Genome skimming elucidates the evolutionary history of Octopoda.

Phylogenies for Octopoda have, until now, been based on morphological characters or a few genes. Here we provide the complete mitogenomes and the nuclear 18S and 28S ribosomal genes of twenty Octopoda specimens, comprising 18 species of Cirrata and Incirrata, representing 13 genera and all five putative families of Cirrata (Cirroctopodidae, Cirroteuthidae, Grimpoteuthidae, Opisthoteuthidae and Stauroteuthidae) and six families of Incirrata (Amphitretidae, Argonautidae, Bathypolypodidae, Eledonidae, Enteroctopodidae, and Megaleledonidae) which were assembled using genome skimming. Phylogenetic trees were built using Maximum Likelihood and Bayesian Inference with several alignment matrices. All mitochondrial genomes had the 'typical' genome composition and gene order previously reported for octopodiforms, except Bathypolypus ergasticus, which appears to lack ND5, two tRNA genes that flank ND5 and two other tRNA genes. Argonautoidea was revealed as sister to Octopodidae by the mitochondrial protein-coding gene dataset, however, it was recovered as sister to all other incirrate octopods with strong support in an analysis using nuclear rRNA genes. Within Cirrata, our study supports two existing classifications suggesting neither is likely in conflict with the true evolutionary history of the suborder. Genome skimming is useful in the analysis of phylogenetic relationships within Octopoda; inclusion of both mitochondrial and nuclear data may be key.

Trace element concentrations, including Cd and Hg, in long-finned pilot whales (Globicephala melas edwardii) mass stranded on the New Zealand coast.

The Southern Hemisphere long-finned pilot whale (Globicephala melas edwardii) is a top predator in the New Zealand pelagic food web, feeding predominantly on arrow squids. This study quantified trace element concentrations (Al, As, Cd, Co, Cr, Cu, Fe, Hg, Ni, Mn, Pb, Se, V, Zn) in four tissues (blubber, kidney, liver, muscle) from 21 individuals from stranding sites in New Zealand. Maximum Cd and Hg concentrations were measured in liver and kidney, respectively. Selenium had a positive correlation with Cd and Hg, suggesting the involvement of Se in Cd and Hg detoxification. Arrow squids from the whales' stomach contents were DNA barcoded and identified as Nototodarus sloanii. Trace element concentrations were measured in squid samples from the whale stomach contents. The significant correlation for Hg between the squid tissue and the whale tissue suggests that arrow squids play a major role in trace element uptake by G. m. edwardii.

Influence of sexual dimorphism on stable isotopes and trace element concentrations in the greater hooked squid Moroteuthopsis ingens from New Zealand waters.

The Chatham Rise, one of the highest offshore-primary production regions in New Zealand waters, hosts a great abundance and diversity of deep-sea cephalopods including the greater hooked squid, Moroteuthopsis ingens. Stable isotope ratios of carbon (δ13C) and nitrogen (δ15N) and trace element concentrations (Ag, As, Cd, Co, Cr, Cu, Fe, Hg, Ni, Pb, Se, V, and Zn) were assessed in female and male specimens of different size classes (89-563 mm mantle length). Values of δ13C and δ15N were overall higher in females and δ13C was further influenced by size and sex. Both muscular mantle (the largest fraction of the total body mass) and digestive gland (the known main storage organ for Ag, Cd, Cu and Zn in many cephalopods) tissues were analysed. Higher levels of Cd were observed in males than in females. A positive effect was found between size and Hg concentrations, which could be related to the ontogenetic descent of larger specimens into deeper waters, where they are exposed to higher Hg concentrations, and/or dietary shifts toward Hg-enriched prey with increasing size. This study provides trace element data for this abundant and ecologically important species, and further reveals higher trace element concentrations (especially Hg) in M. ingens from the Chatham Rise, compared to specimens from the sub-Antarctic zone.

Regional assessment of trace element concentrations in arrow squids (Nototodarus gouldi, N. sloanii) from New Zealand waters: Bioaccessibility and impact on food web and human consumers.

The New Zealand arrow squids, Nototodarus gouldi and N. sloanii, play an important role in the marine food web, and are both economically important fishery species. This study compares trace element concentrations (As, Cd, Co, Cr, Cu, Fe, Hg, Ni, Mn, Pb, U, and Zn) in these animals from different fishing locations within New Zealand's Exclusive Economic Zone (Auckland Islands, Chatham Rise, Dunedin, Golden Bay, Taranaki, and West Coast of the South Island). Muscular mantle (the tissue usually consumed by humans) and digestive gland tissue (the primary organ for trace element accumulation) concentrations were compared among regions, revealing size, species, and sampling location effects. Overall, N. gouldi had higher concentrations of As and Zn. The Taranaki region had the highest concentrations for Cd, Co, Cu, and Zn in both tissues analysed. To assess the bioaccessibility of these trace elements to humans, an in-vitro digestion simulation experiment was conducted, revealing bioavailable concentrations overall below the maximum levels presently considered tolerable for human consumption. However, the in-vitro digestion had no observable effect upon Cd concentrations in arrow squid mantle tissue (implying that the measured concentrations are bioaccessible to humans), and revealed that Cd had the highest potential impact on marine food webs and human health. Digestive gland concentrations in particular indicate that arrow squids can be considered important vectors for trace element transfer to top predators (mean concentration for Cd: 123 ± 148 µg g-1 dw), with region-specific differences.

Preclinical immunogenicity study of trivalent meningococcal AWX-OMV vaccines for the African meningitis belt.

In the recent decade, epidemic meningitis in the African meningitis belt has mostly been caused by Neisseria meningitidis of serogroups A, W and X (MenA, MenW and MenX, respectively). There is at present no licensed vaccine available to prevent MenX meningococcal disease. To explore a trivalent MenAWX vaccine concept, we have studied the immunogenicity in mice of MenX outer membrane vesicles (X-OMV) or MenX polysaccharide (X-PS) when combined with a bivalent A-OMV and W-OMV (AW-OMV) vaccine previously shown to be highly immunogenic in mice. The vaccine antigens were produced from three representative wild type strains of MenA (ST-7), MenW (ST-11) and MenX (ST-751) isolated from patients in the African meningitis belt. Groups of mice were immunized with two doses of X-OMV or X-PS combined with the AW-OMV vaccine or as individual components. All vaccine preparations were adsorbed to Al(OH)3. Sera from immunized mice were tested by ELISA and immunoblotting. Functional antibody responses were measured as serum bactericidal activity (SBA) and opsonophagocytic activity (OPA). Immunization of mice with X-OMV, alone or in combination with AW-OMV induced high levels of anti-X OMV IgG. Moreover, X-OMV alone or in combination with the AW-OMV vaccine induced high SBA and OPA titers against the MenX target strain. X-PS alone was not immunogenic in mice; however, addition of the AW-OMV vaccine to X-PS increased the immunogenicity of X-PS. Both AWX vaccine formulations induced high levels of IgG against A- and W-OMV and high SBA titers against the MenA and MenW vaccine strains. These results suggest that a trivalent AWX vaccine, either as a combination of OMV or OMV with X-PS, could potentially prevent the majority of meningococcal disease in the meningitis belt.

Preclinical immunogenicity and functional activity studies of an A+W meningococcal outer membrane vesicle (OMV) vaccine and comparisons with existing meningococcal conjugate- and polysaccharide vaccines.

Meningococci of serogroups A and W (MenA and MenW) are the main causes of epidemic bacterial meningitis outbreaks in sub-Saharan Africa. In this study we prepared a detergent extracted outer membrane vesicle (dOMV) vaccine from representative African MenA and MenW strains, and compared the immunogenicity of this vaccine with existing meningococcal conjugate and polysaccharide (PS) vaccines in mice. NMRI mice were immunized with preclinical batches of the A+W dOMV vaccine, or with commercially available vaccines; a MenA conjugate vaccine (MenAfriVac(®), Serum Institute of India), ACYW conjugate vaccine (Menveo(®), Novartis) or ACYW PS vaccine (Mencevax(®), GlaxoSmithKline). The mice received 2 doses of 1/10 or 1/50 of a human dose with a three week interval. Immune responses were tested in ELISA, serum bactericidal activity (SBA) and opsonophagocytic activity (OPA) assays. High levels of IgG antibodies against both A and W dOMV were detected in mice receiving the A+W dOMV vaccine. High SBA titers against both MenA and MenW vaccine strains were detected after only one dose of the A+W dOMV vaccine, and the titers were further increased after the second dose. The SBA and OPA titers in mice immunized with dOMV vaccine were significantly higher than in mice immunized with the ACYW-conjugate vaccine or the PS vaccine. Furthermore, the A+W dOMV vaccine was shown to induce SBA and OPA titers against MenA of the same magnitude as the titers induced by the A-conjugate vaccine. In conclusion, the A+W dOMV vaccine induced high levels of functional antibodies to both MenA and MenW strains, levels that were shown to be higher or equal to the levels induced by licensed meningococcal vaccines. Thus, an A+W dOMV vaccine could potentially serve as an alternative or a supplement to existing conjugate and PS vaccines in the African meningitis belt.

Meningococcal omp85 in detergent-extracted outer membrane vesicle vaccines induces high levels of non-functional antibodies in mice.

The vaccine potential of meningococcal Omp85 was studied by comparing the immune responses of genetically modified deoxycholate-extracted outer membrane vesicles, expressing five-fold higher levels of Omp85, with wild-type vesicles. Groups (n = 6-12) of inbred and outbred mouse strains (Balb/c, C57BL/6, OFI and NMRI) were immunized with the two vaccines, and the induced antibody levels and bactericidal and opsonic activities measured. Except for Balb/c mice, which were low responders, the genetically modified vaccine raised high Omp85 antibody levels in all mouse strains. In comparison, the wild-type vaccine gave lower antibody levels, but NMRI mice responded to this vaccine with the same high levels as the modified vaccine in the other strains. Although the vaccines induced strain-dependent Omp85 antibody responses, the mouse strains showed high and similar serum bactericidal titres. Titres were negligible with heterologous or PorA-negative meningococcal target strains, demonstrating the presence of the dominant bactericidal PorA antibodies. The two vaccines induced the same opsonic titres. Thus, the genetically modified vaccine with high Omp85 antibody levels and the wild-type vaccine induced the same levels of functional activities related to protection against meningococcal disease, suggesting that meningococcal Omp85 is a less attractive vaccine antigen.

Seroepidemiological study after a long-distance industrial outbreak of legionnaires' disease.

Following a long-distance outbreak of Legionnaires' disease from an industrial air scrubber in Norway in 2005, a seroepidemiological study measuring levels of immunoglobulin G (IgG) and IgM antibodies to Legionella pneumophila was performed with a polyvalent enzyme-linked immunosorbent assay. One year after the outbreak, IgG levels in employees (n = 213) at the industrial plant harboring the scrubber and in blood donors (n = 398) from the outbreak county were low but significantly higher (P < or = 0.002) than those in blood donors (n = 406) from a nonexposed county. No differences in IgM levels among the three groups were found after adjustment for gender and age. Home addresses of the seroresponders in the exposed county clustered to the city of the outbreak, in contrast to the scattering of addresses of the seroresponding donors in the nonexposed county. Factory employees who operated at an open biological treatment plant had significantly higher IgG and IgM levels (P < or = 0.034) than those working >200 m away. Most of the healthy seroresponders among the factory employees worked near this exposure source. Immunoblotting showed that IgG and IgM antibodies in 82.1% of all seroresponders were directed to the lipopolysaccharide of the L. pneumophila serogroup 1 outbreak strain. In conclusion, 1 year after the long-distance industrial outbreak a small increase in IgG levels of the exposed population was observed. The open biological treatment plant within the industrial premises, however, constituted a short-distance exposure source of L. pneumophila for factory employees working nearby.

Functional and specific antibody responses in adult volunteers in new zealand who were given one of two different meningococcal serogroup B outer membrane vesicle vaccines.

This study presents detailed analyses of total and specific serum antibody levels among 26 and 24 adult volunteers before vaccination and after the third dose of the meningococcal serogroup B outer membrane vesicle (OMV) vaccines MeNZB and MenBvac, respectively, in a clinical trial in New Zealand (V. Thornton, D. Lennon, K. Rasanathan, J. O'Hallahan, P. Oster, J. Stewart, S. Tilman, I. Aaberge, B. Feiring, H. Nokleby, E. Rosenqvist, K. White, S. Reid, K. Mulholland, M. J. Wakefield, and D. Martin, Vaccine 24:1395-1400, 2006). With the homologous vaccine strains as targets, both vaccines induced significant increases in serum bactericidal and opsonophagocytic activities and in the levels of immunoglobulin G (IgG) to OMV antigens in an enzyme-linked immunosorbent assay (ELISA) and to live meningococci by flow cytometry. They also induced high levels of activity against the heterologous strains, particularly in terms of opsonophagocytic activity and IgG binding to live bacteria. The antibody levels with the homologous and heterologous strains in the four assays showed high and significant positive correlations. Specific IgG binding to 10 major OMV antigens in each vaccine was measured by scanning of immunoblots; ELISAs for two antigens, lipopolysaccharide and Neisseria surface protein A (NspA), were also performed. Both vaccines elicited significant increases in IgG binding to all homologous and heterologous OMV antigens except NspA. The total IgG band intensity on the blots correlated significantly with the IgG levels determined by the OMV ELISA and flow cytometry. In conclusion, the results of the various immunological assays showed that both OMV vaccines gave rise to high levels of specific and cross-reacting antibodies.

IgG antibody levels to meningococcal porins in patient sera: comparison of immunoblotting and ELISA measurements.

IgG antibody levels to the meningococcal PorA and PorB proteins in 56 acute and convalescent phase sera from 25 patients with meningococcal disease were compared by immunoblotting and ELISA. Heat-treated outer membrane vesicles from strain 44/76 (B:15:P1.7, 16) served as antigens for immunoblotting, whereas purified P1.7,16 PorA and P15 PorB from the same strain were used as antigens in the ELISA. In the blotting method, IgG binding to the porins was determined by digital scanning of the immunoreactive bands and calculated relative to the PorA binding of a reference serum on each blot. The coefficient of variation for the reference serum was 21.6% (a total of 144 strips) with smaller variations for each day's experiments. Blotting of all 56 sera at the standard 1/200 dilution measured anti-PorA and anti-PorB levels that correlated with those obtained by ELISA (Spearman rank-order correlation coefficient r(s)=0.48; P<0.001). At this dilution, the anti-PorA (r(s)=0.52; P<0. 004) and anti-PorB (r(s)=0.60; P<0.001) levels of the convalescent phase sera (n=29) corresponded with the ELISA measurements, whereas no correlation was found with the results for the acute phase sera, which mostly had low ELISA antibody levels (<2 microg/ml IgG). A corresponding blot analysis of convalescent sera from the seven patients, who had received the 44/76 outer membrane vesicle vaccine, demonstrated a high correlation coefficient for the anti-PorA levels (r(s)=0.95; P<0.001) vs. the ELISA results. No such correlation was observed for the PorB response in these sera, being nine-fold higher than the PorA response, because of a prozone effect on the blots at the standard dilution. However, blotting at a higher serum dilution (1/2000) resulted in anti-PorB levels that also correlated strongly (r(s)=0.93 P<0.001) with the ELISA measurements.

In vivo growth kinetics of P388 and L1210 leukemias.

The P388 lymphocytic leukemia and the L1210 lymphoid leukemia are used as test systems for putative cytotoxic drugs. These leukemias are also used to investigate the perturbation of cell cycle progression of various chemical compounds in more detail. There is little information on the normal growth kinetics in vivo of these leukemias. In the present report we therefore present the results from growth kinetic studies of P388 and L1210 leukemic cells growing in ascites form in mice. We used 3H-TdR autoradiography, DNA flow cytometry and the stathmokinetic method. During exponential growth both leukemias showed a growth fraction of unity. Whereas no significant cell loss was observed during the early growth phase of P388 cells, cell loss was indicated by a discrepancy between potential and actual doubling times during exponential growth of L1210 cells. During the phase of growth retardation, the proportion of G1 and G2 cells increased at the expence of a reduced S phase fraction in the P388 leukemia, whereas only small changes in cell cycle distributions were seen with time after inoculation of L1210 cells. An increasing discrepancy in the reduction of the S phase fraction and the 3H-TdRLI was seen in the P388 cells with time after inoculation. Thus, a majority of P388 cells with S phase DNA content were unlabelled during the late phase of growth restriction, indicating resting cells in S phase. A good correlation was found between the 3H-TdR LI and S phase fraction throughout the life history of L1210 cells, revealing considerable differences in in vivo growth kinetics between the two leukemias. Such differences should be considered when evaluating test results.

Fixation of Dwyer screws for the treatment of scoliosis. A postmortem study.

The strength of fixation of Dwyer screws in seven or eight vertebral bodies in each of six fresh osteoporotic cadavera was measured under the following conditions: with fixation with only the screw; with the staple-screw combination; with each of these modes of fixation combined with polymethylmethacrylate placed in the hole prior to insertion of the screw; and with the screw reinserted after failure and filling the hole with methacrylate. The strength of fixation of the screw-staple combination was increased almost twofold by the methacrylate, and after failure followed by reinsertion of the screw embedding it in methacrylate, strong fixation was restored.

A single unit digital flexor tendon prosthesis.

Historically, the surgical repair of serious injuries to the flexor tendons of the hand have met with less than satisfactory clinical results. In an attempt to solve this problem, studies have been made on the design and testing of a Dacron polyester tendon prosthesis for complete replacement of the digital flexor tendon. The prosthesis attaches proximally at the anatomical musculotendon junction via collagen ingrowth into a fabric structure and in similar manner distally via bone ingrowth at the anatomical insertion site. These fabrics are continuous with the tendon body of the prosthesis consisting of an inextensible braided cord which is silicone rubber coated to prevent tissue adherence in the glide zone of the tendon bed. An added benefit of the prosthesis is a simple means of length adjustment which can easily be carried out on the operating table. Studies have been performed in both dogs and chickens which indicate that sufficient tissue ingrowth occurs at both the distal and proximal anastomoses for retention of full load bearing capability in these animals and that postoperative adhesions are negligible, if present at all. Full prostheses have been implanted in dogs for periods of over 1 year and chickens for 2 months. The chicken is the preferred animal model as a tendency for extensive scar tissue generation was shown in the dog. Both in vivo mechanical testing and histological characterization have been made on sacrificed animals which have received the full prostheses.