Characterization of Aquaporin 4 Protein Expression and Localization in Tissues of the Dogfish (\u3cem\u3eSqualus acanthias\u3c/em\u3e)

The role of aquaporin water channels such as aquaporin 4 (Aqp4) in elasmobranchs such as the dogfish Squalus acanthias is completely unknown. This investigation set out to determine the expression and cellular and sub-cellular localization of Aqp4 protein in dogfish tissues. Two polyclonal antibodies were generated (AQP4/1 and AQP4/2) and these showed somewhat different characteristics in Western blotting and immunohistochemistry. Western blots using the AQP4/1 antibody showed two bands (35.5 and 49.5 kDa) in most tissues in a similar fashion to mammals. Liver had an additional band of 57 kDa and rectal gland two further faint bands of 37.5 and 38.5 kDa. However, unlike in mammals, Aqp4 protein was ubiquitously expressed in all tissues including gill and liver. The AQP4/2 antibody appeared much less specific in Western blots. Both antibodies were used in immunohistochemistry and showed similar cellular localizations, although the AQP4/2 antibody had a more restricted sub-cellular distribution compared to AQP4/1 and therefore appeared to be more specific for Aqp4. In kidney a sub-set of tubules were stained which may represent intermediate tubule segments (In-III–In-VI). AQP4/1 and AQP4/2 antibodies localized to the same tubules segments in serial sections although the intensity and sub-cellular distribution were different. AQP4/2 showed a basal or basolateral membrane distribution whereas AQP4/1 was often distributed throughout the whole cell including the nuclear region. In rectal gland and cardiac stomach Aqp4 was localized to secretory tubules but again AQP/1 and AQP/2 exhibited different sub-cellular distributions. In gill, both antibodies stained large cells in the primary filament and secondary lamellae. Again AQP4/1 antibody stained most or all the cell including the nucleus, whereas AQP4/2 had a plasma membrane or plasma membrane and cytoplasmic distribution. Two types of large mitochondrial rich transport cells are known to exist in elasmobranchs, that express either Na, K-ATPase, or V-type ATPase ion transporters. Using Na, K-ATPase, and V-type ATPase antibodies, Aqp4 was colocalized with these proteins using the AQP4/1 antibody. Results show Aqp4 is expressed in both (and all) branchial Na, K-ATPase, and V-type ATPase expressing cells

Elemental Abundances of Nearby Galaxies through High Signal-to-Noise XMM-Newton Observations of ULXs

(abridged) In this paper, we examined XMM Newton EPIC spectra of 14 ultra-luminous X-ray sources (ULXs)in addition to the XMM RGS spectra of two sources (Holmberg II X-1 and Holmberg IX X-1). We determined oxygen and iron abundances of the host galaxy's interstellar medium (ISM) using K-shell (O) and L-shell (Fe) X-ray photo-ionization edges towards these ULXs. We found that the oxygen abundances closely matched recent solar abundances for all of our sources, implying that ULXs live in similar local environments despite the wide range of galaxy host properties. Also, we compare the X-ray hydrogen column densities (n_H) for 8 ULX sources with column densities obtained from radio H I observations. The X-ray model n_H values are in good agreement with the H I n_H values, implying that the hydrogen absorption towards the ULXs is not local to the source (with the exception of the source M81 XMM1). In order to obtain the column density and abundance values, we fit the X-ray spectra of the ULXs with a combined power law and one of several accretion disk models. We tested the abundances obtained from the XSPEC models bbody, diskbb, grad, and diskpn along with a power law, finding that the abundances were independent of the thermal model used. We comment on the physical implications of these different model fits. We also note that very deep observations allow a breaking of the degeneracy noted by Stobbart et al. (2006) favoring a high mass solution for the absorbed grad + power law model.Comment: 18 pages, accepted to Ap

When Less is More: Validating a Brief Scale to Rate Interprofessional Team Competencies

Background: There is a need for validated and easy-to-apply behavior-based tools for assessing interprofessional team competencies in clinical settings. The seven-item observerbased Modified McMaster-Ottawa scale was developed for the Team Objective Structured Clinical Encounter (TOSCE) to assess individual and team performance in interprofessional patient encounters. Objective: We aimed to improve scale usability for clinical settings by reducing item numbers while maintaining generalizability; and to explore the minimum number of observed cases required to achieve modest generalizability for giving feedback. Design: We administered a two-station TOSCE in April 2016 to 63 students split into 16 newly-formed teams, each consisting of four professions. The stations were of similar difficulty. We trained sixteen faculty to rate two teams each. We examined individual and team performance scores using generalizability (G) theory and principal component analysis (PCA). Results: The seven-item scale shows modest generalizability (.75) with individual scores. PCA revealed multicollinearity and singularity among scale items and we identified three potential items for removal. Reducing items for individual scores from seven to four (measuring Collaboration, Roles, Patient/Family-centeredness, and Conflict Management) changed scale generalizability from .75 to .73. Performance assessment with two cases is associated with reasonable generalizability (.73). Students in newly-formed interprofessional teams show a learning curve after one patient encounter. Team scores from a two-station TOSCE demonstrate low generalizability whether the scale consisted of four (.53) or seven items (.55). Conclusion: The four-item Modified McMaster-Ottawa scale for assessing individual performance in interprofessional teams retains the generalizability and validity of the seven-item scale. Observation of students in teams interacting with two different patients provides reasonably reliable ratings for giving feedback. The four-item scale has potential for assessing individual student skills and the impact of IPE curricula in clinical practice settings

Star Formation in Bright Rimmed Clouds. I. Millimeter and Submillimeter Molecular Line Surveys

We present the results of the first detailed millimeter and submillimeter molecular line survey of bright rimmed clouds, observed at FCRAO in the CO (J=1-0), C18O (J=1-0), HCO+ (J=1-0), H13CO+ (J=1-0), and N2H+ (J=1-0) transitions, and at the HHT in the CO (J=2-1), HCO+ (J=3-2), HCO+ (J=4-3), H13CO+ (J=3-2), and H13CO+ (J=4-3) molecular line transitions. The source list is composed of a selection of bright rimmed clouds from the catalog of such objects compiled by Sugitani et al. (1991). We also present observations of three Bok globules done for comparison with the bright rimmed clouds. We find that the appearance of the millimeter CO and HCO+ emission is dominated by the morphology of the shock front in the bright rimmed clouds. The HCO+ (J=1-0) emission tends to trace the swept up gas ridge and overdense regions which may be triggered to collapse as a result of sequential star formation. Five of the seven bright rimmed clouds we observe seem to have an outflow, however only one shows the spectral line blue-asymmetric signature that is indicative of infall, in the optically thick HCO+ emission. We also present evidence that in bright rimmed clouds the nearby shock front may heat the core from outside-in thereby washing out the normally observed line infall signatures seen in isolated star forming regions. We find that the derived core masses of these bright rimmed clouds are similar to other low and intermediate mass star forming regions.Comment: 67 pages, including 35 figures and 6 tables. Accepted for publication in ApJ. Version with embedded full-resolution figures available at http://www.astro.umass.edu/~devries/brc1

The FAD Cofactor of RebC Shifts to an IN Conformation upon Flavin Reduction†,‡

RebC is a putative flavin hydroxylase functioning together with RebP to carry out a key step in the biosynthesis of rebeccamycin. To probe the mechanism of flavin-based chemistry in RebC, we solved the structure of RebC with reduced flavin. Upon flavin reduction, the RebC crystal undergoes a change in its unit cell dimension concurrent with a 5 Å movement of the isoalloxazine ring, positioning the flavin ring adjacent to the substrate-binding pocket. Additionally, a disordered helix becomes ordered upon flavin reduction, closing off one side of the substrate-binding pocket. This structure, along with previously reported structures, increases our understanding of the RebC enzyme mechanism, indicating that either the reduction of the flavin itself or binding of substrate is sufficient to drive major conformational changes in RebC to generate a closed active site. Our finding that flavin reduction seals the active site such that substrate cannot enter suggests that our reduced flavin RebC structure is off-pathway and that substrate binding is likely to precede flavin reduction during catalysis. Along with kinetic data presented here, these structures suggest that the first cycle of catalysis in RebC may resemble that of p-hydroxybenzoate hydroxylase, with substrate binding promoting flavin reduction

Cyclization of Fungal Nonribosomal Peptides by a Terminal Condensation-Like Domain

Cyclization of linear peptidyl precursors produced by nonribosomal peptide synthetases (NRPSs) is an important step in the biosynthesis of bioactive cyclic peptides. Whereas bacterial NRPSs use thioesterase (TE) domains to perform the cyclization, fungal NRPSs have apparently evolved to use a different enzymatic route. In verified fungal NRPSs that produce macrocyclic peptides, each megasynthetase terminates with a condensation-like (CT) domain that may perform the macrocyclization reaction. To probe the role of such a CT domain, we reconstituted the activities of the Penicillium aethiopicum trimodular NPRS TqaA in Saccharomyces cerevisiae and in vitro. Together with a reconstituted bimodular NRPS AnaPS, we dissected the cyclization steps of TqaA in transforming the linear anthranilate-D-tryptophan-L-alanyl tripeptide into fumiquinazoline F. Extensive biochemical and mutational studies confirmed the essential role of the CT domain in catalyzing cyclization in a thiolation domain-dependent fashion. Our work provided evidence of a likely universal macrocyclization strategy employed by fungal NRPSs

Plastic formulation is an emerging control of its photochemical fate in the ocean

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Walsh, A. N., Reddy, C. M., Niles, S. F., McKenna, A. M., Hansel, C. M., & Ward, C. P. Plastic formulation is an emerging control of its photochemical fate in the ocean. Environmental Science & Technology, 55(18), (2021): 12383–12392, https://doi.org/10.1021/acs.est.1c02272.Sunlight exposure is a control of long-term plastic fate in the environment that converts plastic into oxygenated products spanning the polymer, dissolved, and gas phases. However, our understanding of how plastic formulation influences the amount and composition of these photoproducts remains incomplete. Here, we characterized the initial formulations and resulting dissolved photoproducts of four single-use consumer polyethylene (PE) bags from major retailers and one pure PE film. Consumer PE bags contained 15–36% inorganic additives, primarily calcium carbonate (13–34%) and titanium dioxide (TiO2; 1–2%). Sunlight exposure consistently increased production of dissolved organic carbon (DOC) relative to leaching in the dark (3- to 80-fold). All consumer PE bags produced more DOC during sunlight exposure than the pure PE (1.2- to 2.0-fold). The DOC leached after sunlight exposure increasingly reflected the 13C and 14C isotopic composition of the plastic. Ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry revealed that sunlight exposure substantially increased the number of DOC formulas detected (1.1- to 50-fold). TiO2-containing bags photochemically degraded into the most compositionally similar DOC, with 68–94% of photoproduced formulas in common with at least one other TiO2-containing bag. Conversely, only 28% of photoproduced formulas from the pure PE were detected in photoproduced DOC from the consumer PE. Overall, these findings suggest that plastic formulation, especially TiO2, plays a determining role in the amount and composition of DOC generated by sunlight. Consequently, studies on pure, unweathered polymers may not accurately represent the fates and impacts of the plastics entering the ocean.Funding was provided by the Seaver Institute, the Gerstner Family Foundation, Woods Hole Oceanographic Institution, and the National Science Foundation Graduate Research Fellowship Program (A.N.W.). The Ion Cyclotron Resonance user facility at the National High Magnetic Field Laboratory is supported by the National Science Foundation Division of Chemistry and Division of Materials Research through DMR-1644779 and the State of Florida

Structural elements of an NRPS cyclization domain and its intermodule docking domain

Epothilones are thiazole-containing natural products with anticancer activity that are biosynthesized by polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) enzymes EpoA–F. A cyclization domain of EpoB (Cy) assembles the thiazole functionality from an acetyl group and L-cysteine via condensation, cyclization, and dehydration. The PKS carrier protein of EpoA contributes the acetyl moiety, guided by a docking domain, whereas an NRPS EpoB carrier protein contributes L-cysteine. To visualize the structure of a cyclization domain with an accompanying docking domain, we solved a 2.03-Å resolution structure of this bidomain EpoB unit, comprising residues M1-Q497 (62 kDa) of the 160-kDa EpoB protein. We find that the N-terminal docking domain is connected to the V-shaped Cy domain by a 20-residue linker but otherwise makes no contacts to Cy. Molecular dynamic simulations and additional crystal structures reveal a high degree of flexibility for this docking domain, emphasizing the modular nature of the components of PKS-NRPS hybrid systems. These structures further reveal two 20-Å-long channels that run from distant sites on the Cy domain to the active site at the core of the enzyme, allowing two carrier proteins to dock with Cy and deliver their substrates simultaneously. Through mutagenesis and activity assays, catalytic residues N335 and D449 have been identified. Surprisingly, these residues do not map to the location of the conserved HHxxxDG motif in the structurally homologous NRPS condensation (C) domain. Thus, although both C and Cy domains have the same basic fold, their active sites appear distinct