A multiscale, spatially distributed model of asthmatic airway hyper-responsiveness

We present a multiscale, spatially distributed model of lung and airway behaviour with the goal of furthering the understanding of airway hyper-responsiveness and asthma. The model provides an initial computational framework for linking events at the cellular and molecular levels, such as Ca²⁺ and crossbridge dynamics, to events at the level of the entire organ. At the organ level, parenchymal tissue is modelled using a continuum approach as a compressible, hyperelastic material in three dimensions, with expansion and recoil of lung tissue due to tidal breathing. The governing equations of finite elasticity deformation are solved using a finite element method. The airway tree is embedded in this tissue, where each airway is modelled with its own airway wall, smooth muscle and surrounding parenchyma. The tissue model is then linked to models of the crossbridge mechanics and their control by Ca²⁺ dynamics, thus providing a link to molecular and cellular mechanisms in airway smooth muscle cells. By incorporating and coupling the models at these scales, we obtain a detailed, computational multiscale model incorporating important physiological phenomena associated with asthma.     

A standard vector for the chromosomal integration and characterization of BioBrick™ parts in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background

The chromosomal integration of biological parts in the host genome enables the engineering of plasmid-free stable strains with single-copy insertions of the desired gene networks. Although different integrative vectors were proposed, no standard pre-assembled genetic tool is available to carry out this task. Synthetic biology concepts can contribute to the development of standardized and user friendly solutions to easily produce engineered strains and to rapidly characterize the desired genetic parts in single-copy context.

Results

In this work we report the design of a novel integrative vector that allows the genomic integration of biological parts compatible with the RFC10, RFC23 and RFC12 BioBrick? standards in Escherichia coli. It can also be specialized by using BioBrick? parts to target the desired integration site in the host genome. The usefulness of this vector has been demonstrated by integrating a set of BioBrick? devices in two different loci of the E. coli chromosome and by characterizing their activity in single-copy. Construct stability has also been evaluated and compared with plasmid-borne solutions.

Conclusions

Physical modularity of biological parts has been successfully applied to construct a ready-to-engineer BioBrick? vector, suitable for a stable chromosomal insertion of standard parts via the desired recombination method, i.e. the bacteriophage integration mechanism or homologous recombination. In contrast with previously proposed solutions, it is a pre-assembled vector containing properly-placed restriction sites for the direct transfer of various formats of BioBrick? parts. This vector can facilitate the characterization of parts avoiding copy number artefacts and the construction of antibiotic resistance-free engineered microbes, suitable for industrial use.

     

DOK2 Inhibits EGFR-Mutated Lung Adenocarcinoma

Somatic mutations in the EGFR proto-oncogene occur in ~15% of human lung adenocarcinomas and the importance of EGFR mutations for the initiation and maintenance of lung cancer is well established from mouse models and cancer therapy trials in human lung cancer patients. Recently, we identified DOK2 as a lung adenocarcinoma tumor suppressor gene. Here we show that genomic loss of DOK2 is associated with EGFR mutations in human lung adenocarcinoma, and we hypothesized that loss of DOK2 might therefore cooperate with EGFR mutations to promote lung tumorigenesis. We tested this hypothesis using genetically engineered mouse models and find that loss of Dok2 in the mouse accelerates lung tumorigenesis initiated by oncogenic EGFR, but not that initiated by mutated Kras. Moreover, we find that DOK2 participates in a negative feedback loop that opposes mutated EGFR; EGFR mutation leads to recruitment of DOK2 to EGFR and DOK2-mediated inhibition of downstream activation of RAS. These data identify DOK2 as a tumor suppressor in EGFR-mutant lung adenocarcinoma.     

Contrasting functional trait syndromes underlay woody alien success in the same ecosystem

We performed a comprehensive comparative study of functional traits in coexisting alien and native woody species in order to examine the strategies related to resource use and dispersion underlying alien success in mountain Chaco woodlands of central Argentina. Our approach integrated seemingly contrasting pieces of evidence in the region. We specifically assessed whether (i) the ‘functional acquisitive trend’ previously observed along a broad environmental gradient accounts for woody alien naturalization when considering a single mesic ecosystem; or (ii) more than one trait syndrome is important among alien species, which would be more in line with the context‐dependent nature of biological invasions at a local scale. Fifteen vegetative and regenerative traits were measured on the most common 14 native and 11 alien woody species. We compared the attributes of (i) native and alien species and (ii) between native species and two contrasting groups of alien species identified in the previous analysis. The overall trait comparison (i) showed that, in terms of vegetative attributes, woody alien species tend to be on average more acquisitive than native species. However, (ii) two contrasting syndromes were revealed among alien species: a group of seven deciduous species with acquisitive attributes; and a group of four evergreen species showing markedly more conservative attributes than the first group. The functional attributes of ‘conservative aliens’ completely overlapped with the range observed for native species, except for an exclusive dispersal phenology and a stronger tendency to clonal spread. Acquisitive aliens, in turn, proved to be beyond the range of attributes of native species, at the acquisitive extreme, as they did in previous comparisons. Despite their importance, general trends in plant functional attributes across regions and ecosystems can sometimes obscure trends at more local scales that are nevertheless important for the understanding and management of particular systems. Our study concurs with previous general trends when looking at the overall comparison between native and alien species, but unveils contrasting functional strategies among alien species when examining their attributes more closely, even within the same ecosystem.     

The use of temperature gradient tunnels for studying the combined effect of CO2, temperature and water availability in N2 fixing alfalfa plants

Atmospheric CO₂ concentration and temperature are increasing as a consequence of human activity. Periods of low water availability are expected to increase in Mediterranean ecosystems. Temperature gradient tunnels were used to provide near to ambient conditions and conditions simulating predicted increases in CO₂ and temperature for an alfalfa crop. The performance, construction, and running costs of the tunnels are reported and discussed. Two levels of water supply were included in the treatments. Plants were grown in large, 13 L pots, keeping a fixed soil volumetric water content and with nodule fixation as the only nitrogen source for the plant. Regardless of water regime, the effect of elevated CO₂ on plant growth was temperature dependent. Dry matter was enhanced when elevated CO₂ and temperature were combined. Plant yield improvement was partly a result of increased photosynthetic rates. There were no effects on plant N concentration. Decreased specific nodule activity might suggest that lack of differences in plant N were a consequence of reduced nodule enzyme activity.     

Increasing importance of small phytoplankton in a warmer ocean

The macroecological relationships among marine phytoplankton total cell density, community size structure and temperature have lacked a theoretical explanation. The tiniest members of this planktonic group comprise cyanobacteria and eukaryotic algae smaller than 2 μm in diameter, collectively known as picophytoplankton. We combine here two ecological rules, the temperature–size relationship with the allometric size‐scaling of population abundance to explain a remarkably consistent pattern of increasing picophytoplankton biomass with temperature over the ?0.6 to 22 °C range in a merged dataset obtained in the eastern and western temperate North Atlantic Ocean across a diverse range of environmental conditions. Our results show that temperature alone was able to explain 73% of the variance in the relative contribution of small cells to total phytoplankton biomass regardless of differences in trophic status or inorganic nutrient loading. Our analysis predicts a gradual shift toward smaller primary producers in a warmer ocean. Because the fate of photosynthesized organic carbon largely depends on phytoplankton size, we anticipate future alterations in the functioning of oceanic ecosystems.     

ADAPTATION OF A METHIONINE AUXOTROPH ESCHERICHIA COLI GROWTH ASSAY TO MICROTITER PLATES FOR QUANTITATING METHIONINE

A rapid microtiter methionine assay was developed using a methionine auxotroph E. coli strain. The bacterial strain was first grown on rich media to promote extensive bacterial growth and the cells were depleted to exhaust all endogenous methionine. After depletion, cells were transferred to minimal media with increasing concentrations of methionine and microtiter plates were incubated at 37C for 6 h. Methionine microtiter standard curves yielded linear growth responses to increasing concentrations of methionine in the range of 0 to 26.8 μM. Addition of different antibiotic and antifungal agents to the media did not significantly alter the linear growth response observed in the microtiter assay. This microtiter plate E. coli methionine assay has potential as a rapid in vitro assay method for quantifying methionine.     

GFP‐SCFV: Expression and possible applications as a tool for experimental investigations of atherosclerosis

Experimental studies on atherosclerosis are crucial for investigating its pathophysiology, defining new therapeutic targets, and developing new drugs and diagnostic tools. Thus, many imaging markers have been developed and introduced in experimental studies. The main advantage of these new tools is that they allow the noninvasive diagnosis of atherosclerotic vascular disease. Here, we describe the cloning, expression, purification, and stabilization of a chimeric protein specifically designed to probe cells and tissues for the presence of LDL(?), a relevant marker of atherosclerosis. The DNA sequence that encodes the anti‐LDL(?) scFv, previously obtained from a hybridoma secreting an anti‐LDL(?) monoclonal antibody, was inserted into the bacterial vector pET‐28a(+) in tandem with a DNA sequence encoding GFP. The recombinant protein was expressed in high yields in E. coli as inclusion bodies. The applicability of GFP‐scFv was assessed by ELISA, which determined its affinity for LDL(?) and confocal microscopy, that showed macrophage uptake of the protein along with LDL(?). In conclusion, our data suggest that the anti‐LDL(?) GFP‐scFv chimeric protein could be useful in studies on atherogenesis as well as for developing diagnostic tools for atherosclerosis. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1206–1213, 2014     

Functional diversity and trait–environment relationships of stream fish assemblages in a large tropical catchment

1. The species composition of stream fish assemblages changes across the longitudinal fluvial gradient of large river basins. These changes may reflect both zonation in species distributions and environmental filtering of fish traits as stream environments change from the uplands to the lowlands of large catchments. Previous research has shown that taxonomic diversity generally increases in larger, lowland streams, and the River Continuum Concept, the River Habitat Template and other frameworks have provided expectations for what functional groups of fishes should predominate in certain stream types. However, studies addressing the functional trait composition of fish assemblages across large regions are lacking, particularly in tropical river basins. 2. We examined functional trait–environment relationships and functional diversity of stream fish assemblages in the Río Grijalva Basin in southern Mexico. Traits linked to feeding, locomotion and life history strategy were measured in fishes from streams throughout the catchment, from highland headwaters to broad, lowland streams. Relationships between functional traits and environmental variables at local and landscape scales were examined using multivariate ordination, and the convex hull volume of trait space occupied by fish assemblages was calculated as a measure of functional diversity. 3. Although there were a few exceptions, functional diversity of assemblages increased with species richness along the gradient from uplands to lowlands within the Grijalva Basin. Traits related to swimming, habitat preference and food resource use were associated with both local (e.g. substratum type, pool availability) and landscape‐scale (e.g. forest cover) environmental variables. 4. Along with taxonomic structure and diversity, the functional composition of fish assemblages changed across the longitudinal fluvial gradient of the basin. Trait–environment relationships documented in this study partially confirmed theoretical expectations and revealed patterns that may help in developing a better understanding of general functional responses of fish assemblages to environmental change.     

Monoclonal antibodies conjugated with superparamagnetic iron oxide particles allow magnetic resonance imaging detection of lymphocytes in the mouse brain

We investigated the potential of antibody-vectorialized superparamagnetic iron oxide (SPIO) particles as cellular specific magnetic resonance contrast agents to image lymphocyte populations within the central nervous system (CNS), with the final goal of obtaining a reliable tool for noninvasively detecting and tracking specific cellular populations in vivo. We used superparamagnetic particles bound to a monoclonal antibody. The particle is the contrast agent, by means of its T?* relaxation properties; the antibody is the targeting vector, responsible for homing the particle to target a surface antigen. To investigate the efficiency of particle vectorialization by these antibodies, we compared two types of antibody-vectorialized CD3-specific particles in vivo. We successfully employed vectorialized SPIO particles to image B220? cells in a murine model of B-cell lymphoma. Likewise, we were able to identify CD3? infiltrates in a murine model of multiple sclerosis. The specificity of the technique was confirmed by immunohistochemistry and electron microscopy of corresponding sections. Our findings suggest that indirect binding of the antibody to a streptavidinated particle allows for enhanced particle vectorialization compared to covalent binding of the antibody to the particle.