Osteoblastic phenotype expression of MC3T3-E1 cultured on electrospun polycaprolactone fiber mats filled with hydroxyapatite nanoparticles

Electrospun (e-spun) fiber mats of polycaprolactone (PCL; Mn = 80 000 g mol-1) with or without the presence of hydroxyapatite (HAp) nanoparticles (at 1% w/v based on the volume of the PCL solution) were successfully fabricated. The potential for use of these e-spun fiber mats as bone scaffolds was assessed by mouse calvaria-derived pre-osteoblastic cells, MC3T3-E1, in terms of attachment, proliferation, differentiation, and mineralization. Despite the lower number of cells attached at early time points, both the fibrous scaffolds supported the proliferation of MC3T3-E1 at similar levels to tissue-culture polystyrene plate (TCPS), with the cells growing on the PCL/HAp fiber mat (i.e., PCL/HAp-FS) showing the greatest proliferation rate on day 3 after the initial attachment period of 16 h. Alkaline phosphatase (ALP) activity of the cells grown on TCPS was the greatest on day 3 after cell culturing, while that of the cells grown on PCL/HAp-FS reached a maximum on day 5. On the other hand, the ALP activity of the cells grown on the neat PCL fiber mat (i.e., PCL-FS) was the lowest at any given time point. MC3T3-E1 cultured on the surface of PCL/HAp-FS expressed the greatest amount of osteocalcin (OC) gene on day 14 after cell culturing and OC protein on day 21 after cell culturing, respectively, when compared with those cultured on the surfaces of PCL-FS and TCPS. This corresponded to the greatest extent of mineralization for the cells grown on the surface of PCL/HAp-FS on day 21, followed by that for the cells grown on PCL-FS and TCPS, respectively.     

Global and comparative proteomic profiling of overwintering and developing mountain pine beetle,Dendroctonus ponderosae (Coleoptera: Curculionidae), larvae

BackgroundMountain pine beetles, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae), are native to western North America, but have recently begun to expand their range across the Canadian Rocky Mountains. The requirement for larvae to withstand extremely cold winter temperatures and potentially toxic host secondary metabolites in the midst of their ongoing development makes this a critical period of their lives.ResultsWe have uncovered global protein profiles for overwintering mountain pine beetle larvae. We have also quantitatively compared the proteomes for overwintering larvae sampled during autumn cooling and spring warming using iTRAQ methods. We identified 1507 unique proteins across all samples. In total, 33 proteins exhibited differential expression (FDR < 0.05) when compared between larvae before and after a cold snap in the autumn; and 473 proteins exhibited differential expression in the spring when measured before and after a steady incline in mean daily temperature. Eighteen proteins showed significant changes in both autumn and spring samples.ConclusionsThese first proteomic data for mountain pine beetle larvae show evidence of the involvement of trehalose, 2-deoxyglucose, and antioxidant enzymes in overwintering physiology; confirm and expand upon previous work implicating glycerol in cold tolerance in this insect; and provide new, detailed information on developmental processes in beetles. These results and associated data will be an invaluable resource for future targeted research on cold tolerance mechanisms in the mountain pine beetle and developmental biology in coleopterans.     

Dose-dependent and isoform-specific modulation of Ca2+ channels by RGK GTPases

Although inhibition of voltage-gated calcium channels by RGK GTPases (RGKs) represents an important mode of regulation to control Ca(2+) influx in excitable cells, their exact mechanism of inhibition remains controversial. This has prevented an understanding of how RGK regulation can be significant in a physiological context. Here we show that RGKs-Gem, Rem, and Rem2-decreased Ca(V)1.2 Ca(2+) current amplitude in a dose-dependent manner. Moreover, Rem2, but not Rem or Gem, produced dose-dependent alterations on gating kinetics, uncovering a new mode by which certain RGKs can precisely modulate Ca(2+) currents and affect Ca(2+) influx during action potentials. To explore how RGKs influence gating kinetics, we separated the roles mediated by the Ca(2+) channel accessory beta subunit's interaction with its high affinity binding site in the pore-forming alpha(1C) subunit (AID) from its other putative contact sites by utilizing an alpha(1C)*beta3 concatemer in which the AID was mutated to prevent beta subunit interaction. This mutant concatemer generated currents with all the hallmarks of beta subunit modulation, demonstrating that AID-beta-independent interactions are sufficient for beta subunit modulation. Using this construct we found that although inhibition of current amplitude was still partially sensitive to RGKs, Rem2 no longer altered gating kinetics, implicating different determinants for this specific mode of Rem2-mediated regulation. Together, these results offer new insights into the molecular mechanism of RGK-mediated Ca(2+) channel current modulation.     

Proteomic methods applied to the analysis of immobilized biocatalysts

Methods adapted from proteomics can directly characterize proteins present in immobilized biocatalysts. Complete hydrolysis followed by HPLC analysis of Tyr and Phe estimates total protein bound, and is preferable to conventional difference methods, as tested with subtilisin Carlsberg on silica. This new method shows that various treatments give quantitative desorption of proteins immobilized by adsorption. Intact desorbed proteins may be analyzed by electrospray mass spectrometry. The Candida antarctica lipase B from Novozyme 435 was shown to be heavily glycosylated, while the lipase from Lipozyme RM IM was a mixture of four N-terminally truncated forms. Peptides from selective cleavage were analyzed by tandem mass spectrometry, leading to automatic identification of proteins present. A second major protein present in Lipozyme RM IM was thus found to be alpha-amylase from Aspergillus oryzae. These methods should be valuable complements to activity measurements in understanding immobilized enzyme activity and stability.     

The C-terminus of Raf-1 acts as a 14-3-3-dependent activation switch

The Raf-1 protein kinase is a major activator of the ERK MAPK pathway, which links signaling by a variety of cell surface receptors to the regulation of cell proliferation, survival, differentiation and migration. Signaling by Raf-1 is regulated by a complex and poorly understood interplay between phosphorylation events and protein–protein interactions. One important mode of Raf-1 regulation involves the phosphorylation-dependent binding of 14-3-3 proteins. Here, we have examined the mechanism whereby the C-terminal 14-3-3 binding site of Raf-1, S621, controls the activation of MEK-ERK signaling. We show that phosphorylation of S621 turns over rapidly and is enriched in the activated pool of endogenous Raf-1. The phosphorylation on this site can be mediated by Raf-1 itself but also by other kinase(s). Mutations that prevent the binding of 14-3-3 proteins to S621 render Raf-1 inactive by specifically disrupting its capacity to bind to ATP, and not by gross conformational alteration as indicated by intact MEK binding. Phosphorylation of S621 correlates with the inhibition of Raf-1 catalytic activity in vitro, but 14-3-3 proteins can completely reverse this inhibition. Our findings suggest that 14-3-3 proteins function as critical cofactors in Raf-1 activation, which induce and maintain the protein in a state that is competent for both ATP binding and MEK phosphorylation.     

Pseudomonas aeruginosa Population Structure Revisited

At present there are strong indications that Pseudomonas aeruginosa exhibits an epidemic population structure; clinical isolates are indistinguishable from environmental isolates, and they do not exhibit a specific (disease) habitat selection. However, some important issues, such as the worldwide emergence of highly transmissible P. aeruginosa clones among cystic fibrosis (CF) patients and the spread and persistence of multidrug resistant (MDR) strains in hospital wards with high antibiotic pressure, remain contentious. To further investigate the population structure of P. aeruginosa, eight parameters were analyzed and combined for 328 unrelated isolates, collected over the last 125 years from 69 localities in 30 countries on five continents, from diverse clinical (human and animal) and environmental habitats. The analysed parameters were: i) O serotype, ii) Fluorescent Amplified-Fragment Length Polymorphism (FALFP) pattern, nucleotide sequences of outer membrane protein genes, iii) oprI, iv) oprL, v) oprD, vi) pyoverdine receptor gene profile (fpvA type and fpvB prevalence), and prevalence of vii) exoenzyme genes exoS and exoU and viii) group I pilin glycosyltransferase gene tfpO. These traits were combined and analysed using biological data analysis software and visualized in the form of a minimum spanning tree (MST). We revealed a network of relationships between all analyzed parameters and non-congruence between experiments. At the same time we observed several conserved clones, characterized by an almost identical data set. These observations confirm the nonclonal epidemic population structure of P. aeruginosa, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise. One of these clones is the renown and widespread MDR serotype O12 clone. On the other hand, we found no evidence for a widespread CF transmissible clone. All but one of the 43 analysed CF strains belonged to a ubiquitous P. aeruginosa “core lineage” and typically exhibited the exoS ⁺/exoU ⁻ genotype and group B oprL and oprD alleles. This is to our knowledge the first report of an MST analysis conducted on a polyphasic data set.     

A biohybrid artificial lung prototype with active mixing of endothelialized microporous hollow fibers

Acute respiratory distress syndrome (ARDS) affects nearly 150,000 patients per year in the US, and is associated with high mortality (≈40%) and suboptimal options for patient care. Mechanical ventilation and extracorporeal membrane oxygenation are limited to short‐term use due to ventilator‐induced lung injury and poor biocompatibility, respectively. In this report, we describe the development of a biohybrid lung prototype, employing a rotating endothelialized microporous hollow fiber (MHF) bundle to improve blood biocompatibility while MHF mixing could contribute to gas transfer efficiency. MHFs were surface modified with radio frequency glow discharge (RFGD) and protein adsorption to promote endothelial cell (EC) attachment and growth. The MHF bundles were placed in the biohybrid lung prototype and rotated up to 1,500 revolutions per minute (rpm) using speed ramping protocols to condition ECs to remain adherent on the fibers. Oxygen transfer, thrombotic deposition, and EC p‐selectin expression were evaluated as indicators of biohybrid lung functionality and biocompatibility. A fixed aliquot of blood in contact with MHF bundles rotated at either 250 or 750 rpm reached saturating pO₂ levels more quickly with increased rpm, supporting the concept that fiber rotation would positively contribute to oxygen transfer. The presence of ECs had no effect on the rate of oxygen transfer at lower fiber rpm, but did provide some resistance with increased rpm when the overall rate of mass transfer was higher due to active mixing. RFGD followed by fibronectin adsorption on MHFs facilitated near confluent EC coverage with minimal p‐selectin expression under both normoxic and hyperoxic conditions. Indeed, even subconfluent EC coverage on MHFs significantly reduced thrombotic deposition adding further support that endothelialization enhances, blood biocompatibility. Overall these findings demonstrate a proof‐of‐concept that a rotating endothelialized MHF bundle enhances gas transfer and biocompatibility, potentially producing safer, more efficient artificial lungs. Biotechnol. Bioeng. 2010; 106: 490–500. © 2010 Wiley Periodicals, Inc.     

Differential risk effects of wolves on wild versus domestic prey have consequences for conservation

Predators play integral roles in shaping ecosystems through cascading effects to prey and vegetation. Such effects occur when prey species alter their behavior to avoid predators, a phenomenon called the risk effects of predators. Risk effects of wild predators such as wolves are well documented for wild prey, but not for free ranging domestic animals such as cattle despite their importance for ecosystem function and conservation. We compared risk effects of satellite‐collared wolves (n = 16) on habitat selection by global‐positioning‐system‐collared elk (n = 10) and cattle (n = 31). We calculated resource selection functions (RSFs) in periods before, during and after wolf visits in elk home ranges or cattle pastures. The habitat variables tested included: distance to roads and trails, terrain ruggedness, food‐quality and distance to forest. When wolves were present, elk stayed closer to forest cover and selected less for high‐quality‐food habitat. Thus, the risk effects of wolf presence on elk produced a change in the tradeoff between food and cover selection. Cattle responded by avoiding high‐quality‐food habitat and selecting areas closer to roads and trails (where people likely provided security), but these effects manifested only after wolves had left. Artificial selection in cattle may have attenuated natural anti‐predator behaviors. The effects of predators on ecosystems are likely different when mediated through risk effects on domestic compared to wild animals. Furthermore, predator control in response to livestock predation, an important conservation issue, may produce broad ecosystem effects triggered by decrease of an important predator species. Conservation planners should consider these effects where domestic herbivores are dominant species in the ecosystem.     

Filter-well technology for advanced three-dimensional cell culture: perspectives for respiratory research

Cell culture has long been a valuable tool for studying cell behaviour. Classical plastic substrates are two-dimensional, and usually promote cellular proliferation and inhibit differentiation. Understanding cell behaviour within complex multicellular tissues requires the systematic study of cells within the context of specific model microenvironments. A model system must mimic, to a certain degree, the in vivo situation, but, at the same time, can significantly reduce its complexity. There is increasing agreement that moving up to the third dimension provides a more physiologically-relevant and predictive model system. Moreover, many cellular processes (morphogenesis, organogenesis and pathogenesis) have been confirmed to occur exclusively when cells are ordered in a three-dimensional (3-D) manner. In order to achieve the desired in vivo phenotype, researchers can use microporous membranes for improved in vitro cell culture experiments. In the present review, we discuss the applications of filter-well technology for the advanced 3-D cell culture of human pulmonary cells.