An active intracellular device to prevent lethal disease outcomes in virus-infected bacterial cells

Synthetic biology includes an effort to logically control cellular behavior. One long-term goal is to implement medical interventions inside living cells, creating intracellular "disease fighters"; one may imagine a system that detects viral infection and responds to halt the spread of the virus. Here, we explore a system designed to display some of the qualitative features that such disease prevention systems should have, while not claiming that the system itself has any medical application. An intracellular disease prevention mechanism should: lie dormant in the absence of the disease state; detect the onset of a lethal disease pathway; respond to halt or mitigate the disease's effects; and be subject to external deactivation when required. We have created a device that displays these properties, in the highly simplified case of a bacterial viral disease. Our system detects the onset of the lytic phase of bacteriophage lambda in Escherichia coli, responds by preventing this lethal pathway from being followed, and is deactivated by a temperature shift. We have formulated a mathematical model of the engineered system, using parameters obtained from the literature and by local experimental measurement, and shown that the model captures the essential experimental behavior of the system in most parameter regimes.     

Growth kinetics and mathematical modeling of Synechocystis sp. PCC 6803 under flashing light

In photobioreactors and natural systems, microalgae are subjected to rapidly changing light intensities (LI) due to light attenuation and mixing. A controlled way to study the effect of rapidly changing LI is to subject cultures to flashing light. In this study, series of flashing-light experiments were conducted using Synechocystis sp. PCC6803 with constant overall average LI of approximately 84 μmol·m⁻²·s⁻¹ and relative times in the light and dark varied. The results were also compared with simulated results using a mathematical model including an absorbed pool of light energy, photoacclimation, and photoinhibition. With equal time in light and dark, the specific growth rate (μ) systematically decreased with increasing light duration, and µ decreased further when the ratio of light to dark was decreased. The model captured both trends with the mechanistic explanation that when the light duration was very short the changes in the pool of absorbed LI were smoothed out across the light and dark periods, whereas longer durations caused the biomass to experience discrete light and dark conditions that lead to reduced light absorption, more energy loss to nonphotochemical quenching, and more photodamage. These growth effects were accentuated as the ratio of light to dark decreased.     

Dependence of Asian honeybee on deciduous woody plants for pollen resource during spring to mid‐summer in northern Japan

Apis cerana japonica Radoszkowski, endemic to Japan, is known to be one of the most important pollinators for wild plants and crops, such as buckwheat, in cool to warm temperate Japan. To determine the degree of dependence of A. cerana japonica on forest resources, we analyzed pollen brought back to nests in a typical “Satoyama” landscape with relatively high deciduous forest coverage in northern Japan. We divided the landscape elements of the study area into three types: deciduous forest, conifer plantation and open land according to landcover digital data, and each pollen taxon was assigned to one of these three types of landscape elements. We collected total pollen loads of 15.75 g (total of colonies A and B) in May (spring), 1.57 g (total of colonies A and C) in June (early summer), 19.03 g (total of colonies A, B and C) in July (mid‐summer) and 45.61 g (total of colonies A, B and D) in September (autumn). Deciduous forests are the most important foraging habitats for A. cerana japonica in the “Satoyama” landscape especially from spring to mid‐summer when mass flowering of tall trees and shrubs species provides rich floral resources for developing bee colonies. On the other hand, the bees frequently foraged from herbaceous plant species in autumn when flowering of tree species reduces and herbaceous plant species have flowering peaks. In turn, the bees provide pollination services to a number of wild flowers blooming in various forest layers ranging from the canopy to the understory layer.     

Climate‐driven increases in storm frequency simplify kelp forest food webs

Climate models predict a dramatic increase in the annual frequency and severity of extreme weather events during the next century. Here we show that increases in the annual frequency of severe storms lead to a decrease in the diversity and complexity of food webs of giant kelp forests, one of the most productive habitats on Earth. We demonstrate this by linking natural variation in storms with measured changes in kelp forest food web structure in the Santa Barbara Channel using structural equation modeling (SEM). We then match predictions from statistical models to results from a multiyear kelp removal experiment designed to simulate frequent large storms. Both SEM models and experiments agree: if large storms remain at their current annual frequency (roughly one major kelp‐removing storm every 3.5 years), periodic storms help maintain the complexity of kelp forest food webs. However, if large storms increase in annual frequency and begin to occur year after year, kelp forest food webs become less diverse and complex as species go locally extinct. The loss of complexity occurs primarily due to decreases in the diversity and complexity of higher trophic levels. Our findings demonstrate that shifts in climate‐driven disturbances that affect foundation species are likely to have impacts that cascade through entire ecosystems.     

Bio-optical modeling of photosynthetic pigments in corals

The spectral reflectance of coral is inherently related to the amounts of photosynthetic pigments present in the zooxanthellae. There are no studies, however, showing that the suite of major photosynthetic pigments can be predicted from optical reflectance spectra. In this study, we measured cm-scale in vivo and in situ spectral reflectance for several colonies of the massive corals Porites lobata and Porites lutea, two colonies of the branching coral Porites compressa, and one colony of the encrusting coral Montipora flabellata in Kaneohe Bay, Oahu, Hawaii. For each reflectance spectrum, we collected a tissue sample and utilized high-performance liquid chromatography to quantify six major photosynthetic pigments, located in the zooxanthellae. We used multivariate multiple regression analysis with cross-validation to build and test an empirical linear model for predicting pigment concentrations from optical reflectance spectra. The model accurately predicted concentrations of chlorophyll a, chlorophyll c ₂, peridinin, diadinoxanthin, diatoxanthin and β-carotene, with correlation coefficients of 0.997, 0.941, 0.995, 0.996, 0.980 and 0.984, respectively. The relationship between predicted and actual concentrations was 1:1 for each pigment, except chlorophyll c ₂. This simple empirical model demonstrates the potential for routine, rapid, non-invasive monitoring of coral-zooxanthellae status, and ultimately for remote sensing of reef biogeochemical processes.     

Homology models of two isozymes of manganese peroxidase: Prediction of a Mn(II) binding site

The three-dimensional structures of two isozymes of manganese peroxidase (MnP) have been predicted from homology modeling using lignin peroxidase as a template. Although highly homologous, MnP differs from LiP by the requirement of Mn(II) as an intermediate in its oxidation of substrates. The Mn(II) site is absent in LiP and unique to the MnP family of peroxidases. The model structures were used to identify the unique Mn(II) binding sites, to determine to what extent they were conserved in the two isozymes, and to provide insight into why this site is absent in LiP. For each isozyme of MnP, three candidate Mn(II) binding sites were identified. Energy optimizations of the three possible Mn(II) enzyme complexes allowed the selection of the most favorable Mn(II) binding site as one with the most anionic oxygen moieties best configured to act as ligands for the Mn(II). At the preferred site, the Mn(II) is coordinated to the carboxyl oxygens of Glu-35, Glu-39, and Asp-179, and a propionate group of the heme. The predicted Mn(II) binding site is conserved in both isozymes. Comparison between the residues at this site in MnP and the corresponding residues in LiP shows that two of the three anionic residues in MnP are replaced by neutral residues in LiP, explaining why LiP does not bind Mn(II). © 1994 Wiley-Liss, Inc.     

MOLS sampling and its applications in structural biophysics

This review describes the MOLS method and its applications. This computational method has been developed in our laboratory primarily to explore the conformational space of small peptides and identify features of interest, particularly the minima, i.e., the low energy conformations. A systematic “brute-force” search through the vast conformational space for such features faces the insurmountable problem of combinatorial explosion, whilst other techniques, e.g., Monte Carlo searches, are somewhat limited in their region of exploration and may be considered inexhaustive. The MOLS method, on the other hand, uses a sampling technique commonly employed in experimental design theory to identify a small sample of the conformational space that nevertheless retains information about the entire space. The information is extracted using a technique that is a variant of the self-consistent mean field technique, which has been used to identify, for example, the optimal set of side-chain conformations in a protein. Applications of the MOLS method to understand peptide structure, predict the structures of loops in proteins, predict three-dimensional structures of small proteins, and arrive at the best conformation, orientation, and positions of a small molecule ligand in a protein receptor site have all yielded satisfactory results.