Forced and natural convective drying of trehalose/water thin films: implication in the desiccation preservation of Mammalian cells

Trehalose is believed to offer desiccation protection to mammalian cells by forming stable glassy matrices. The goal of the current study was to explore the desiccation kinetics of thin films of trehalose-water solution under forced and natural convective conditions and to investigate the thermophysical state of mammalian cells at the bottom of the thin film. We developed a finite difference model based on the mass and energy conservation equations coupled to the water transport model from the cells. The boundary conditions were obtained from correlations or experimental measurements and the Gordon-Taylor equation was used to predict the glass transition temperature at every location. Results indicated that there are three distinct regimes for drying for both forced and natural convection, characterized by the slope of the moisture content plot as a function of time. Our results also indicate that the surface of the solution reached the glassy state in less than 10 min for the Reynolds (forced) numbers explored and approximately 30 min for some Rayleigh (natural convective) numbers; however, significant water was trapped at this instant. Larger drying force hastened quicker glass formation but trapped more water. The numerical model was capable of predicting the drying kinetics for the dilute region accurately, but deviated while predicting the other regimes. Based on these experimental validations of the model, the osmotic response of different cells located at the bottom of the solution with orders of magnitude difference in their membrane permeability (Lp) was predicted. The results suggested that extracellular glass formed around cells at the bottom of a trehalose-water solution by the propagation of glass into the solution; however it takes more than an order of magnitude time (approximately 7 min to >100 min for forced convective drying) to remove sufficient water to form glass around cells from the time when the first surface glass is formed. This is attributed to low diffusivity of water through the glass. In addition, the water transport from the glassy matrix could be either diffusion or Lp limited. For diffusion-limited transport, lowering the film thickness at the beginning of drying by half almost lowers the drying time by an order of magnitude. In summary, the optimal design of convective desiccation protocols requires accounting for the size of the cell, their membrane permeability (Lp) and the starting thickness of the solution.     

Common Human 5′ Dopamine Transporter (SLC6A3) Haplotypes Yield Varying Expression Levels In Vivo

1. Individuals display significant differences in their levels of expression of the dopamine transporter (DAT; SLC6A3). These differences in DAT are strong candidates to contribute to individual differences in motor, mnemonic and reward functions. To identify “cis”-acting genetic mechanisms for these individual differences, we have sought variants in 5′ aspects of the human DAT gene and identified the haplotypes that these variants define.2. We report (i) significant relationships between 5′ DAT haplotypes and human individual differences in ventral striatal DAT expression assessed in vivo using [¹¹C] cocaine PET and (ii) apparent confirmation of these results in studies of DAT expression in postmortem striatum using [³H] carboxyflurotropane binding.3. These observations support the idea that cis-acting variation in 5′ aspects of the human DAT/SLC6A3 locus contributes to individual differences in levels of DAT expression in vivo. 5′ DAT variation is thus a good candidate to contribute to individual differences in a number of human phenotypes.These authors contributed equally to this article     

The Drosophila protein palmitoylome: characterizing palmitoyl-thioesterases and DHHC palmitoyl-transferases

Palmitoylation is the post-translational addition of a palmitate moiety to a cysteine residue through a covalent thioester bond. The addition and removal of this modification is controlled by both palmitoyl acyl-transferases and thioesterases. Using bioinformatic analysis, we identified 22 DHHC family palmitoyl acyl-transferase homologs in the Drosophila genome. We used in situ hybridization,RT-PCR, and published FlyAtlas microarray data to characterize the expression patterns of all 22 fly homologs. Our results indicate that all are expressed genes, but several, including CG1407, CG4676, CG5620, CG6017/dHIP14, CG6618, CG6627 and CG17257 appear to be enriched in neural tissues suggesting that they are important for neural function. Furthermore, we have found that several may be expressed in a sex-specific manner with adult male specific expression of CG4483 and CG17195. Using tagged versions of the DHHC genes, we demonstrate that fly DHHC proteins are primarily located in either the Golgi Apparatus or Endoplasmic Reticulum in S2 cells, except for CG1407, which was found on the plasma membrane. We also characterized the subcellular localization and expression of the three known thioesterases: Palmitoyl-protein Thioesterase 1 (Ppt1), Palmitoyl-protein Thioesterase 2 (Ppt2)and Acyl-protein Thioesterase 1 (APT1). Our results indicate that Ppt1 and Ppt2 are the major lysosomal thioesterases while APT1 is the likely cytoplasmic thioesterase. Finally, in vivo rescue experiments show that Ppt2 expression cannot rescue the neural inclusion phenotypes associated with loss of Ppt1, further supporting distinct functions and substrates for these two thioesterases. These results will serve as the basis for a more complete understanding of the protein palmitoylome's normal cellular functions in the fly and will lead to further insights into the molecular etiology of diseases associated with the mis-regulation of palmitoylation.     

Developmental Regulation of Low-temperature Tolerance in Winter Wheat

Vernalization and photoperiod genes have wide-ranging effectson the timing of gene expression in plants. The objectives ofthis study were to (1) determine if expression of low-temperature(LT) tolerance genes is developmentally regulated and (2) establishthe interrelationships among the developmental stages and LTtolerance gene expression. LT response curves were determinedfor three photoperiod-sensitive LT tolerant winter wheat (Triticumaestivum L. em Thell) genotypes acclimated at 4 °C under8 h short-day (SD) and 20 h long-day (LD) photoperiods from0 to 112 d. Also, three de-acclimation and re-acclimation cycleswere used that bridged the vegetative/reproductive transitionpoint for each LD and SD photoperiod treatment. A vernalizationperiod of 49 d at 4 °C was sufficient for all genotypesto reach vernalization saturation as measured by minimum finalleaf number (FLN) and confirmed by examination of shoot apicesdissected from crowns that had been de-acclimated at 20 °CLD. Before the vegetative/reproductive transition, both theLD- and SD-treated plants were able to re-acclimate to similarLT₅₀(temperature at which 50% of the plants are killed by LTstress) levels following de-acclimation at 20 °C. De-acclimationof LD plants after vernalization saturation resulted in rapidprogression to the reproductive phase and limited ability tore-acclimate. The comparative development of the SD (non-flowering-inductivephotoperiod) de-acclimated plants was greatly delayed relativeto LD plants, and this delay in development was reflected inthe ability of SD plants to re-acclimate to a lower temperature.These observations confirm the hypothesis that the point oftransition to the reproductive stage is pivotal in the expressionof LT tolerance genes, and the level and duration of LT acclimationare related to the stage of phenological development as regulatedby vernalization and photoperiod requirements. Copyright 2001Annals of Botany Company Triticum aestivum L., wheat, low-temperature tolerance, vernalization, photoperiod, phenological development     

Temperature shock during development fails to increase the fluctuating asymmetry of a sexual trait in stalk-eyed flies.

The fluctuating asymmetry (FA) of bilateral traits is claimed to be a general indicator of environmental stress. Exaggerated sexual ornaments are thought to show elevated levels of FA and a greater response to stress than other traits. Previous work with stalk-eyed flies (Cyrtodiopsis dalmanni) has shown that the FA of the sexual trait (male eye stalks), wing length and wing width were unaffected by a continually applied food stress. Here we tested whether a transient stress (24-h heat shock at 31 degrees C during development) affected the FA of these traits. A second experiment tested the combined stresses of transient heat shock at 31 degrees C with continuous exposure to desiccation. In each experiment, temperature shock reduced the trait size, confirming that the treatments were stressful. However, stress had no effect on the FA of individual traits or the FA summed across all traits. Exposure to the combined stresses significantly elevated mortality and reduced trait size compared to the single-stress regime. However, FA did not differ significantly between flies from the two experiments. We found no evidence that FA in sexual and non-sexual traits reflects transient stress during the development of C. dalmanni.     

Genetic approaches to sustainable pest management in sugar beet (Beta vulgaris)

Sugar beet (Beta vulgaris) is an important arable crop, traditionally used for sugar extraction, but more recently, for biofuel production. A wide range of pests, including beet cyst nematode (Heterodera schachtii), root‐knot nematodes (Meloidogyne spp.), green peach aphids (Myzus persicae) and beet root maggot (Tetanops myopaeformis), infest the roots or leaves of sugar beet, which leads to yield loss directly or through transmission of beet pathogens such as viruses. Conventional pest control approaches based on chemical application have led to high economic costs. Development of pest‐resistant sugar beet varieties could play an important role towards sustainable crop production while minimising environmental impact. Intensive Beta germplasm screening has been fruitful, and genetic lines resistant to nematodes, aphids and root maggot have been identified and integrated into sugar beet breeding programmes. A small number of genes responding to pest attack have been cloned from sugar beet and wild Beta species. This trend will continue towards a detailed understanding of the molecular mechanism of insect–host plant interactions and host resistance. Molecular biotechnological techniques have shown promise in developing transgenic pest resistance varieties at an accelerated speed with high accuracy. The use of transgenic technology is discussed with regard to biodiversity and food safety.     

Helix-helix interactions in membrane proteins: coarse-grained simulations of glycophorin a helix dimerization

Oligomerization of transmembrane (TM) helices is a key stage in the folding of membrane proteins. Glycophorin A (GpA) is a well-documented test system for this process. Coarse-grained molecular dynamics (CG-MD) allows us to simulate the self-assembly of TM helices into dimers, for both wild-type (WT) and mutant GpA sequences. For the WT sequences, dimers formed rapidly and remained stable in all simulations. The resultant dimers exhibited right-handed crossing and the same interhelix contacts as in NMR structures. Simulations of disruptive mutants revealed the dimers were less stable, with values of DeltaDelta G dimerization consistent with experimental data. The dimers of disruptive mutants were distorted relative to the WT and showed left-handed crossing of their helices. CG-MD can therefore be used to explore the interactions of TM helices, an important stage in the folding of membrane proteins. In particular, CG-MD has been shown to be sensitive enough to detect disruptions introduced by mutation. Future refinement of such models via atomistic simulations will enable a multiscale approach to predict the folding of membrane proteins.     

Challenges in discovering bioactives for the food industry

Nutritional interventions are associated with transient deviations of homeostasis within the human body. The role of a balanced nutrition is, firstly, to ensure an adequate intake of nutrients to efficiently enable all metabolic processes and, secondly, to contribute to sustained human health. This is a new challenge for the food industry because consumers demand taste and convenience in addition to healthy food. The attempts of the food industry to omit health-adverse compounds, and incorporate constituents with identified beneficial health effects has coined the term 'functional food', encompassing fresh or processed food with health-promoting and/or disease-preventing properties. The active ingredients of these products are known as 'nutraceuticals'. This paper outlines scientific concepts applied to the identification of novel bioactive food ingredients.