Light Environments Differently Affect Parasitoid Wasps and their Hosts’ Locomotor Activity

In natural environments, organisms must adapt to changing light conditions. Significant research has been done on diurnal pollinating insects’ vision. However, little is known on parasitoid insects. Here, we studied how locomotor activity of the parasitoid wasp Aphidius ervi and its main host, the pea aphid Acyrthosiphon pisum, is affected under controlled artificial illumination. Using LEDs of 5 different wavelengths (361, 450, 500-600, 626 and 660 nm), we created different artificial light spectra that parasitoids and host aphids can encounter in natural environment including leaf-shade and direct sunlight. We found that pea aphid probability of walking depended on interactions between illumination, developmental stage and genotype as expressed in clonal variation. Artificial light intensity did not affect the parasitoid’s probability of walking as opposed to wavelength, and activity depended on the sex of individuals. Males were more active than females under all monochromatic wavelength spectra tested. Virgin females were much less active under the artificial leaf-shade illumination and artificial sunlight, as compared to males and mated females. Delay before flight for females was favored by sunlight illumination whereas the light environment did not affect flight delay for males. We demonstrated that locomotor activity of A. pisum (walking) and of A. ervi (walking and flight) vary according to the light environment. This study should help develop better understanding of the effects of illumination on host-parasitoid interactions, which in turn may help control insect pest populations.     

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs^1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.     

Alternate Binding Modes for a Ubiquitin-SH3 Domain Interaction Studied by NMR Spectroscopy

Surfaces of many binding domains are plastic, enabling them to interact with multiple targets. An understanding of how they bind and recognize their partners is therefore predicated on characterizing such dynamic interfaces. Yet, these interfaces are difficult to study by standard biophysical techniques that often ‘freeze’ out conformations or that produce data averaged over an ensemble of conformers. In this study, we used NMR spectroscopy to study the interaction between the C-terminal SH3 domain of CIN85 and ubiquitin that involves the ‘classical’ binding sites of these proteins. Notably, chemical shift titration data of one target with another and relaxation dispersion data that report on millisecond time scale exchange processes are both well fit to a simple binding model in which free protein is in equilibrium with a single bound conformation. However, dissociation constants and chemical shift differences between free and bound states measured from both classes of experiment are in disagreement. It is shown that the data can be reconciled by considering three-state binding models involving two distinct bound conformations. By combining titration and dispersion data, kinetic and thermodynamic parameters of the three-state binding reaction are obtained along with chemical shifts for each state. A picture emerges in which one bound conformer has increased entropy and enthalpy relative to the second and chemical shifts similar to that of the free state, suggesting a less packed interface. This study provides an example of the interplay between entropy and enthalpy to fine-tune molecular interactions involving the same binding surfaces.     

Genetic Ancestry and Asthma and Rhinitis Occurrence in Hispanic Children: Findings from the Southern California Children’s Health Study

BackgroundAsthma and rhinitis are common childhood health conditions. Being an understudied and rapidly growing population in the US, Hispanic children have a varying risk for these conditions that may result from sociocultural (including acculturative factors), exposure and genetic diversities. Hispanic populations have varying contributions from European, Amerindian and African ancestries. While previous literature separately reported associations between genetic ancestry and acculturation factors with asthma, whether Amerindian ancestry and acculturative factors have independent associations with development of early-life asthma and rhinitis in Hispanic children remains unknown. We hypothesized that genetic ancestry is an important determinant of early-life asthma and rhinitis occurrence in Hispanic children independent of sociodemographic, acculturation and environmental factors.MethodsSubjects were Hispanic children (5–7 years) who participated in the southern California Children’s Health Study. Data from birth certificates and questionnaire provided information on acculturation, sociodemographic and environmental factors. Genetic ancestries (Amerindian, European, African and Asian) were estimated based on 233 ancestry informative markers. Asthma was defined by parental report of doctor-diagnosed asthma. Rhinitis was defined by parental report of a history of chronic sneezing or runny or blocked nose without a cold or flu. Sample sizes were 1,719 and 1,788 for investigating the role of genetic ancestry on asthma and rhinitis, respectively.ResultsChildren had major contributions from Amerindian and European ancestries. After accounting for potential confounders, per 25% increase in Amerindian ancestry was associated with 17.6% (95% confidence interval [CI]: 0.74–0.99) and 13.6% (95% CI: 0.79–0.98) lower odds of asthma and rhinitis, respectively. Acculturation was not associated with either outcome.ConclusionsEarlier work documented that Hispanic children with significant contribution from African ancestry are at increased asthma risk; however, in Hispanic children who have little contribution from African ancestry, Amerindian ancestry was independently associated with lower odds for development of early-childhood asthma and rhinitis.     

Is it possible to produce succinic acid at a low pH?

Bio-based succinate is still a matter of special emphasis in biotechnology and adjacent research areas. The vast majority of natural and engineered producers are bacterial strains that accumulate succinate under anaerobic conditions. Recently, we succeeded in obtaining an aerobic yeast strain capable of producing succinic acid at low pH. Herein, we discuss some difficulties and advantages of microbial pathways producing "succinic acid" rather than "succinate." It was concluded that the peculiar properties of the constructed yeast strain could be clarified in view of a distorted energy balance. There is evidence that in an acidic environment, the majority of the cellular energy available as ATP will be spent for proton and anion efflux. The decreased ATP:ADP ratio could essentially reduce the growth rate or even completely inhibit growth. In the same way, the preference of this elaborated strain for certain carbon sources could be explained in terms of energy balance. Nevertheless, the opportunity to exclude alkali and mineral acid waste from microbial succinate production seems environmentally friendly and cost-effective.     

Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy

Quantitative single-cell time-lapse microscopy is a powerful method for analyzing gene circuit dynamics and heterogeneous cell behavior. We describe the application of this method to imaging bacteria by using an automated microscopy system. This protocol has been used to analyze sporulation and competence differentiation in Bacillus subtilis, and to quantify gene regulation and its fluctuations in individual Escherichia coli cells. The protocol involves seeding and growing bacteria on small agarose pads and imaging the resulting microcolonies. Images are then reviewed and analyzed using our laboratory's custom MATLAB analysis code, which segments and tracks cells in a frame-to-frame method. This process yields quantitative expression data on cell lineages, which can illustrate dynamic expression profiles and facilitate mathematical models of gene circuits. With fast-growing bacteria, such as E. coli or B. subtilis, image acquisition can be completed in 1 d, with an additional 1-2 d for progressing through the analysis procedure.     

Production of recombinant Rhizopus oryzae lipase by the yeast Yarrowia lipolytica results in increased enzymatic thermostability

The gene encoding Rhizopus oryzae lipase (ROL) was expressed in the non-conventional yeast Yarrowia lipolytica under the control of the strong inducible XPR2 gene promoter. The effects of three different preprosequence variants were examined: a preprosequence of the Y. lipolytica alkaline extracellular protease (AEP) encoded by XPR2, the native preprosequence of ROL, and a hybrid variant of the presequence of AEP and the prosequence of ROL. Lipase production was highest (7.6 U/mL) with the hybrid prepropeptide. The recombinant protein was purified by ion-exchange chromatography. The ROL included 28 amino acids of the C-terminal region of the prosequence, indicating that proteolytic cleavage occurred below the KR site through the activity of the Kex2-like endoprotease. The optimum temperature for recombinant lipase activity was between 30 and 40 °C, and the optimum pH was 7.5. The enzyme was shown not to be glycosylated. Furthermore, recombinant ROL exhibited greater thermostability than previously reported, with the enzyme retaining 64% of its hydrolytic activity after 30 min of incubation at 55 °C.     

Origins of Pressure-Induced Protein Transitions

The molecular mechanisms underlying pressure-induced protein denaturation can be analyzed based on the pressure-dependent differences in the apparent volume occupied by amino acids inside the protein and when they are exposed to water in an unfolded conformation. We present here an analysis for the peptide group and the 20 naturally occurring amino acid side chains based on volumetric parameters for the amino acids in the interior of the native state, the micelle-like interior of the pressure-induced denatured state, and the unfolded conformation modeled by N-acetyl amino acid amides. The transfer of peptide groups from the protein interior to water becomes increasingly favorable as pressure increases. Thus, solvation of peptide groups represents a major driving force in pressure-induced protein denaturation. Polar side chains do not appear to exhibit significant pressure-dependent changes in their preference for the protein interior or solvent. The transfer of nonpolar side chains from the protein interior to water becomes more unfavorable as pressure increases. We conclude that a sizeable population of nonpolar side chains remains buried inside a solvent-inaccessible core of the pressure-induced denatured state. At elevated pressures, this core may become packed almost as tightly as the interior of the native state. The presence and partial disappearance of large intraglobular voids is another driving force facilitating pressure-induced denaturation of individual proteins. Our data also have implications for the kinetics of protein folding and shed light on the nature of the folding transition state ensemble.     

Partial molar volumes,expansibilities, and compressibilities of oligoglycines in aqueous solutions at 18–55°C

We have determined the partial molar volumes, expansibilities, and adiabatic compressibilities of glycine, diglycine, triglycine, tetraglycine, and pentaglycine over the temperature range 18–55°C. These data were analyzed and interpreted in terms of the hydration of these short oligoglycines and their constituent groups. From our results, we have estimated the contributions of the peptide group to the partial molar volume and the partial molar adiabatic compressibility of these oligoglycines. Based on these data, we propose that each of the polar atomic groups of the peptide bond forms approximately two hydrogen bonds with adjacent water molecules. Furthermore, the temperature dependence of the partial molar volume suggests that water that solvates the polar groups of a peptide linkage behaves more like a “normal” liquid than does bulk water, which exhibits its well-known anomalous liquid properties. © 1994 John Wiley & Sons, Inc.