Hexamers of the Type II Secretion ATPase GspE from Vibrio cholerae with Increased ATPase Activity

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Determination of hyaluronan molecular mass distribution in human breast milk

Hyaluronan (HA) in human milk mediates host responses to microbial infection via TLR4- and CD44-dependent signaling. Signaling by HA is generally size specific. Because pure HA with average molecular mass (M) of 35 kDa can elicit a protective response in intestinal epithelial cells, it has been proposed that human milk HA may have a bioactive low-M component. Here we report the size distribution of HA in human milk samples from 20 unique donors. A new method for HA analysis, employing ion exchange (IEX) chromatography to fractionate HA by size and specific quantification of each size fraction by competitive enzyme-linked sorbent assay (ELSA), was developed. When separated into four fractions, milk HA with M ? 20 kDa, M ∼ 20 to 60 kDa, and M ∼ 60 to 110 kDa comprised averages of 1.5, 1.4, and 2.0% of the total HA, respectively. The remaining 95% was HA with M ? 110 kDa. Electrophoretic analysis of the higher M HA from 13 samples showed nearly identical M distributions, with an average M of approximately 440 kDa. This higher M HA component in human milk is proposed to bind to CD44 and to enhance human beta defensin 2 (HBD2) induction by the low-M HA components.     

Agarose and polyacrylamide gel electrophoresis methods for molecular mass analysis of 5- to 500-kDa hyaluronan

Agarose and polyacrylamide gel electrophoresis systems for the molecular mass-dependent separation of hyaluronan (HA) in the size range of approximately 5–500 kDa were investigated. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffer systems was determined. Using chemoenzymatically synthesized HA standards of low polydispersity, the molecular mass range was determined for each gel composition over which the relationship between HA mobility and logarithm of the molecular mass was linear. Excellent linear calibration was obtained for HA molecular mass as low as approximately 9 kDa in agarose gels. For higher resolution separation, and for extension to molecular masses as low as approximately 5 kDa, gradient polyacrylamide gels were superior. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in a sample as well as calculation of weight-average and number-average values. The methods were validated for polydisperse HA samples with viscosity-average molecular masses of 112, 59, 37, and 22 kDa at sample loads of 0.5 μg (for polyacrylamide) to 2.5 μg (for agarose). Use of the methods for electrophoretic mobility shift assays was demonstrated for binding of the HA-binding region of aggrecan (recombinant human aggrecan G1–IGD–G2 domains) to a 150-kDa HA standard.     

Increased FGF-2 secretion and ability to support neurite outgrowth by astrocytes cultured on polyamide nanofibrillar matrices

An electrospun nonwoven matrix of polyamide nanofibers was employed as a new model for the capillary basement membrane at the blood-brain barrier (BBB). The basement membrane separates astrocytes from endothelial cells and is associated with growth factors, such as fibroblast growth factor-2 (FGF-2). FGF-2 is produced by astrocytes and induces specialized functions in endothelial cells, but also has actions on astrocytes. To investigate potential autocrine actions of FGF-2 at the BBB, astrocytes were cultured on unmodified nanofibers or nanofibers covalently modified with FGF-2. The former assumed an in vivo-like stellate morphology that was enhanced in the presence of cross-linked FGF-2. Furthermore, astrocyte monolayers established on unmodified nanofibers were more permissive for neurite outgrowth when cultured with an overlay of neurons than similar monolayers established on standard tissue culture surfaces, while astrocytes cultured on FGF-2-modifed nanofibers were yet more permissive. The observed differences were due in part to progressively increasing amounts of FGF-2 secreted by the astrocytes into the medium; hence FGF-2 increases its own expression in astrocytes to modulate astrocyte–neuron interactions. Soluble FGF-2 was unable to replicate the effects of cross-linked FGF-2. Nanofibers alone up-regulated FGF-2, albeit to a lesser extent than nanofibers covalently modified with FGF-2. These results underscore the importance of both surface topography and growth factor presentation on cellular function. Moreover, these results indicate that FGF-2-modified nanofibrillar scaffolds may demonstrate utility in tissue engineering applications for replacement and regeneration of lost tissue following central nervous system (CNS) injury or disease.     

VPM tokens: virtual machine-aware power budgeting in datacenters

Power consumption and cooling overheads are becoming increasingly significant for enterprise datacenters, affecting overall costs and the ability to extend resource capacities. To help mitigate these issues, active power management technologies are being deployed aggressively, including power budgeting, which enables improved power provisioning and can address critical periods when power delivery or cooling capabilities are temporarily reduced. Given the use of virtualization to encapsulate application components into virtual machines (VMs), however, such power management capabilities must address the interplay between budgeting physical resources and the performance of the virtual machines used to run these applications. This paper proposes a set of management components and abstractions for use by software power budgeting policies. The key idea is to manage power from a VM-centric point of view, where the goal is to be aware of global utility tradeoffs between different virtual machines (and their applications) when maintaining power constraints for the physical hardware on which they run. Our approach to VM-aware power budgeting uses multiple distributed managers integrated into the VirtualPower Management (VPM) framework whose actions are coordinated via a new abstraction, termed VPM tokens. An implementation with the Xen hypervisor illustrates technical benefits of VPM tokens that include up to 43% improvements in global utility, highlighting the ability to dynamically improve cluster performance while still meeting power budgets. We also demonstrate how VirtualPower based budgeting technologies can be leveraged to improve datacenter efficiency in the context of cooling infrastructure management.

Drosophila Follistatin exhibits unique structural modifications and interacts with several TGF-beta family members

Follistatin (FS) is one of several secreted proteins that modulate the activity of TGF-beta family members during development. The structural and functional analysis of Drosophila Follistatin (dFS) reveals important differences between dFS and its vertebrate orthologues: it is larger, more positively charged, and proteolytically processed. dFS primarily inhibits signaling of Drosophila Activin (dACT) but can also inhibit other ligands like Decapentaplegic (DPP). In contrast, the presence of dFS enhances signaling of the Activin-like protein Dawdle (DAW), indicating that dFS exhibits a dual function in promoting and inhibiting signaling of TGF-beta ligands. In addition, FS proteins may also function in facilitating ligand diffusion. We find that mutants of daw are rescued in significant numbers by expression of vertebrate FS proteins. Since two PiggyBac insertions in dfs are not lethal, it appears that the function of dFS is non-essential or functionally redundant.     

Mice lacking Homer 1 exhibit a skeletal myopathy characterized by abnormal transient receptor potential channel activity

Transient receptor potential (TRP) channels are nonselective cation channels, several of which are expressed in striated muscle. Because the scaffolding protein Homer 1 has been implicated in TRP channel regulation, we hypothesized that Homer proteins play a significant role in skeletal muscle function. Mice lacking Homer 1 exhibited a myopathy characterized by decreased muscle fiber cross-sectional area and decreased skeletal muscle force generation. Homer 1 knockout myotubes displayed increased basal current density and spontaneous cation influx. This spontaneous cation influx in Homer 1 knockout myotubes was blocked by reexpression of Homer 1b, but not Homer 1a, and by gene silencing of TRPC1. Moreover, diminished Homer 1 expression in mouse models of Duchenne's muscular dystrophy suggests that loss of Homer 1 scaffolding of TRP channels may contribute to the increased stretch-activated channel activity observed in mdx myofibers. These findings provide direct evidence that Homer 1 functions as an important scaffold for TRP channels and regulates mechanotransduction in skeletal muscle.     

Human Milk Hyaluronan Enhances Innate Defense of the Intestinal Epithelium

Breast-feeding is associated with enhanced protection from gastrointestinal disease in infants, mediated in part by an array of bioactive glycan components in milk that act through molecular mechanisms to inhibit enteric pathogen infection. Human milk contains hyaluronan (HA), a glycosaminoglycan polymer found in virtually all mammalian tissues. We have shown that synthetic HA of a specific size range promotes expression of antimicrobial peptides in intestinal epithelium. We hypothesize that hyaluronan from human milk also enhances innate antimicrobial defense. Here we define the concentration of HA in human milk during the first 6 months postpartum. Importantly, HA isolated from milk has a biological function. Treatment of HT-29 colonic epithelial cells with human milk HA at physiologic concentrations results in time- and dose-dependent induction of the antimicrobial peptide human β-defensin 2 and is abrogated by digestion of milk HA with a specific hyaluronidase. Milk HA induction of human β-defensin 2 expression is also reduced in the presence of a CD44-blocking antibody and is associated with a specific increase in ERK1/2 phosphorylation, suggesting a role for the HA receptor CD44. Furthermore, oral administration of human milk-derived HA to adult, wild-type mice results in induction of the murine Hβ D2 ortholog in intestinal mucosa and is dependent upon both TLR4 and CD44 in vivo. Finally, treatment of cultured colonic epithelial cells with human milk HA enhances resistance to infection by the enteric pathogen Salmonella typhimurium. Together, our observations suggest that maternally provided HA stimulates protective antimicrobial defense in the newborn.     

Genetic determinants of QT interval variation and sudden cardiac death

Electrocardiographic QT interval prolongation or shortening is a risk factor for sudden cardiac death. The study of Mendelian syndromes in families with extreme long and short QT interval duration and ventricular arrhythmias has led to the identification of genes encoding ion channel proteins important in myocardial repolarization. Rare mutations in such ion channel genes do not individually contribute substantially to the population burden of ventricular arrhythmias and sudden cardiac death. Only now are studies systematically testing the relationship between common variants in these genes--or elsewhere in the genome--and QT interval variation and sudden cardiac death. Identification of genetic variation underlying myocardial repolarization could have important implications for the prevention of both sporadic and drug-induced arrhythmias.