Capturing complex 3D tissue physiology in vitro

The emergence of tissue engineering raises new possibilities for the study of complex physiological and pathophysiological processes in vitro. Many tools are now available to create 3D tissue models in vitro, but the blueprints for what to make have been slower to arrive. We discuss here some of the 'design principles' for recreating the interwoven set of biochemical and mechanical cues in the cellular microenvironment, and the methods for implementing them. We emphasize applications that involve epithelial tissues for which 3D models could explain mechanisms of disease or aid in drug development.     

Ezrin functionality and hypothermic preservation injury in LLC-PK1 cells

Renal epithelial cells from donor kidneys are susceptible to hypothermic preservation injury, which is attenuated when they over express the cytoskeletal linker protein ezrin. This study was designed to characterize the mechanisms of this protection. Renal epithelial cell lines were created from LLC-PK1 cells, which expressed mutant forms of ezrin with site directed alterations in membrane binding functionality. The study used cells expressing wild type ezrin, T567A, and T567D ezrin point mutants. The A and D mutants have constitutively inactive and active membrane binding conformations, respectively. Cells were cold stored (4 °C) for 6-24 h and reperfused for 1h to simulate transplant preservation injury. Preservation injury was assessed by mitochondrial activity (WST-1) and LDH release. Cells expressing the active ezrin mutant (T567D) showed significantly less preservation injury compared to wild type or the inactive mutant (T567A), while ezrin-specific siRNA knockdown and the inactive mutant potentiated preservation injury. Ezrin was extracted and identified from purified mitochondria. Furthermore, isolated mitochondria specifically bound anti-ezrin antibodies, which were reversed with the addition of exogenous recombinant ezrin. Recombinant wild type ezrin significantly reduced the sensitivity of the mitochondrial permeability transition pore (mPTP) to calcium, suggesting ezrin may modify mitochondrial function. In conclusion, the cytoskeletal linker protein ezrin plays a significant role in hypothermic preservation injury in renal epithelia. The mechanisms appear dependent on the molecule's open configuration (traditional linker functionality) and possibly a novel mitochondrial specific role, which may include modulation of mPTP function or calcium sensitivity.     

Variation in phenotypic plasticity and selection patterns in blue tit breeding time: between- and within-population comparisons

1.?Phenotypic plasticity, the response of individual phenotypes to their environment, can allow organisms to cope with spatio-temporal variation in environmental conditions. Recent studies have shown that variation exists among individuals in their capacity to adjust their traits to environmental changes and that this individual plasticity can be under strong selection. Yet, little is known on the extent and ultimate causes of variation between populations and individuals in plasticity patterns. 2.?In passerines, timing of breeding is a key life-history trait strongly related to fitness and is known to vary with the environment, but few studies have investigated the within-species variation in individual plasticity. 3.?Here, we studied between- and within-population variation in breeding time, phenotypic plasticity and selection patterns for this trait in four Mediterranean populations of blue tits (Cyanistes caeruleus) breeding in habitats varying in structure and quality. 4.?Although there was no significant warming over the course of the study, we found evidence for earlier onset of breeding in warmer years in all populations, with reduced plasticity in the less predictable environment. In two of four populations, there was significant inter-individual variation in plasticity for laying date. Interestingly, selection for earlier laying date was significant only in populations where there was no inter-individual differences in plasticity. 5.?Our results show that generalization of plasticity patterns among populations of the same species might be challenging even at a small spatial scale and that the amount of within-individual variation in phenotypic plasticity may be linked to selective pressures acting on these phenotypic traits.     

Engineered mutations change the structure and stability of a virus-like particle

The single-coat protein (CP) of bacteriophage Qβ self-assembles into T = 3 icosahedral virus-like particles (VLPs), of interest for a wide range of applications. These VLPs are very stable, but identification of the specific molecular determinants of this stability is lacking. To investigate these determinants along with manipulations that confer more capabilities to our VLP material, we manipulated the CP primary structure to test the importance of various putative stabilizing interactions. Optimization of a procedure to incorporate fused CP subunits allowed for good control over the average number of covalent dimers in each VLP. We confirmed that the disulfide linkages are the most important stabilizing elements for the capsid and that acidic conditions significantly enhance the resistance of VLPs to thermal degradation. Interdimer interactions were found to be less important for VLP assembly than intradimer interactions. Finally, a single point mutation in the CP resulted in a population of smaller VLPs in three distinct structural forms.     

How insects survive the cold: molecular mechanisms—a review

Insects vary considerably in their ability to survive low temperatures. The tractability of these organisms to experimentation has lead to considerable physiology-based work investigating both the variability between species and the actual mechanisms themselves. This has highlighted a range of strategies including freeze tolerance, freeze avoidance, protective dehydration and rapid cold hardening, which are often associated with the production of specific chemicals such as antifreezes and polyol cryoprotectants. But we are still far from identifying the critical elements behind over-wintering success and how some species can regularly survive temperatures below −20°C. Molecular biology is the most recent tool to be added to the insect physiologist’s armoury. With the public availability of the genome sequence of model insects such as Drosophila and the production of custom-made molecular resources, such as EST libraries and microarrays, we are now in a position to start dissecting the molecular mechanisms behind some of these well-characterised physiological responses. This review aims to provide a state-of-the-art snapshot of the molecular work currently being conducted into insect cold tolerance and the very interesting preliminary results from such studies, which provide great promise for the future.     

Organic carbon cycling in Taylor Valley, Antarctica: quantifying soil reservoirs and soil respiration

Organic carbon reservoirs and respiration rates in soils have been calculated for most major biomes on Earth revealing patterns related to temperature, precipitation, and location. Yet data from one of the Earth's coldest, driest, and most southerly soil ecosystems, that of the McMurdo Dry Valleys of Antarctica, are currently not a part of this global database. In this paper, we present the first regional calculations of the soil organic carbon reservoirs in a dry valley ecosystem (Taylor Valley) and report measurements of CO₂ efflux from Antarctic soils. Our analyses indicate that, despite the absence of visible accumulations of organic matter in most of Taylor Valley's arid soils, this soil environment contained a significant percentage (up to 72%) of the seasonally unfrozen organic carbon reservoir in the terrestrial ecosystem. Field measurements of soil CO₂‐efflux in Taylor Valley soils were used to evaluate biotic respiration and averaged 0.10 ± 0.08 μmol CO₂ m^?2 s^?1. Laboratory soil microcosms suggested that this respiration rate was sensitive to increases in temperature, moisture, and carbon addition. Finally, a steady‐state calculation of the mean residence time for organic carbon in Taylor Valley soils was 23 years. Because this value contradicts all that is currently known about carbon cycling rates in the dry valleys, we suggest that the dry valley soil carbon dynamics is not steady state. Instead, we suggest that the dynamic is complex, with at least two (short‐ and long‐term) organic carbon reservoirs. We also suggest that organic carbon in the dry valley soil environment may be more important, and play a more active role in long‐term ecosystem processes, than previously believed.     

Infectivity of Four Species of Nematodes (Rhabditoidea: Steinernematidae, Heterorhabditidae) to the Asian Longhorn Beetle, Anoplophora glabripennis (Motchulsky) (Coleoptera: Cerambycidae)

Four species of entomopathogenic nematodes, Steinernema carpocapsae , Heterorhabditis bacteriophora , H. indica and H. marelatus , were tested for their ability to kill and reproduce in larvae of the Asian longhorn beetle, Anoplophora glabripennis (Motchulsky). The larvae were permissive to all four species but mortality was higher and production of infective juveniles was greater for S. carpocapsae and H. marelatus . The lethal dosage of H. marelatus was determined to be 19 infective juveniles for second and third instar larvae and 347 infective juveniles for fourth and fifth instar larvae. H. marelatus infective juveniles, applied via sponges to oviposition sites on cut logs, located and killed host larvae within 30 cm galleries and reproduced successfully in several of the larvae.