Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented

Hierarchical B-spline complexes of discrete differential forms

In this paper, we introduce the hierarchical B-spline complex of discrete differential forms for arbitrary spatial dimension. This complex may be applied to the adaptive isogeometric solution of problems arising in electromagnetics and uid mechanics. We derive a sufficient and necessary condition guaranteeing exactness of the hierarchical B-spline complex for arbitrary spatial dimension, and we derive a set of local, easy-to-compute, and sufficient exactness conditions for the two-dimensional setting. We examine the stability properties of the hierarchical B-spline complex, and we find that it yields stable approximations of both the Maxwell eigenproblem and Stokes problem provided that the local exactness conditions are satisfied. We conclude by providing numerical results showing the promise of the hierarchical B-spline complex in an adaptive isogeometric solution framework

CMBPol Mission Concept Study: Foreground Science Knowledge and Prospects

We report on our knowledge of Galactic foregrounds, as well as on how a CMB satellite mission aiming at detecting a primordial B-mode signal (CMBPol) will contribute to improving it. We review the observational and analysis techniques used to constrain the structure of the Galactic magnetic field, whose presence is responsible for the polarization of Galactic emissions. Although our current understanding of the magnetized interstellar medium is somewhat limited, dramatic improvements in our knowledge of its properties are expected by the time CMBPol flies. Thanks to high resolution and high sensitivity instruments observing the whole sky at frequencies between 30 GHz and 850 GHz, CMBPol will not only improve this picture by observing the synchrotron emission from our galaxy, but also help constrain dust models. Polarized emission from interstellar dust indeed dominates over any other signal in CMBPol's highest frequency channels. Observations at these wavelengths, combined with ground-based studies of starlight polarization, will therefore enable us to improve our understanding of dust properties and of the mechanism(s) responsible for the alignment of dust grains with the Galactic magnetic field. CMBPol will also shed new light on observations that are presently not well understood. Morphological studies of anomalous dust and synchrotron emissions will indeed constrain their natures and properties, while searching for fluctuations in the emission from heliospheric dust will test our understanding of the circumheliospheric interstellar medium. Finally, acquiring more information on the properties of extra-Galactic sources will be necessary in order to maximize the cosmological constraints extracted from CMBPol's observations of CMB lensing. (abridged)Comment: 43 pages, 7 figures, 2 table

CMBPol Mission Concept Study: Prospects for polarized foreground removal

In this report we discuss the impact of polarized foregrounds on a future CMBPol satellite mission. We review our current knowledge of Galactic polarized emission at microwave frequencies, including synchrotron and thermal dust emission. We use existing data and our understanding of the physical behavior of the sources of foreground emission to generate sky templates, and start to assess how well primordial gravitational wave signals can be separated from foreground contaminants for a CMBPol mission. At the estimated foreground minimum of ~100 GHz, the polarized foregrounds are expected to be lower than a primordial polarization signal with tensor-to-scalar ratio r=0.01, in a small patch (~1%) of the sky known to have low Galactic emission. Over 75% of the sky we expect the foreground amplitude to exceed the primordial signal by about a factor of eight at the foreground minimum and on scales of two degrees. Only on the largest scales does the polarized foreground amplitude exceed the primordial signal by a larger factor of about 20. The prospects for detecting an r=0.01 signal including degree-scale measurements appear promising, with 5 sigma_r ~0.003 forecast from multiple methods. A mission that observes a range of scales offers better prospects from the foregrounds perspective than one targeting only the lowest few multipoles. We begin to explore how optimizing the composition of frequency channels in the focal plane can maximize our ability to perform component separation, with a range of typically 40 < nu < 300 GHz preferred for ten channels. Foreground cleaning methods are already in place to tackle a CMBPol mission data set, and further investigation of the optimization and detectability of the primordial signal will be useful for mission design.Comment: 42 pages, 14 figures, Foreground Removal Working Group contribution to the CMBPol Mission Concept Study, v2, matches AIP versio

Hemodialysis acutely improves hepatic CYP3A4 metabolic activity

The uremic syndrome remains poorly understood despite the widespread availability of dialysis for almost four decades. To date, assessment of the biologic activity of uremic toxins has focused primarily on in vitro effects, rather than on specific biochemical pathways or enzymatic activity in vivo. The activity of cytochrome P450 (CYP) 3A4, the most important enzyme in human drug metabolism, is decreased in uremia. The purpose of this study was to assess the effect of hemodialysis and hence varying concentrations of uremic toxins on CYP3A4 activity using the 14 C-erythromycin breath test and the traditional phenotypic trait measure, 20-min 14 CO 2 flux. CYP3A4 activity increased by 27% postdialysis (P ‫؍‬ 0.002 compared with predialysis) and was significantly inversely related to plasma blood urea nitrogen concentration (r s ‫؍‬ ؊0.50, P ‫؍‬ 0.012), but not to several middle molecules. This is the first study in humans characterizing uremia as a state in which hepatic CYP3A4 activity is acutely improved by hemodialysis. J Am Soc Nephrol 17: 2363 -2367 The regulation of CYP3A4 in ESRD patients undergoing hemodialysis has not been well studied. Alterations in CYP3A4 expression and/or activity have been observed in experimental models of uremia (2,3,8 to 13) and a recent report using the 14 C-erythromycin breath test demonstrates that CYP3A4 activity is reduced in ESRD patients compared to healthy subjects (14). Although restoration of kidney function after transplantation leads to a sustained improvement in the uremic state and in hepatic drug metabolism (2,15), hemodialysis therapy only temporarily improves uremia and does not appear to generate long-term improvements in CYP3A function (2). However, the acute effect of hemodialysis on CYP3A4 activity in vivo has not been studied to date. We hypothesized that hepatic CYP3A4 activity would be inversely related to the level of uremic toxins, and that removal of uremic toxins via hemodialysis would lead to acute changes in CYP3A4 activity. Thus, the purpose of this study was to assess the effect of conventional hemodialysis on hepatic CYP3A4 metabolic activity in ESRD patients using the erythromycin breath test and the phenotypic trait measure 20-min 14 CO 2 flux, and to evaluate the relationship between CYP3A4 activity and the concentrations of several uremic toxins. Materials and Methods Study Subjects Twelve patients with ESRD and undergoing chronic hemodialysis participated in this study after providing written informed consent. All subjects underwent a screening evaluation that was based on a complete medical history, physical examination, medication history, and conventional biochemical tests. Eligibility criteria included normal hepatic function, body weight within 40% of ideal weight for height, body frame size, and sex according to the 1983 Metropolitan Life Insurance Company weight tables (16), documented compliance with dialysis prescriptions as determined by a Kt/V Ն1.20 within the 28-d period before the study day, and a negative pregnancy test for women of child-bearing potential. Subjects taking drugs known to inhibit or induce CYP3A4 or with a known sensitivity or previous adverse reaction to erythromycin were excluded. All participants were instructed to abstain from grapefruit products and herbal supplements/teas for at least 72 h before and during the study day

Accelerating tropicalization and the transformation of temperate seagrass meadows

Climate-driven changes are altering production and functioning of biotic assemblages in terrestrial and aquatic environments. In temperate coastal waters, rising sea temperatures, warm water anomalies and poleward shifts in the distribution of tropical herbivores have had a detrimental effect on algal forests. We develop generalized scenarios of this form of tropicalization and its potential effects on the structure and functioning of globally significant and threatened seagrass ecosystems, through poleward shifts in tropical seagrasses and herbivores. Initially, we expect tropical herbivorous fishes to establish in temperate seagrass meadows, followed later by megafauna. Tropical seagrasses are likely to establish later, delayed by more limited dispersal abilities. Ultimately, food webs are likely to shift from primarily seagrass-detritus to more directconsumption- based systems, thereby affecting a range of important ecosystem services that seagrasses provide, including their nursery habitat role for fishery species, carbon sequestration, and the provision of organic matter to other ecosystems in temperate regions

Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant

To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function

Association between canine leishmaniosis and Ehrlichia canis co-infection: a prospective case-control study

Abstract Background In the Mediterranean basin, Leishmania infantum is a major cause of disease in dogs, which are frequently co-infected with other vector-borne pathogens (VBP). However, the associations between dogs with clinical leishmaniosis (ClinL) and VBP co-infections have not been studied. We assessed the risk of VBP infections in dogs with ClinL and healthy controls. Methods We conducted a prospective case-control study of dogs with ClinL (positive qPCR and ELISA antibody for L. infantum on peripheral blood) and clinically healthy, ideally breed-, sex- and age-matched, control dogs (negative qPCR and ELISA antibody for L. infantum on peripheral blood) from Paphos, Cyprus. We obtained demographic data and all dogs underwent PCR on EDTA-blood extracted DNA for haemoplasma species, Ehrlichia/Anaplasma spp., Babesia spp., and Hepatozoon spp., with DNA sequencing to identify infecting species. We used logistic regression analysis and structural equation modelling (SEM) to evaluate the risk of VBP infections between ClinL cases and controls. Results From the 50 enrolled dogs with ClinL, DNA was detected in 24 (48%) for Hepatozoon spp., 14 (28%) for Mycoplasma haemocanis, 6 (12%) for Ehrlichia canis and 2 (4%) for Anaplasma platys. In the 92 enrolled control dogs, DNA was detected in 41 (45%) for Hepatozoon spp., 18 (20%) for M. haemocanis, 1 (1%) for E. canis and 3 (3%) for A. platys. No Babesia spp. or “Candidatus Mycoplasma haematoparvum” DNA was detected in any dog. No statistical differences were found between the ClinL and controls regarding age, sex, breed, lifestyle and use of ectoparasitic prevention. A significant association between ClinL and E. canis infection (OR = 12.4, 95% CI: 1.5–106.0, P = 0.022) was found compared to controls by multivariate logistic regression. This association was confirmed using SEM, which further identified that younger dogs were more likely to be infected with each of Hepatozoon spp. and M. haemocanis, and dogs with Hepatozoon spp. were more likely to be co-infected with M. haemocanis. Conclusions Dogs with ClinL are at a higher risk of co-infection with E. canis than clinically healthy dogs. We recommend that dogs diagnosed with ClinL should be tested for E. canis co-infection using PCR