Interannual variability in reproductive traits of the Patagonian toothfish Dissostichus eleginoides around the sub-Antarctic island of South Georgia.

Commercial fisheries data, collected as part of an observer programme and covering the period 1997-2014, were utilized in order to define key reproductive traits and spawning dynamics of the Patagonian toothfish Dissostichus eleginoides at South Georgia. Multi-year spawning site fidelity of D. eleginoides was revealed through the identification of previously unknown spawning hotspots. Timing of female spawning was shown to have shifted later, leading to a shorter spawning duration. A decrease in length and mass of female and male spawning fish and a reduced number of large spawning fish was found, evidence of a change in size structure of spawning D. eleginoides. During the study period fewer later maturity stage females (including spawning stage) were observed in conjunction with increased proportions of early stage female D. eleginoides. The findings are discussed in the context of reproductive success, with consideration of the possible effects such spawning characteristics and behaviours may have on egg and larval survival. This work presents the first long-term assessment of D. eleginoides spawning dynamics at South Georgia and provides valuable knowledge for both the ecology of the species and for future fisheries management of this commercially important species.

Inhaled Pyrazinoic Acid Esters for the Treatment of Tuberculosis.

PURPOSE: Analog development of existing drugs and direct drug delivery to the lungs by inhalation as treatments for multiple and extensively drug resistant (MDR and XDR) tuberculosis (TB) represent new therapeutic strategies. Pyrazinamide (PZA) is critical to drug sensitive TB therapy and is included in regimens for MDR TB. However, PZA-resistant Mycobacterium tuberculosis (Mtb) strains threaten its use. Pyrazinoic acid esters (PAEs) are PZA analogs effective against Mtb in vitro, including against the most common PZA resistant strains. However, PAEs require testing for TB efficacy in animal models. METHODS: PAEs were delivered daily as aqueous dispersions from a vibrating mesh nebulizer to Mtb infected guinea pigs for 4 weeks in a regimen including orally administered first-line TB drugs. RESULTS: PAEs tested as a supplement to oral therapy significantly reduced the organ bacterial burden in comparison to infected, untreated control animals. Thus, PAE aerosol therapy is a potentially significant addition to the regimen for PZA resistant MDR-TB and XDR-TB treatment. Interestingly, low dose oral PZA treatment combined with standard therapy also reduced bacterial burden. This observation may be important for PZA susceptible disease treatment. CONCLUSION: The present study justifies further evaluation of PZA analogs and their lung delivery to treat TB.

Discovery of two new satellites of Pluto.

Pluto's first known satellite, Charon, was discovered in 1978. It has a diameter (approximately 1,200 km) about half that of Pluto, which makes it larger, relative to its primary, than any other moon in the Solar System. Previous searches for other satellites around Pluto have been unsuccessful, but they were not sensitive to objects less, similar150 km in diameter and there are no fundamental reasons why Pluto should not have more satellites. Here we report the discovery of two additional moons around Pluto, provisionally designated S/2005 P 1 (hereafter P1) and S/2005 P 2 (hereafter P2), which makes Pluto the first Kuiper belt object known to have multiple satellites. These new satellites are much smaller than Charon, with estimates of P1's diameter ranging from 60 km to 165 km, depending on the surface reflectivity; P2 is about 20 per cent smaller than P1. Although definitive orbits cannot be derived, both new satellites appear to be moving in circular orbits in the same orbital plane as Charon, with orbital periods of approximately 38 days (P1) and approximately 25 days (P2).

A giant impact origin for Pluto's small moons and satellite multiplicity in the Kuiper belt.

The two newly discovered satellites of Pluto (P1 and P2) have masses that are small compared to both Pluto and Charon-that is, between 5 x 10(-4) and 1 x 10(-5) of Pluto's mass, and between 5 x 10(-3) and 1 x 10(-4) of Charon's mass. This discovery, combined with the constraints on the absence of more distant satellites of Pluto, reveal that Pluto and its moons comprise an unusual, highly compact, quadruple system. These facts naturally raise the question of how this puzzling satellite system came to be. Here we show that P1 and P2's proximity to Pluto and Charon, the fact that P1 and P2 are on near-circular orbits in the same plane as Pluto's large satellite Charon, along with their apparent locations in or near high-order mean-motion resonances, all probably result from their being constructed from collisional ejecta that originated from the Pluto-Charon formation event. We also argue that dust-ice rings of variable optical depths form sporadically in the Pluto system, and that rich satellite systems may be found--perhaps frequently--around other large Kuiper belt objects.

Differentiation of the asteroid Ceres as revealed by its shape.

The accretion of bodies in the asteroid belt was halted nearly 4.6 billion years ago by the gravitational influence of the newly formed giant planet Jupiter. The asteroid belt therefore preserves a record of both this earliest epoch of Solar System formation and variation of conditions within the solar nebula. Spectral features in reflected sunlight indicate that some asteroids have experienced sufficient thermal evolution to differentiate into layered structures. The second most massive asteroid--4 Vesta--has differentiated to a crust, mantle and core. 1 Ceres, the largest and most massive asteroid, has in contrast been presumed to be homogeneous, in part because of its low density, low albedo and relatively featureless visible reflectance spectrum, similar to carbonaceous meteorites that have suffered minimal thermal processing. Here we show that Ceres has a shape and smoothness indicative of a gravitationally relaxed object. Its shape is significantly less flattened than that expected for a homogeneous object, but is consistent with a central mass concentration indicative of differentiation. Possible interior configurations include water-ice-rich mantles over a rocky core.

Localization of phospholamban in smooth muscle using immunogold electron microscopy.

Phospholamban, the putative regulator of the Ca2+-ATPase in cardiac sarcoplasmic reticulum, was immunolocalized in canine visceral and vascular smooth muscle. Gently disrupted tissues were labeled with an affinity-purified phospholamban polyclonal antibody and indirect immunogold, using preembedding techniques. The sarcoplasmic reticulum of smooth muscle cells was specifically labeled with patches of immunogold distributed in a nonuniform fashion, while the sarcolemma did not appear to contain any phospholamban. The outer nuclear envelopes were also observed to be heavily labeled with the affinity-purified phospholamban polyclonal antibody. These findings suggest that phospholamban may play a role in the regulation of cytoplasmic and intranuclear calcium levels in smooth muscle cells.

A dry powder combination of pyrazinoic acid and its n-propyl ester for aerosol administration to animals.

Combining the advantage of higher efficacy due to local pulmonary administration of pyrazinoic acid (POA) and potent effect of pyrazinoic acid ester (PAE) delivered as an aerosol would aid in tuberculosis therapy. A combination spray dried dry powder, composed of POA, PAE (n-propyl POA), maltodextrin and leucine, was prepared for aerosol delivery to animals. Solid-state characteristics of morphology (scanning electron microscopy) crystallinity (X-ray powder diffraction), thermal properties (thermogravimetric analysis and differential scanning calorimetry) and moisture content (Karl Fisher) were evaluated. Particle size distributions, by volume (laser diffraction) for the dispersed powder and by mass (inertial impaction) were determined. Efficient delivery of the powder to a nose only animal exposure chamber employed a novel rotating brush/micro-fan apparatus. Spherical, crystalline particles were prepared. The volume median diameter, ∼1.5µm, was smaller than the mass median aerodynamic diameter, ∼3.0µm, indicating modest aggregation. Drug content variations were observed across the particle size distribution and may be explained by PAE evaporative losses. Delivery to the nose-only exposure chamber indicated that boluses could be administered at approximately 3min intervals to avoid aerosol accumulation and effect uniform dose delivery with successive doses suitable for future pharmacokinetic and pharmacodynamic studies.

Dengue Virus Envelope Dimer Epitope Monoclonal Antibodies Isolated from Dengue Patients Are Protective against Zika Virus.

UNLABELLED: Zika virus (ZIKV) is a mosquito-borne flavivirus responsible for thousands of cases of severe fetal malformations and neurological disease since its introduction to Brazil in 2013. Antibodies to flaviviruses can be protective, resulting in lifelong immunity to reinfection by homologous virus. However, cross-reactive antibodies can complicate flavivirus diagnostics and promote more severe disease, as noted after serial dengue virus (DENV) infections. The endemic circulation of DENV in South America and elsewhere raises concerns that preexisting flavivirus immunity may modulate ZIKV disease and transmission potential. Here, we report on the ability of human monoclonal antibodies and immune sera derived from dengue patients to neutralize contemporary epidemic ZIKV strains. We demonstrate that a class of human monoclonal antibodies isolated from DENV patients neutralizes ZIKV in cell culture and is protective in a lethal murine model. We also tested a large panel of convalescent-phase immune sera from humans exposed to primary and repeat DENV infection. Although ZIKV is most closely related to DENV compared to other human-pathogenic flaviviruses, most DENV immune sera (73%) failed to neutralize ZIKV, while others had low (50% effective concentration [EC50], <1:100 serum dilution; 18%) or moderate to high (EC50, >1:100 serum dilution; 9%) levels of cross-neutralizing antibodies. Our results establish that ZIKV and DENV share epitopes that are targeted by neutralizing, protective human antibodies. The availability of potently neutralizing human monoclonal antibodies provides an immunotherapeutic approach to control life-threatening ZIKV infection and also points to the possibility of repurposing DENV vaccines to induce cross-protective immunity to ZIKV. IMPORTANCE: ZIKV is an emerging arbovirus that has been associated with severe neurological birth defects and fetal loss in pregnant women and Guillain-Barré syndrome in adults. Currently, there is no vaccine or therapeutic for ZIKV. The identification of a class of antibodies (envelope dimer epitope 1 [EDE1]) that potently neutralizes ZIKV in addition to all four DENV serotypes points to a potential immunotherapeutic to combat ZIKV. This is especially salient given the precedent of antibody therapy to treat pregnant women infected with other viruses associated with microcephaly, such as cytomegalovirus and rubella virus. Furthermore, the identification of a functionally conserved epitope between ZIKV and DENV raises the possibility that a vaccine may be able to elicit neutralizing antibodies against both viruses.

Interrogation of the rat mammary gland using intraductal impedance spectroscopy.

Extant technologies for the detection of breast cancer exploit changes in the morphology of the mammary ductal epithelial network and can involve ionizing radiation. Intraductal surveillance of mammary epithelium has the potential to allow for earlier detection based on changes in function of the epithelium. This study investigated the feasibility of using intraductal impedance spectroscopy (IIS) to assess changes in resistance in the mammary epithelium in a small group of female rats in resting, pregnant and ultimately lactating states. In resting rats, intraductal surveillance was able to detect only a single resistive capacitance (RC). In pregnant animals, a second RC became evident in the frequency range between 1 and 190 Hz. The real resistance of this low frequency RC increased when measurements were made after the animals had begun lactating. Equivalent circuit modeling revealed this increase to be a 1.7-fold change from pregnancy to lactation. A model of tight junction closure in the context of ductal expansion is proposed. These results suggest that physiologic measurements can be made in rodent mammary epithelium using this technique allowing for assessment of function in normal and disease states.

Solid state studies by means of Fourier transform spectroscopy.

A commercially available Michelson interferometer has been adapted to meet the needs of our research. Transmission and reflection measurements of solid state materials can be made over the wavelength range of 25-1000 micro (400-10 cm(-1)). Provision has been made for the samples in each case to be studied from 4 degrees K to 700 degrees K with suitable cryostats and heated sample holders. These devices can easily be interchanged in the interferometer and also in a single beam far ir vacuum gratings pectrometer so that the measurements can be duplicated when necessary, using conventional spectroscopic techniques. The interferometer has the unique advantage of covering a broad frequency range without changing the operating conditions. A suitable choice of beam splitter thickness and ionic crystal powder filter combinations provides maximum efficiency in the area of interest. The region beyond 250 micro shows a rapid deterioration in signal to noise, and the relative merits of various cooled detectors and the Golay detector have been studied. Polarization measurements on solids have also been of interest, and various polarizers have been investigated. Applications of interferometry applied to a number of solid state problems are described. These include the dielectric dispersion in perovskites, compound semiconductors, internal molecular and lattice vibrations in inorganic materials and other related compounds. The relative advantages of single-sided vs double-sided interferograms in terms of accurate intensity measurements are compared. Examples of each are discussed.

Structural characterization of phospholamban in cardiac sarcoplasmic reticulum membranes by cross-linking.

The native form of phospholamban in cardiac sarcoplasmic reticulum membranes was investigated using photosensitive heterobifunctional cross-linkers, both cleavable and noncleavable, and common protein modifiers. The photosensitive heterobifunctional cleavable cross-linker ethyl 4-azidophenyl-1, 4-dithiobutyrimidate was used in native SR vesicles and it cross-linked phospholamban into an apparent phospholamban-phospholamban dimer and into an approximately 110,000-Da species. The phospholamban dimer migrated at approximately 12,000 Da on sodium dodecyl sulfate-polyacrylamide gels, and upon cleavage of the cross-linker before electrophoresis the dimer disappeared. The approximately 110,000-Da cross-linked species was not affected by boiling in sodium dodecyl sulfate prior to electrophoresis. This cross-linked form of phospholamban migrated approximately 5500 Da above the Ca2(+)-ATPase, which was visualized using fluorescein 5'-isothiocynate, a fluorescent marker that binds specifically to the Ca2(+)-ATPase. p-Azidophenacyl bromide, iodoacetic acid, and N-ethylmaleimide, all of which react with sulfhydryl groups, were also employed to further characterize phospholamban in native sarcoplasmic reticulum membranes. Cross-linking with p-azidophenacyl bromide resulted in only monomeric and dimeric forms of phospholamban as observed on sodium dodecyl sulfate-polyacrylamide gels. Iodoacetic acid and N-ethylmalemide were found to be effective in disrupting the pentameric form of phospholamban only when reacted with sodium dodecyl sulfate solubilized sarcoplasmic reticulum. In view of these findings, the amino acid sequence of phospholamban was examined for possible protein-protein interaction sites. Analysis by hydropathic profiling and secondary structure prediction suggests that the region of amino acids 1-14 may form an amphipathic alpha helix and the hydrophobic surface on one of its sites could interact with the reciprocal hydrophobic surface of another protein, such as the Ca2(+)-ATPase.

Regulation of rat cardiac nuclei-associated Mg(2+)-NTPase by phosphorylation.

A nucleoside triphosphatase (NTPase) activity appeared to be associated with a highly purified nuclear preparation from rat cardiac ventricles. Different nucleoside triphosphates (UTP greater than GTP greater than ITP greater than CTP) supported this enzymic activity, which was stimulated by Mg2+ but not by Ca2+. The nuclear NTPase activity could be down regulated by endogenous phosphorylation of a 55,000 Mr protein. Maximal phosphorylation of the 55,000 Mr protein occurred in the presence of Mg(2+)-ATP. Addition of cAMP, cGMP, Ca2+, Ca2+/phospholipid, Ca2+/calmodulin, and catalytic subunit of cAMP-dependent protein kinase was not associated with any further phosphorylation of the 55,000 Mr protein. However, in the presence of Ca2+/calmodulin or the catalytic subunit of the cAMP-dependent protein kinase additional proteins became phosphorylated, but these had no effect on the Mg(2+)-NTPase activity. These results indicate that a protein with Mr 55,000 may be involved in the regulation the Mg(2+)-NTPase activity associated with rat cardiac nuclei.

Topological mapping of acetylcholine receptor: evidence for a model with five transmembrane segments and a cytoplasmic COOH-terminal peptide.

Antibodies were raised against two synthetic peptides whose sequences correspond respectively to the COOH-terminal end (residues 501-516) of the protein encoded by the gene for the delta chain and to a proposed cytoplasmic region (residues 350-358) of the beta chain of the acetylcholine receptor from Torpedo californica. Binding of the COOH-terminal antibody to the acetylcholine receptor in intact, receptor-rich vesicles was tested by radioimmunoassay and by precipitation with immobilized protein A. In both cases, binding was detected only after treatment of the vesicles with detergent, suggesting that the segment of the receptor that is recognized by this antibody is on the cytoplasmic side of the membrane. Electron microscopy of tissue from Torpedo electric organ labeled with colloidal gold-conjugated second antibodies established that both anti-receptor antibodies bind to the cytoplasmic surface of the postsynaptic membrane. These experiments give ultrastructural evidence that the COOH-terminal segment of the delta chain as well as residues 350-358 of the beta chain are on the cytoplasmic surface. They strongly support a model in which each of the receptor subunits crosses the membrane five times in which one transmembrane segment of each chain contributes to the formation of a central ion channel.

The role of phospholamban in the regulation of calcium transport by cardiac sarcoplasmic reticulum.

The calcium transport mechanism of cardiac sarcoplasmic reticulum (SR) is (SR) is regulated by a phosphoregulatory mechanism involving the phosphorylation-dephosphorylation of an integral membrane component, termed phospholamban. Phospholamban, a 27,000 Da proteolipid, contains phosphorylation sites for three independent protein kinases: 1) cAMP-dependent, 2) Ca2(+)-calmodulin-dependent, and 3) Ca2(+)-phospholipid-dependent. Phosphorylation of phospholamban by any one of these kinases is associated with stimulation of the calcium transport rates in isolated SR vesicles. Dephosphorylation of phosphorylated phospholamban results in the reversal of the stimulatory effects produced by the protein kinases. Studies conducted on perfused hearts have shown that during exposure to beta-adrenergic agents, a good correlation exists between the in situ phosphorylation of phospholamban and the relaxation of the left ventricle. Phosphorylation of phospholamban in situ is associated with stimulation of calcium transport rates by cardiac SR, similar to in vitro findings. Removal of beta-adrenergic agents results in the reversal of the inotropic response and this is associated with dephosphorylation of phospholamban. These findings indicate that a phospho-regulatory mechanism involving phospholamban may provide at least one of the controls for regulation of the contractile properties of the myocardium.