Cell spinpods are a simple inexpensive suspension culture device to deliver fluid shear stress to renal proximal tubular cells.

Rotating forms of suspension culture allow cells to aggregate into spheroids, prevent the de-differentiating influence of 2D culture, and, perhaps most importantly of all, provide physiologically relevant, in vivo levels of shear stress. Rotating suspension culture technology has not been widely implemented, in large part because the vessels are prohibitively expensive, labor-intensive to use, and are difficult to scale for industrial applications. Our solution addresses each of these challenges in a new vessel called a cell spinpod. These small 3.5 mL capacity vessels are constructed from injection-molded thermoplastic polymer components. They contain self-sealing axial silicone rubber ports, and fluoropolymer, breathable membranes. Here we report the two-fluid modeling of the flow and stresses in cell spinpods. Cell spinpods were used to demonstrate the effect of fluid shear stress on renal cell gene expression and cellular functions, particularly membrane and xenobiotic transporters, mitochondrial function, and myeloma light chain, cisplatin and doxorubicin, toxicity. During exposure to myeloma immunoglobulin light chains, rotation increased release of clinically validated nephrotoxicity cytokine markers in a toxin-specific pattern. Addition of cisplatin or doxorubicin nephrotoxins reversed the enhanced glucose and albumin uptake induced by fluid shear stress in rotating cell spinpod cultures. Cell spinpods are a simple, inexpensive, easily automated culture device that enhances cellular functions for in vitro studies of nephrotoxicity.

Effects of Space Flight on Mouse Liver versus Kidney: Gene Pathway Analyses.

Understanding genome wide, tissue-specific, and spaceflight-induced changes in gene expression is critical to develop effective countermeasures. Transcriptome analysis has been performed on diverse tissues harvested from animals flown in space, but not the kidney. We determined the genome wide gene expression using a gene array analysis of kidney and liver tissue from mice flown in space for 12 days versus ground based control animals. By comparing the transcriptome of liver and kidney from animals flown in space versus ground control animals, we tested a unique hypothesis: Are there common gene expression pathways activated in multiple tissue types in response to spaceflight stimuli? Although there were tissue-specific changes, both liver and kidney overexpressed genes in the same four areas: (a) cellular responses to peptides, hormones, and nitrogen/organonitrogen compounds; (b) apoptosis and cell death; (c) fat cell differentiation and (d) negative regulation of protein kinase.

Physical Forces Modulate Oxidative Status and Stress Defense Meditated Metabolic Adaptation of Yeast Colonies: Spaceflight and Microgravity Simulations.

Baker's yeast (Saccharomyces cerevisiae) has broad genetic homology to human cells. Although typically grown as 1-2mm diameter colonies under certain conditions yeast can form very large (10 + mm in diameter) or 'giant' colonies on agar. Giant yeast colonies have been used to study diverse biomedical processes such as cell survival, aging, and the response to cancer pharmacogenomics. Such colonies evolve dynamically into complex stratified structures that respond differentially to environmental cues. Ammonia production, gravity driven ammonia convection, and shear defense responses are key differentiation signals for cell death and reactive oxygen system pathways in these colonies. The response to these signals can be modulated by experimental interventions such as agar composition, gene deletion and application of pharmaceuticals. In this study we used physical factors including colony rotation and microgravity to modify ammonia convection and shear stress as environmental cues and observed differences in the responses of both ammonia dependent and stress response dependent pathways We found that the effects of random positioning are distinct from rotation. Furthermore, both true and simulated microgravity exacerbated both cellular redox responses and apoptosis. These changes were largely shear-response dependent but each model had a unique response signature as measured by shear stress genes and the promoter set which regulates them These physical techniques permitted a graded manipulation of both convection and ammonia signaling and are primed to substantially contribute to our understanding of the mechanisms of drug action, cell aging, and colony differentiation.

Genes required for survival in microgravity revealed by genome-wide yeast deletion collections cultured during spaceflight.

Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses. However, the more subtle biological effects of spaceflight on cells and organisms are difficult to measure in a systematic, unbiased manner. Here we test the utility of the molecularly barcoded yeast deletion collection to provide a quantitative assessment of the effects of microgravity on a model organism. We developed robust hardware to screen, in parallel, the complete collection of ~4800 homozygous and ~5900 heterozygous (including ~1100 single-copy deletions of essential genes) yeast deletion strains, each carrying unique DNA that acts as strain identifiers. We compared strain fitness for the homozygous and heterozygous yeast deletion collections grown in spaceflight and ground, as well as plus and minus hyperosmolar sodium chloride, providing a second additive stressor. The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported. We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state, suggesting mechanisms by which spaceflight may negatively affect cell fitness.

Effects of microgravity on the virulence of Listeria monocytogenes, Enterococcus faecalis, Candida albicans, and methicillin-resistant Staphylococcus aureus.

To evaluate effects of microgravity on virulence, we studied the ability of four common clinical pathogens--Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, and Candida albicans--to kill wild type Caenorhabditis elegans (C. elegans) nematodes at the larval and adult stages. Simultaneous studies were performed utilizing spaceflight, clinorotation in a 2-D clinorotation device, and static ground controls. The feeding rate of worms for killed E. coli was unaffected by spaceflight or clinorotation. Nematodes, microbes, and growth media were separated until exposed to true or modeled microgravity, then mixed and grown for 48 h. Experiments were terminated by paraformaldehyde fixation, and optical density measurements were used to assay residual microorganisms. Spaceflight was associated with reduced virulence for Listeria, Enterococcus, MRSA, and Candida for both larval and adult C. elegans. These are the first data acquired with a direct in vivo assay system in space to demonstrate virulence. Clinorotation reproduced the effects of spaceflight in some, but not all, virulence assays: Candida and Enterococcus were less virulent for larval worms but not adult worms, whereas virulence of MRSA and Listeria were unaffected by clinorotation in tests with both adult and larval worms. We conclude that four common clinical microorganisms are all less virulent in space.

Optimism bias and parental views on unintentional injuries and safety: improving anticipatory guidance in early childhood.

The purpose of this integrative literature review is to improve anticipatory guidance in early childhood by reviewing the influence of optimism bias on parents' views about safety and beliefs about their children's risk for unintentional injuries. This article reviews the theory of optimism bias and recent research utilizing optimism bias to explain parental health-related behaviors. The three articles in this literature review find a link between optimism bias and parents' failure to implement safety behaviors. Currently, there is no tool to measure a parent's level of optimism bias concerning the risk of unintentional injury to his or her child. It is important for primary care providers to try and identify optimism bias in parents and address it as a barrier to implementation of safety recommendations. More research should be dedicated to developing screening tools to identify optimism bias in parents and interventions to help them accept their children's vulnerability.

Novel Sfp1 transcriptional regulation of Saccharomyces cerevisiae gene expression changes during spaceflight.

This study identifies transcriptional regulation of stress response element (STRE) genes in space in the model eukaryotic organism, Saccharomyces cerevisiae. To determine transcription-factor dependence, gene expression changes in space were examined in strains bearing green fluorescent protein-tagged (GFP-tagged) reporters for YIL052C (Sfp1 dependent with stress), YST-2 (Sfp1/Rap1 dependent with stress), or SSA4 (Msn4 dependent with stress), along with strains of SSA4-GFP and YIL052C-GFP with individual deletions of the Msn4 or Sfp1. When compared to parallel ground controls, spaceflight induces significant gene expression changes in SSA4 (35% decrease) and YIL052C (45% decrease), while expression of YST-2 (0.08% decrease) did not change. In space, deletion of Sfp1 reversed the SSA4 gene expression effect (0.00% change), but Msn4 deletion yielded a similar decrease in SSA4 expression (34% change), which indicates that SSA4 gene expression is dependent on the Sfp1 transcription factor in space, unlike other stresses. For YIL052C, deletion of Sfp1 reversed the effect (0.01% change), and the Msn4 deletion maintained the decrease in expression (30% change), which indicates that expression of YIL052C is also dependent on Sfp1 in space. Spaceflight has selective and specific effects on SSA4 and YIL052C gene expression, indicated by novel dependence on Sfp1.

Transcriptional regulation of changes in growth, cell cycle, and gene expression of Saccharomyces cerevisiae due to changes in buoyancy.

To understand the cellular effects of magnetic traps requires independent analysis of the effects of magnetic field, gravity, and buoyancy. In the current study, buoyancy is manipulated by addition of Ficoll, a viscous substance that can create gradients of buoyancy without significantly affecting osmolality. Specifically, we investigated whether Ficoll induces concentration dependent changes in cell growth, cell cycle, and gene expression in Saccharomyces cerevisiae, with special attention paid to the neutrally buoyant concentration of 35% Ficoll. Cell growth and cell cycle analysis were examined in three strains: wild-type (WT) yeast and strains with deletions in transcription factors Msn4 (Msn4Delta) or Sfp1 (Sfp1Delta). Changes in growth were observed in all three strains with WT and Msn4Delta strains showing strong concentration dependence. In addition, these changes in growth were supported by changes in the cell cycle of all three strains. Gene expression changes were observed in seven GFP-reporter strains including: SSA4, YIL052C, YST2, Msn4DeltaSSA4, Sfp1DeltaSSA4, Msn4DeltaYIL052C, and Sfp1DeltaYIL052C. Buoyancy forces had selective concentration dependent effects on gene expression of SSA4 and YIL052C with transcription factor dependence on Msn4. Additionally, SSA4 expression was dependent on Sfp1. YST2 gene expression was not dependent on changes in buoyancy force. This study shows that buoyancy has selective and concentration dependent effects on growth, cell cycle and gene expression, some of which are Msn4 and Sfp1 dependent. For the first time, SSA4 gene expression is shown to be dependent on Sfp1 and YIL052C gene expression is dependent on Msn4.

Diamagnetic levitation changes growth, cell cycle, and gene expression of Saccharomyces cerevisiae.

Inhomogeneous magnetic fields are used in magnetic traps to levitate biological specimens by exploiting the natural diamagnetism of virtually all materials. Using Saccharomyces cerevisiae, this report investigates whether magnetic field (B) induces changes in growth, cell cycle, and gene expression. Comparison to the effects of gravity and temperature allowed determination of whether the responses are general pathways or stimulus specific. Growth and cell cycle analysis were examined in wild-type (WT) yeast and strains with deletions in transcription factors Msn4 or Sfp1. Msn4, Sfp1, and Rap1 have been implicated in responses to physical forces, but only Msn4 and Sfp1 deletions are viable. Gene expression changes were examined in strains bearing GFP-tagged reporters for YIL052C (Sfp1-dependent), YST-2 (Sfp1/Rap1-dependent), or SSA4 (Msn4-dependent). The cell growth and gene expression responses were highly stimulus specific. B increased growth only following Msn4 or Sfp1 deletion, associated with decreased G1 and G2/M and increased S phase of the cell cycle. In addition, B suppressed expression of both YIL052C and YST2. Gravity decreased growth in an Sfp1 but not Msn4-dependent manner, in association with decreased G2/M and increased S phase of the cell cycle. Additionally, gravity decreased expression of SSA4 and YIL052C genes. Temperature increased cell growth in an Msn4- and Sfp1-dependent manner in association with increased G1 and G2/M with decreased S phase of the cell cycle. In addition, temperature increased YIL052C gene expression. This study shows that B has selective effects on cell growth, cell cycle, and gene expression that are stimulus specific.

Leaves of three, let them be: if it were only that easy!

Humans of all races and skin color are susceptible and uniquely sensitive to poison ivy, oak, and sumac. Contact with the plant oil, urushiol, found not only in the leaves but in the stems and roots, results in an allergic contact dermatitis in 50% to 60% of people. Clinical manifestations, differential diagnosis, complications, and treatments are discussed, with a special emphasis on the pediatric population.

Gene expression and survival changes in Saccharomyces cerevisiae during suspension culture.

This study explores the connection between changes in gene expression and the genes that determine strain survival during suspension culture, using the model eukaryotic organism, Saccharomyces cerevisiae. The Saccharomyces cerevisiae homozygous diploid deletion pool (HDDP), and the BY4743 parental strain were grown for 18 h in a rotating wall vessel (RWV), a suspension culture device optimized to minimize the delivered shear. In addition to the reduced shear conditions, the RWVs were also placed in a static position or in a shaker in order to change the amount of shear stress on the cells. Using simple linear regression, it was found that there were 140 differentially expressed genes for which >70% of the variation can be explained by shear stress alone. A significant number of these genes are involved in catalytic activity. In the HDDP, shear stress was associated with significant survival changes in 15 deletion strains (R(2>) > 0.7) Interestingly, both analyses uncovered changes in the ribosomal protein machinery. Comparing the changes in gene expression and strain survival under the different shear conditions allows for the insights into the molecular mechanisms behind the cells response to shear stress. This in turn can provide information for the optimization of suspension culture.

Megalin binds and internalizes angiotensin-(1-7).

Megalin is a multiligand receptor heavily involved in protein endocytosis. We recently demonstrated that megalin binds and mediates internalization of ANG II. Although there is a strong structural resemblance between ANG II and ANG-(1-7), their physiological actions and their affinity for the angiotensin type 1 receptor (AT(1)R) are dissimilar. Therefore, the hypothesis of the present work was to test whether megalin binds and internalizes ANG-(1-7). The uptake of ANG-(1-7) was determined by exposure of confluent monolayers of BN/MSV cells (a model representative of the yolk sac epithelium) to fluorescently labeled ANG-(1-7) (100 nM) and measurement of the amount of cell-associated fluorescence after 4 h by flow cytometry. Anti-megalin antisera and an AT(1)R blocker (olmesartan) were used to interfere with uptake via megalin and the AT(1)R, respectively. ANG-(1-7) uptake was prevented by anti-megalin antisera (63%) to a higher degree than olmesartan (13%) (P < 0.001). In analysis by flow cytometry of binding experiments performed in brush-border membrane vesicles isolated from kidneys of CD-1 mice, anti-megalin antisera interfered with ANG-(1-7) binding more strongly than olmesartan (P < 0.05 against positive control). Interactions of megalin with ANG-(1-7) at a molecular level were studied by surface plasmon resonance, demonstrating that ANG-(1-7) binds megalin dose and time dependently and with an affinity similar to ANG II. These results show that the scavenger receptor megalin binds and internalizes ANG-(1-7).

Homozygous diploid deletion strains of Saccharomyces cerevisiae that determine lag phase and dehydration tolerance.

This study identifies genes that determine length of lag phase, using the model eukaryotic organism, Saccharomyces cerevisiae. We report growth of a yeast deletion series following variations in the lag phase induced by variable storage times after drying-down yeast on filters. Using a homozygous diploid deletion pool, lag times ranging from 0 h to 90 h were associated with increased drop-out of mitochondrial genes and increased survival of nuclear genes. Simple linear regression (R2 analysis) shows that there are over 500 genes for which > 70% of the variation can be explained by lag alone. In the genes with a positive correlation, such that the gene abundance increases with lag and hence the deletion strain is suitable for survival during prolonged storage, there is a strong predominance of nucleonic genes. In the genes with a negative correlation, such that the gene abundance decreases with lag and hence the strain may be critical for getting yeast out of the lag phase, there is a strong predominance of glycoproteins and transmembrane proteins. This study identifies yeast deletion strains with survival advantage on prolonged storage and amplifies our understanding of the genes critical for getting out of the lag phase.

Shifts in relative tissue delta15N values in snowy egret nestlings with dietary mercury exposure: a marker for increased protein degradation.

Shifts in tissue nitrogen isotope composition may be a more sensitive general indicator of stress than measurement of high-turnover defensive biomolecules such as metallothionein and glutathione. As a physical resource transmitted along the trophic web, perturbations in protein nitrogen metabolism may also help resolve issues concerning the effects of contaminants on organisms and their consequential hierarchical linkages in ecotoxicology. Snowy egret nestlings (Egretta thula) fed mercury-contaminated diets of constant nitrogen isotope composition exhibited increased relative delta15N values in whole liver (p = 0.0011) and the acid-soluble fraction (ASF) of the liver (p = 0.0005) when compared to nestlings fed a reference diet. When nitrogen isotope data were adjusted for the source term of the diet, liver mercury concentrations corresponded with both whole liver relative 15N enrichment (r2 = 0.79, slope 0.009, p < 0.0001) and relative 15N enrichment in the acid-soluble fraction of the liver (r2 = 0.85, slope 0.026, p < 0.0001). Meanwhile, significant differences were not observed in hepatic levels of the metal-binding peptides metallothionein and glutathione despite a nearly 3-fold difference in liver mercury content. Because increases in tissue delta15N values result from increased rates of protein breakdown relative to synthesis, we propose that the increased relative liver delta15N values reflect a shift in protein metabolism. The relationship between ASF and mercury was significantly stronger (p < 0.0001) than that for whole liver, suggesting that the relationship is driven by an increase in bodily derived amino acids in the acid-soluble, free amino acid pool.

Megalin binds and internalizes angiotensin II.

Megalin is an abundant membrane protein heavily involved in receptor-mediated endocytosis. The major functions of megalin in vivo remain incompletely defined as megalin typically faces specialized milieus such as glomerular filtrate, airways, epididymal fluid, thyroid colloid, and yolk sac fluid, which lack many of its known ligands. In the course of studies on ANG II internalization, we were surprised when only part of the uptake of labeled ANG II into immortalized yolk sac cells (BN-16 cells) was blocked by specific peptide inhibitors and direct competitors of the angiotensin type 1 receptor. This led us to test if megalin was a receptor for ANG II. Four lines of direct evidence demonstrate that megalin and, to a lesser extent, its chaperone protein cubilin are receptors for ANG II. First, in BN-16 cells anti-megalin and anti-cubilin antisera interfere with ANG II uptake. Second, also in BN-16 cells, pure ANG II competes for uptake of a known megalin ligand. Third, in proximal tubule cell brush-border membrane vesicles extracted from mice, anti-megalin antisera interfere with ANG II binding. Fourth, purified megalin binds ANG II directly in surface plasmon resonance experiments. The finding that megalin is a receptor for ANG II suggests a major new function for the megalin pathway in vivo. These results also indicate that ANG II internalization in some tissues is megalin dependent and that megalin may play a role in regulating proximal tubule ANG II levels.

Light chains are a ligand for megalin.

Cubilin and megalin are giant glycoprotein receptors abundant on the luminal surface of proximal tubular cells of the kidney. We showed previously that light chains are a ligand for cubilin. As cubilin and megalin share a number of common ligands, we further investigated the ligand specificity of these receptors. Three lines of evidence suggest that light chains can also bind megalin: 1) anti-megalin antiserum largely displaces brush-border light chain binding and megalin-expressing BN-16 cell uptake more than anti-cubilin antiserum, 2) direct binding studies on isolated proteins using surface plasmon resonance techniques confirm that megalin binds light chains, and 3) light chains compete with known megalin ligands for brush-border membrane binding and BN-16 cell uptake. The megalin-light chain interaction is divalent ion dependent and similar for both kappa- and lambda-light chains. A fit of the data on light chain binding to megalin over a concentration range 0.078-2.5 mg/ml leads to an estimated dissociation constant of 6 x 10(-5) M, corresponding approximately to one light chain-binding site per megalin and in the same range for dissociation constants for cubilin binding. These data suggest that light chains bind the tandem megalin-cubilin complex. Megalin is the major mediator of light chain entry into megalin-expressing membrane such as the apical surface of proximal tubular epithelial cells.