Sequential Statistical Improvement of the Liquid Cultivation of Helicobacter pylori

Background: Colonization of the gastric mucosa by Helicobacter pylori is one of the most important causes of acute and chronic gastric pathologies in humans. Achieving the growth of H. pylori in liquid media is of great importance in the development of clinical studies. In this study, we developed a sequential optimization strategy based on statistical models to improve the conditions of liquid culture of H. pylori. Materials and Methods: Four statistical models were sequentially used. First, a Box‐Behnken design was used to select the best process conditions (shaking speed, inoculum concentration, and final volume of culture). Secondly, a general factorial design was used to evaluate the influence of adding gel blocks or gel beads (shape and composition). Then a D‐optimal reduce design was carried out to allow the selection of the most influential factors in increasing the cell concentration (culture media components). Finally, another Box‐Behnken design was used to optimize the concentration of the culture media components previously selected. Results: After 12 hours of liquid culture a concentration of 25 × 10⁸ cells per mL (9.4 log₁₀ cells per mL) of H. pylori was obtained, compared with a predicted 32 × 10⁸ (9.5 log₁₀ cells per mL), which means between 1 and 5 log₁₀ units higher than some previous reports. Conclusions: The sequential statistical approach increased the planktonic H. pylori cell culture. The final culture media and conditions were: Brain Heart Infusion, blood agarose (1.5% w/v), lamb’s blood (3.18% v/v), DENT (0.11% v/v), and Vitox (0.52% v/v) at 60 rpm and 37 °C with filtered CO₂ (5% v/v) bubbled directly into the culture media in a final volume of 76.22 mL.     

Restoration of a Mediterranean forest after a fire: bioremediation and rhizoremediation field‐scale trial

Forest fires pose a serious threat to countries in the Mediterranean basin, often razing large areas of land each year. After fires, soils are more likely to erode and resilience is inhibited in part by the toxic aromatic hydrocarbons produced during the combustion of cellulose and lignins. In this study, we explored the use of bioremediation and rhizoremediation techniques for soil restoration in a field‐scale trial in a protected Mediterranean ecosystem after a controlled fire. Our bioremediation strategy combined the use of Pseudomonas putida strains, indigenous culturable microbes and annual grasses. After 8 months of monitoring soil quality parameters, including the removal of monoaromatic and polycyclic aromatic hydrocarbons as well as vegetation cover, we found that the site had returned to pre‐fire status. Microbial population analysis revealed that fires induced changes in the indigenous microbiota and that rhizoremediation favours the recovery of soil microbiota in time. The results obtained in this study indicate that the rhizoremediation strategy could be presented as a viable and cost‐effective alternative for the treatment of ecosystems affected by fires.     

Large-Scale Patterns of Turnover and Basal Area Change in Andean Forests

General patterns of forest dynamics and productivity in the Andes Mountains are poorly characterized. Here we present the first large-scale study of Andean forest dynamics using a set of 63 permanent forest plots assembled over the past two decades. In the North-Central Andes tree turnover (mortality and recruitment) and tree growth declined with increasing elevation and decreasing temperature. In addition, basal area increased in Lower Montane Moist Forests but did not change in Higher Montane Humid Forests. However, at higher elevations the lack of net basal area change and excess of mortality over recruitment suggests negative environmental impacts. In North-Western Argentina, forest dynamics appear to be influenced by land use history in addition to environmental variation. Taken together, our results indicate that combinations of abiotic and biotic factors that vary across elevation gradients are important determinants of tree turnover and productivity in the Andes. More extensive and longer-term monitoring and analyses of forest dynamics in permanent plots will be necessary to understand how demographic processes and woody biomass are responding to changing environmental conditions along elevation gradients through this century.     

Age of spent Octopus vulgaris and stress mark analysis using beaks of wild individuals

Age estimation of the cephalopod Octopus vulgaris by using beaks has improved in recent years, but maximum age and longevity in the wild have not been confirmed due to the low availability of senescent wild octopuses. In this study, a beak analysis of lateral wall surfaces (LWS) from 20 spent specimens confirmed the 1-year lifecycle of the species in Central East Atlantic waters. Stress marks (checks) were clearly located in the daily increment sequence of rostrum sagittal sections (RSS). The highest daily variations in sea surface temperature (ΔT) that occurred during the last months of their lifetimes coincided with the locations of the marks on the beak, enabling confirmation of O. vulgaris beaks as life recorders for the first time. It also supports the daily deposition of RSS beak increments in the wild. Individuals were grouped into two main zones, at 20ºN and 18ºN, respectively. Both groups showed different thermal check patterns, in accordance with the oceanographic differences. Two other checks (not coinciding with high values of ΔT) were observed in RSS at averages of 15 and 28 days before death, respectively, which were interpreted as responding to senescent-related events.     

Inhibition of fatty acid biosynthesis prevents adipocyte lipotoxicity on human osteoblasts in vitro

Although increased bone marrow fat in age‐related bone loss has been associated with lower trabecular mass, the underlying mechanism responsible remains unknown. We hypothesized that marrow adipocytes exert a lipotoxic effect on osteoblast function and survival through the reversible biosynthesis of fatty acids (FA) into the bone marrow microenvironment. We have used a two‐chamber system to co‐culture normal human osteoblasts (NHOst) with differentiating pre‐adipocytes in the absence or presence of an inhibitor of FA synthase (cerulenin) and separated by an insert that allowed unidirectional trafficking of soluble factors only and prevented direct cell–cell contact. Supernatants were assayed for the presence of FA using mass spectophotometry. After 3 weeks in co‐culture, NHOst showed significantly lower levels of differentiation and function based on lower mineralization and expression of alkaline phosphatase, osterix, osteocalcin and Runx2. In addition, NHOst survival was affected by the presence of adipocytes as determined by MTS‐formazan and TUNEL assays as well as higher activation of caspases 3/7. These toxic effects were inhibited by addition of cerulenin. Furthermore, culture of NHOst with either adipocyte‐conditioned media alone in the absence of adipocytes themselves or with the addition of the most predominant FA (stearate or palmitate) produced similar toxic results. Finally, Runx2 nuclear binding was affected by addition of either adipocyte conditioned media or FA into the osteogenic media. We conclude that the presence of FA within the marrow milieu can contribute to the age‐related changes in bone mass and can be prevented by the inhibition of FA synthase.     

Effect of exogenous circulating anti-bPL antibodies on bovine placental lactogen measurements in foetal samples

Background  

The involvement of placental lactogen (PL) in the regulation of foetal growth has been investigated in different species by in vivo immunomodulation techniques. However, when circulating antibodies are present together with the hormone, the procedure for hormonal measurement becomes considerably complex. The aim of this study was the immunoneutralization of bovine placental lactogen (bPL) concentrations in bovine foetal circulation by direct infusion of rabbit anti-bPL purified immunoglobulins (IgG) via a foetal catheter (in vivo study). The ability of a RIA based on guinea pig anti-bPL antiserum, for the measurement of bPL concentrations in samples containing exogenous rabbit anti-bPL immunoglobulins, was also analyzed in in vitro and in vivo conditions.     

A population genetics study of Anopheles darlingi (Diptera: Culicidae) from Colombia based on random amplified polymorphic DNA-polymerase chain reaction and amplified fragment lenght polymorphism markers

The genetic variation and population structure of three populations of Anopheles darlingi from Colombia were studied using random amplified polymorphic markers (RAPDs) and amplified fragment length polymorphism markers (AFLPs). Six RAPD primers produced 46 polymorphic fragments, while two AFLP primer combinations produced 197 polymorphic fragments from 71 DNA samples. Both of the evaluated genetic markers showed the presence of gene flow, suggesting that Colombian An. darlingi populations are in panmixia. Average genetic diversity, estimated from observed heterozygosity, was 0.374 (RAPD) and 0.309 (AFLP). RAPD and AFLP markers showed little evidence of geographic separation between eastern and western populations; however, the F ST values showed high gene flow between the two western populations (RAPD: F ST = 0.029; Nm: 8.5; AFLP: F ST = 0.051; Nm: 4.7). According to molecular variance analysis (AMOVA), the genetic distance between populations was significant (RAPD:phiST = 0.084; AFLP:phiST = 0.229, P < 0.001). The F ST distances and AMOVAs using AFLP loci support the differentiation of the Guyana biogeographic province population from those of the Chocó-Magdalena. In this last region, Chocó and Córdoba populations showed the highest genetic flow.     

Effects of human versus mouse leukemia inhibitory factor on the in vitro development of bovine embryos

Leukemia inhibitory factor (LIF) is a cytokine that shows conflicting effects on in vitro produced (IVP) bovine embryos. Bovine LIF (bLIF) has been cloned and used in culture, but there is no commercially available bLIF. Thus, researchers use human LIF (hLIF) to supplement the culture medium for bovine embryos because of its greater sequence homology compared to murine LIF (mLIF). We compared the effects of mLIF and hLIF on the development of bovine embryos in culture with the effects described for bLIF. Oocytes were matured and fertilized in vitro and cultured in modified synthetic oviduct fluid with BSA. On Day 6 post-insemination, morulae were cultured for 48h in the presence of: (1) mLIF, 100ngml(-1); (2) hLIF, 100ngml(-1); or (3) no LIF. Reduced blastocyst rates were observed on Day 8 for hLIF at the middle and expanded stages, while mLIF had no effect. In contrast, Day 8 blastocysts showed decreased cell counts both in terms of inner cell mass (ICM) and ICM/total cell proportions in the presence of mLIF, while hLIF had no effect. No changes were seen in trophectoderm (TE) and total cell counts. The increased hatching rates and TE cell counts previously described for bLIF, together with the disparate effects exhibited by hLIF and mLIF during blastocyst formation indicate these compounds are inappropriate to replace bLIF. We recommend that heterospecific LIF should not be used to supplement the culture medium for bovine embryo or embryonic stem cells.     

Adsorption of fluorobenzene onto granular activated carbon: isotherm and bioavailability studies

The adsorption of a recalcitrant fluoroaromatic compound, fluorobenzene (FB), onto granular activated carbon (GAC) was evaluated. The respective isotherm was obtained and the Langmuir, Freundlich and Redlich-Peterson models were fitted to the experimental data, with the Redlich-Peterson model giving the best fitting. Freundlich model also provided a good fit but the Langmuir model could not adequately fit the experimental data, especially at high FB concentrations. Maximal adsorption capacity of FB onto GAC was found to be 388mg of FB per gram of GAC. The reversibility of the adsorption of FB onto GAC was investigated, both in the absence and presence of microorganisms. Abiotic desorption of FB occurred to a small extent (between 3% and 22%, for amounts of FB initially adsorbed to the GAC between 37 and 388mgg(-1)), and bioregeneration of GAC was shown to occur when the matrix was exposed to a FB degrading culture, with 58-80% of the adsorbed FB being biodegraded. A residual amount of FB showed not to be bioavailable, suggesting that part of the adsorbed FB may be irreversibly bound. The fraction of the non-bioavailable FB increased at higher amounts of adsorbed FB, from 19% to 33%. The results indicate that the GAC employed in this study has a good capacity to adsorb FB and that bioregeneration of this matrix is a feasible process.