1-bromododecane or 2-bromododecane were substrates, overall incorporations of bromofatty acids into the lipid fraction were very similar, demonstrating that the position of halogen substitution in the haloalkane was not critical in determining the extent of incorporation of the haloacids into cellular lipids. The results of the study indicate a mechanism by which degradation products of chlorinated paraffins could enter the biological food chain.

A MOLECULAR AND MORPHOLOGICAL ANALYSIS OF THE GYMNOGONGRUS-DEVONIENSIS (RHODOPHYTA) COMPLEX IN THE NORTH-ATLANTIC

The Gymnogongrus devoniensis (Greville) Schotter complex in the North Atlantic Ocean was elucidated by comparative molecular, morphological, and culture studies. Restriction fragment length patterns and hybridization data on organellar DNA revealed two distinct taxa in samples from Europe and eastern Canada. Nucleotide sequences for the intergenic spacer between the large and small subunit genes of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and the adjoining regions of both genes, differed by 12.5-13.4% between the two taxa. One of the taxa, which included material from the type locality of G. devoniensis at Torbay, Devon, England, was taken to represent authentic G. devoniensis. Within this taxon, samples from Ireland, England, northern France, northern Spain, and southern Portugal showed great morphological variation, particularly in habit, but their Rubisco spacer sequences were identical or differed by only a single nucleotide. Constant morphological features included the development, from a single auxiliary cell, of the spherical cystocarp with a thick mucilage sheath that appears to be typical of Gymnogongrus species with internal cystocarps. Two life-history types were found. Northern isolates underwent a direct-type life history, recycling apomictic females by carpospores, whereas the Portuguese isolate followed a heteromorphic life history in which carpospores gave rise to a crustose tetrasporophyte.

Evidence of bacteriophage-mediated horizontal transfer of bacterial 16S rRNA genes in the viral metagenome of the marine sponge Hymeniacidon perlevis

Marine sponges have never been directly examined with respect to the presence of viruses or their potential involvement in horizontal gene transfer. Here we demonstrate for the first time, the presence of viruses in the marine sponge Hymeniacidon perlevis. Moreover, bacterial 16s rDNA was detected in DNA isolated from these viruses, indicating that phage-derived transduction appears to occur in H. perlevis. Phylogenetic analysis revealed that bacterial 16s rDNA isolated from sponge-derived viral and total DNA differed significantly, indicating that not all species are equally involved in transduction.

Allometric analysis of Sitka spruce branches:Mechanical versus hydraulic design principles

The geometry of tree branches can have considerable effect on their efficiency in terms of carbon export per unit carbon investment in structure. The purpose of this study was to evaluate different design criteria using data describing the form of Picea sitchensis branches. Allometric analysis of the data suggests that resources are distributed to favour shoots with the greatest opportunity for extension into new space, with priority to the extension of the leader. The distribution of allometric relations of links (branch elements) was tested against two models: the pipe model, based on hydraulic transport requirements, and a static load model based on the requirement of shoots to provide mechanical resistance to static loads. Static load resistance required the load parameter to be proportional to the link radius raised to the power of 4. This was shown to be true within a 95% statistical confidence limit. The pipe model would require total distal length to be proportional to link radius squared but the measured branches did not conform well to this model. The comparison suggests that the diameters of branch elements were more related to the requirements for mechanical load. The cost of following a hydraulic design principle (the pipe model) in terms of mechanical efficiency was estimated and suggested that the pipe model branch would not be mechanically compromised but would use structural resources inefficiently. Resource allocation among branch elements was found to be consistent with mechanical stability criteria but also indicated the possibility of allocation based on other criteria, such as potential light interception by shoots. The evidence suggests that whilst branch topology increments by reiteration of units of morphogenesis, the geometry follows a functional design pattern.

Rotavirus enterotoxin NSP4 has mucosal adjuvant properties

Rotavirus nonstructural protein 4 (NSP4) is a protein with pleiotropic properties. It functions in rotavirus morphogenesis, pathogenesis, and is the first described viral enterotoxin. Since many bacterial toxins function as potent mucosal adjuvants, we evaluated whether baculovirus-expressed recombinant simian rotavirus SA11 NSP4 possesses adjuvant activity by co-administering NSP4 with keyhole limpet hemocyanin (KLH), tetanus toxoid (TT) or ovalbumin (OVA) as model antigens in mice. Following intranasal immunization, NSP4 significantly enhanced both systemic and mucosal immune responses to model immunogens, as compared to the control group, in an antigen-specific manner. Both full-length and a cleavage product of SA11 NSP4 had adjuvant activity, localizing this activity to the C-terminus of the protein. NSP4 forms from virulent and avirulent porcine rotavirus OSU strain, and SA11 NSP4 localized within a 2/6-virus-like particle (VLP) also exhibited adjuvant effects. These studies suggest that the rotavirus enterotoxin NSP4 can function as an adjuvant to enhance immune responses for a co-administered antigen.

Re-organisation of the cytoskeleton during developmental programmed cell death in Picea abies embryos

Cell and tissue patterning in plant embryo development is well documented. Moreover, it has recently been shown that successful embryogenesis is reliant on programmed cell death (PCD). The cytoskeleton governs cell morphogenesis. However, surprisingly little is known about the role of the cytoskeleton in plant embryogenesis and associated PCD. We have used the gymnosperm, Picea abies , somatic embryogenesis model system to address this question. Formation of the apical-basal embryonic pattern in P. abies proceeds through the establishment of three major cell types: the meristematic cells of the embryonal mass on one pole and the terminally differentiated suspensor cells on the other, separated by the embryonal tube cells. The organisation of microtubules and F-actin changes successively from the embryonal mass towards the distal end of the embryo suspensor. The microtubule arrays appear normal in the embryonal mass cells, but the microtubule network is partially disorganised in the embryonal tube cells and the microtubules disrupted in the suspensor cells. In the same embryos, the microtubule-associated protein, MAP-65, is bound only to organised microtubules. In contrast, in a developmentally arrested cell line, which is incapable of normal embryonic pattern formation, MAP-65 does not bind the cortical microtubules and we suggest that this is a criterion for proembryogenic masses (PEMs) to passage into early embryogeny. In embryos, the organisation of F-actin gradually changes from a fine network in the embryonal mass cells to thick cables in the suspensor cells in which the microtubule network is completely degraded. F-actin de-polymerisation drugs abolish normal embryonic pattern formation and associated PCD in the suspensor, strongly suggesting that the actin network is vital in this PCD pathway.

Actin-binding proteins in the Arabidopsis genome database: properties of functionally distinct plant actin-depolymerizing factors/cofilins

The plant actin cytoskeleton is a highly dynamic, fibrous structure essential in many cellular processes including cell division and cytoplasmic streaming. This structure is stimulus responsive, being affected by internal stimuli, by biotic and abiotic stresses mediated in signal transduction pathways by actin-binding proteins. The completion of the Arabidopsis genome sequence has allowed a comparative identification of many actin-binding proteins. However, not all are conserved in plants, which possibly reflects the differences in the processes involved in morphogenesis between plant and other cells. Here we have searched for the Arabidopsis equivalents of 67 animal/fungal actin-binding proteins and show that 36 are not conserved in plants. One protein that is conserved across phylogeny is actin-depolymerizing factor or cofilin and we describe our work on the activity of vegetative tissue and pollen-specific isoforms of this protein in plant cells, concluding that they are functionally distinct.

Green tea supplementation increases glutathione and plasma antioxidant capacity in adults with the metabolic syndrome

Green tea, a popular polyphenol-containing beverage, has been shown to alleviate clinical features of the metabolic syndrome. However, its effects in endogenous antioxidant biomarkers are not clearly understood. Thus, we tested the hypothesis that green tea supplementation will upregulate antioxidant parameters (enzymatic and nonenzymatic) in adults with the metabolic syndrome. Thirty-five obese participants with the metabolic syndrome were randomly assigned to receive one of the following for 8 weeks: green tea (4 cups per day), control (4 cups water per day), or green tea extract (2 capsules and 4 cups water per day). Blood samples and dietary information were collected at baseline (0 week) and 8 weeks of the study. Circulating carotenoids (a-carotene, ß-carotene, lycopene) and tocopherols (a-tocopherol, ?-tocopherol) and trace elements were measured using high-performance liquid chromatography and inductively coupled plasma mass spectroscopy, respectively. Serum antioxidant enzymes (glutathione peroxidase, glutathione, catalase) and plasma antioxidant capacity were measured spectrophotometrically. Green tea beverage and green tea extract significantly increased plasma antioxidant capacity (1.5 to 2.3 µmol/L and 1.2 to 2.5 µmol/L, respectively; P <.05) and whole blood glutathione (1783 to 2395 µg/g hemoglobin and 1905 to 2751 µg/g hemoglobin, respectively; P <.05) vs controls at 8 weeks. No effects were noted in serum levels of carotenoids and tocopherols and glutathione peroxidase and catalase activities. Green tea extract significantly reduced plasma iron vs baseline (128 to 92 µg/dL, P <.02), whereas copper, zinc, and selenium were not affected. These results support the hypothesis that green tea may provide antioxidant protection in the metabolic syndrome.

Genetic and genomic analyses of musculoskeletal differences between BEH and BEL strains

Berlin high (BEH) and Berlin low (BEL) strains selected for divergent growth differ 3-fold in body weight. We aimed at examining muscle mass, which is a major contributor to body weight, by exploring anatomical characteristics of the soleus muscle, its fiber numbers and their cross sectional area (CSA), by analysing transcriptome of the gastrocnemius and by initiating quantitative trait locus (QTL) mapping. BEH muscles were 4-to-8 times larger compared to BEL strain. In sub-strain BEH+/+, mutant myostatin was replaced with a wild type allele, however, BEH+/+muscles still were 2-to-4 times larger compared to the BEL strain. BEH soleus contained 2-times more (P<0.0001) and 2-times larger in CSA (P<0.0001) fibers compared to BEL strain. In addition, soleus femoral attachment anomaly (SFAA) was observed in all BEL mice. One significant (chromosome 1) and four suggestive (chromosomes 3, 4, 6 and 9) muscle weight QTLs were mapped in 21-day old F2 intercross (n=296) between BEH and BEL strains. The frequency of SFAA incidence in the F2 and in the backcross to BEL strain (BCL) suggested the presence of more than one causative gene. Two suggestive SFAA QTLs were mapped in BCL, however, their peak markers were not associated with the phenotype in F2. RNA-Seq analysis revealed 2,148 differentially expressed (P<0.1) genes and 45,673 SNPs and >2,000 indels between BEH+/+ and BEL males. In conclusion, contrasting muscle traits, genomic and gene expression differences between BEH and BEL strains provide a promising model for the search of genes involved in muscle growth and musculoskeletal morphogenesis.

PTEN Phosphatase-Independent Maintenance of Glandular Morphology in a Predictive Colorectal Cancer Model System

Organotypic models may provide mechanistic insight into colorectal cancer (CRC) morphology. Three-dimensional (3D) colorectal gland formation is regulated by phosphatase and tensin homologue deleted on chromosome 10 (PTEN) coupling of cell division cycle 42 (cdc42) to atypical protein kinase C (aPKC). This study investigated PTEN phosphatase-dependent and phosphatase-independent morphogenic functions in 3D models and assessed translational relevance in human studies. Isogenic PTEN-expressing or PTEN-deficient 3D colorectal cultures were used. In translational studies, apical aPKC activity readout was assessed against apical membrane (AM) orientation and gland morphology in 3D models and human CRC. We found that catalytically active or inactive PTEN constructs containing an intact C2 domain enhanced cdc42 activity, whereas mutants of the C2 domain calcium binding region 3 membrane-binding loop (M-CBR3) were ineffective. The isolated PTEN C2 domain (C2) accumulated in membrane fractions, but C2 M-CBR3 remained in cytosol. Transfection of C2 but not C2 M-CBR3 rescued defective AM orientation and 3D morphogenesis of PTEN-deficient Caco-2 cultures. The signal intensity of apical phospho-aPKC correlated with that of Na/H exchanger regulatory factor-1 (NHERF-1) in the 3D model. Apical NHERF-1 intensity thus provided readout of apical aPKC activity and associated with glandular morphology in the model system and human colon. Low apical NHERF-1 intensity in CRC associated with disruption of glandular architecture, high cancer grade, and metastatic dissemination. We conclude that the membrane-binding function of the catalytically inert PTEN C2 domain influences cdc42/aPKC-dependent AM dynamics and gland formation in a highly relevant 3D CRC morphogenesis model system.

Microbial weeds in hypersaline habitats: the enigma of the weed-like Haloferax mediterranei

Heterotrophic prokaryotic communities that inhabit saltern crystallizer ponds are typically dominated by two species, the archaeon Haloquadratum walsbyi and the bacterium Salinibacter ruber, regardless of location. These organisms behave as ‘microbial weeds’ as defined by Cray et al. (Microb Biotechnol 6: 453–492, 2013) that possess the biological traits required to dominate the microbiology of these open habitats. Here, we discuss the enigma of the less abundant Haloferax mediterranei, an archaeon that grows faster than any other, comparable extreme halophile. It has a wide window for salt tolerance, can grow on simple as well as on complex substrates and degrade polymeric substances, has different modes of anaerobic growth, can accumulate storage polymers, produces gas vesicles, and excretes halocins capable of killing other Archaea. Therefore, Hfx. mediterranei is apparently more qualified as a ‘microbial weed’ than Haloquadratum and Salinibacter. However, the former differs because it produces carotenoid pigments only in the lower salinity range and lacks energy-generating retinal-based, light-driven ion pumps such as bacteriorhodopsin and halorhodopsin. We discuss these observations in relation to microbial weed biology in, and the open-habitat ecology of, hypersaline systems.

Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells from Oxidative Stress through Interaction with Histone Deacetylase 3

It is well-known that atherosclerosis occurs geographically at branch points where disturbed flow predisposes to the development of plaque via triggering of oxidative stress and inflammatory reactions. In this study, we found that disturbed flow activated anti-oxidative reactions via up-regulating heme oxygenase 1 (HO-1) in an X-box binding protein 1 (XBP1) and histone deacetylase 3 (HDAC3)-dependent manner. Disturbed flow concomitantly up-regulated the unspliced XBP1 (XBP1u) and HDAC3 in a vascular endothelial growth factor receptor (VEGFR) and PI3K/Akt dependent manner. The presence of XBP1 was essential for the up-regulation of HDAC3 protein. Over-expression of XBP1u and/or HDAC3 activated Akt1 phosphorylation, Nrf2 protein stabilization and nuclear translocation, and HO-1 expression. Knockdown of XBP1u decreased the basal level and disturbed flow-induced Akt1 phosphorylation, Nrf2 stabilization and HO-1 expression. Knockdown of HDAC3 ablated XBP1u-mediated effects. The mammalian target of rapamycin complex 2 (mTORC2) inhibitor, AZD2014, ablated XBP1u or HDAC3 or disturbed flow-mediated Akt1 phosphorylation, Nrf2 nuclear translocation and HO-1 expression. Neither actinomycin D nor cycloheximide affected disturbed flow-induced up-regulation of Nrf2 Protein. Knockdown of Nrf2 abolished XBP1u or HDAC3 or disturbed flow-induced HO-1 up-regulation. Co-immunoprecipitation assays demonstrated that XBP1u physically bound to HDAC3 and Akt1. The region of amino acids 201 to 323 of the HDAC3 protein was responsible for the binding to XBP1u. Double immunofluorescence staining revealed that the interactions between Akt1 and mTORC2, Akt1 and HDAC3, Akt1 and XBP1u, HDAC3 and XBP1u occurred in the cytosol. Thus, we demonstrate that XBP1u and HDAC3 exert a protective effect on disturbed flow-induced oxidative stress via up-regulation of mTORC2-dependent Akt1 phosphorylation and Nrf2-mediated HO-1 expression.