Role of invariant water molecules and water-mediated ionic interactions in D-xylose isomerase from Streptomyces rubiginosus

The enzyme, D-xylose isomerase (D-xylose keto-isomerase; EC 5.3.1.5) is a soluble enzyme that catalyzes the conversion of the aldo-sugar D-xylose to the keto-sugar D-xylulose. A total of 27 subunits of D-xylose isomerase from Streptomyces rubiginosus were analyzed in order to identify the invariant water molecules and their water-mediated ionic interactions. A total of 70 water molecules were found to be invariant. The structural and/or functional roles of these water molecules have been discussed. These invariant water molecules and their ionic interactions may be involved in maintaining the structural stability of the enzyme D-xylose isomerase. Fifty-eight of the 70 invariant water molecules (83%) have at least one interaction with the main chain polar atom.

Small-Angle Neutron Scattering Studies of Hemoglobin Confined Inside Silica Tubes of Varying Sizes

In addition to the chemical nature of the surface, the dimensions of the confining host exert a significant influence on confined protein structures; this results in immense biological implications, especially those concerning the enzymatic activities of the protein. This study probes the structure of hemoglobin (Hb), a model protein, confined inside silica tubes with pore diameters that vary by one order of magnitude (approximate to 20-200 nm). The effect of confinement on the protein structure is probed by comparison with the structure of the protein in solution. Small-angle neutron scattering (SANS), which provides information on protein tertiary and quaternary structures, is employed to study the influence of the tube pore diameter on the structure and configuration of the confined protein in detail. Confinement significantly influences the structural stability of Hb and the structure depends on the Si-tube pore diameter. The high radius of gyration (R-g) and polydispersity of Hb in the 20 nm diameter Si-tube indicates that Hb undergoes a significant amount of aggregation. However, for Si-tube diameters greater or equal to 100 nm, the R-g of Hb is found to be in very close proximity to that obtained from the protein data bank (PDB) reported structure (R-g of native Hb=23.8 angstrom). This strongly indicates that the protein has a preference for the more native-like non-aggregated state if confined inside tubes of diameter greater or equal to 100 nm. Further insight into the Hb structure is obtained from the distance distribution function, p(r), and ab initio models calculated from the SANS patterns. These also suggest that the Si-tube size is a key parameter for protein stability and structure.

First-Principles Study of Structure, Vibrational, and Elastic Properties of Stoichiometric and Calcium-Deficient Hydroxyapatite

Hydroxyapatite (HAp), a primary constituent of human bone, is usually nonstoichiometric with varying Ca/P molar ratios, with the well-known fact that Ca deficiency can cause marked reductions in its mechanical properties. To gain insights into the mechanism of this degradation, we employ first-principles calculations based on density functional theory and determine the effects of Ca deficiency on structure, vibrational, and elastic properties of HAp. Our simulation results confirm a considerable reduction in the elastic constants of HAp due to Ca deficiency, which was experimentally reported earlier. Stress-induced transformation of the Ca-deficient defected structure into a metastable state upon the application of stress could be a reason for this. Local structural stability of HAp and Ca-deficient HAp structures is assessed with full phonon dispersion studies. Further, specific signatures in the computed vibrational spectra for Ca deficiency in HAp can be utilized in experimental characterization of different types of defected HAp.

Exploiting oriented attachment in stabilizing La3+-doped gallium oxide nano-spindles

Retaining the morphology of gallium oxide nanostructures during structural transformations or after doping with lanthanide ions is not facile. Here we report on the sonochemical synthesis of nearly monodisperse similar to 550 nm long nano-spindles of undoped and La-doped alpha-GaOOH. The transformation of as-prepared undoped and La-doped alpha-GaOOH powders into the corresponding undoped and La-doped Ga2O3 phases (alpha and beta) was achieved by carrying out controlled annealing at elevated temperatures under optimized conditions. The formation of gallium oxide nano-spindles is explained by invoking the phenomenon of oriented attachment, as amply supported by electron microscopy. Interestingly, the morphology of the gallium oxide nano-spindles remained conserved even after doping them with more than 1.4 at% of La3+ ions. Such robust structural stability could be attributed to the oriented attachment-type growth observed in the nano-spindles. The as-prepared samples and the corresponding annealed ones were thoroughly characterized by powder X-ray diffraction (PXRD), electron microscopy (SEM, TEM, and STEM-EDS) and X-ray photoelectron spectroscopy (XPS). Finally, photoluminescence from the single-crystalline undoped and La-doped beta-Ga2O3 was explored.

Achieving Highly Efficient, Selective, and Stable CO2 Reduction on Nitrogen-Doped Carbon Nanotubes

The challenge in the electrosynthesis of fuels from CO2 is to achieve durable and active performance with cost-effective catalysts. Here, we report that carbon nanotubes (CNTs), doped with nitrogen to form resident electron-rich defects, can act as highly efficient and, more importantly, stable catalysts for the conversion of CO2 to CO. The unprecedented overpotential (-0.18 V) and selectivity (80%) observed on nitrogen-doped CNTs (NCNTs) are attributed to their unique features to facilitate the reaction, including (i) high electrical conductivity, (ii) preferable catalytic sites (pyridinic N defects), and (iii) low free energy for CO2 activation and high barrier for hydrogen evolution. Indeed, DFT calculations show a low free energy barrier for the potential-limiting step to form key intermediate COOH as well as strong binding energy of adsorbed CON and weak binding energy for the adsorbed CO. The highest selective site toward CO production is pyridinic N, and the NCNT-based electrodes exhibit no degradation over 10 h of continuous operation, suggesting the structural stability of the electrode.

Molecular Dynamics Simulation of Barnacle Cement

Barnacle cement is an underwater adhesive that is used for permanent settlement. Its main components are insoluble protein complexes that have not been fully studied. In present article, we chose two proteins of barnacle cement for study, 36-KD protein and Mrcp-100K protein. In order to investigate the characteristic of above two proteins, we introduced the method of molecular modeling. And the simulation package GROMACS was used to simulate the behavior of these proteins. In this article, before the simulations, we introduce some theories to predict the time scale for polymer relaxation. During the simulation, we mainly focus on two properties of these two proteins: structural stability and adhesive force to substrate. First, we simulate the structural stability of two proteins in water, and then the stability of 36-KD protein in seawater environment is investigated.We find that the stability varies in the different environments. Next, to study adhesive ability of two proteins, we simulate the process of peeling the two proteins from the substrate (graphite). Then, we analyze the main reasons of these results. We find that hydrogen bonds in proteins play an important role in the protein stability. In the process of the peeling, we use Lennard–Jones 12-6 potential to calculate the van der Waals interactions between proteins and substrate.

Molecular Dynamics Simulation of Barnacle Cement

Barnacle cement is an underwater adhesive that is used for permanent settlement. Its main components are insoluble protein complexes that have not been fully studied. In present article, we chose two proteins of barnacle cement for study, 36-KD protein and Mrcp-100K protein. In order to investigate the characteristic of above two proteins, we introduced the method of molecular modeling. And the simulation package GROMACS was used to simulate the behavior of these proteins. In this article, before the simulations, we introduce some theories to predict the time scale for polymer relaxation. During the simulation, we mainly focus on two properties of these two proteins: structural stability and adhesive force to substrate. First, we simulate the structural stability of two proteins in water, and then the stability of 36-KD protein in seawater environment is investigated. We find that the stability varies in the different environments. Next, to study adhesive ability of two proteins, we simulate the process of peeling the two proteins from the substrate (graphite). Then, we analyze the main reasons of these results. We find that hydrogen bonds in proteins play an important role in the protein stability. In the process of the peeling, we use Lennard-Jones 12-6 potential to calculate the van der Waals interactions between proteins and substrate.

M/V ELPIS Coral Reef Restoration Monitoring Report Monitoring Events 2004-2007 Florida Keys National Marine Sanctuary, Monroe County, Florida

This document presents the results of the first three monitoring events to track the recovery of a repaired coral reef injured by the M/V Elpis vessel grounding incident of November 11, 1989. This grounding occurred within the boundaries of what at the time was designated the Key Largo National Marine Sanctuary (NMS), now designated the Key Largo NMS Existing Management Area within the Florida Keys National Marine Sanctuary (FKNMS). Pursuant to the National Marine Sanctuaries Act (NMSA) 16 U.S.C. 1431 et seq., and the Florida Keys National Marine Sanctuary and Protection Act (FKNMSPA) of 1990, NOAA is the federal trustee for the natural and cultural resources of the FKNMS. Under Section 312 of the NMSA, NOAA has the authority to recover monetary damages for injury, destruction, or loss of Sanctuary resources, and to use the recovered monies to restore injured or lost sanctuary resources within the FKNMS. The restoration monitoring program tracks patterns of biological recovery, determines the success of restoration measures, and assesses the resiliency to environmental and anthropogenic disturbances of the site over time. To evaluate restoration success, reference habitats adjacent to the restoration site are concurrently monitored to compare the condition of restored reef areas with natural coral reef areas unimpacted by the vessel grounding. Restoration of the site was completed September 1995, and thus far three monitoring events have occurred; one in the summer of 2004, one in the summer of 2005, and the latest in the summer of 2007. The monitoring in 2004 was in the nature of a “pilot project,” or proof of concept. Only the quantitative results of the 2005 and 2007 monitoring are presented and discussed. Monitoring has consisted of assessment of the structural stability of limestone boulders used in the restoration and comparison of the coral communities on the boulders and reference areas. Corals are divided into Gorgonians, Milleporans, and Scleractinians. Coral densities at the Restored and Reference areas for the 2005 and 2007 events are compared, and it is shown that the densities of all taxa in the Restored area are greater by 2007, though not significantly so. For the Scleractinians, number and percentage of colonies by species, as well as several common biodiversity indices are provided. The greater biodiversity of the Restored area is evidenced. Also, size-class frequency distributions for Agaricia spp. (Scleractinia) are presented. These demonstrate the approaching convergence of the Restored and Reference areas in this regard. An inter-annual comparison of densities, within both areas, for all three Orders, is presented. The most noteworthy finding was the relative consistency across time for all taxa in each area. Finally, certain anomalies regarding species settlement patterns are presented. (PDF contains 48 pages.)

M/V WELLWOOD Coral Reef Restoration Monitoring Report, Monitoring Events 2004-2006. Florida Keys National Marine Sanctuary Monroe County, Florida

This document presents the results of the first two monitoring events to track the recovery of a repaired coral reef injured by the M/V Wellwood vessel grounding incident of August 4, 1984. This grounding occurred within the boundaries of what at the time was designated the Key Largo National Marine Sanctuary (NMS), now designated the Key Largo NMS Existing Management Area within the Florida Keys National Marine Sanctuary (FKNMS). Pursuant to the National Marine Sanctuaries Act (NMSA) 16 U.S.C. 1431 et seq., and the Florida Keys National Marine Sanctuary and Protection Act (FKNMSPA) of 1990, NOAA is the federal trustee for the natural and cultural resources of the FKNMS. Under Section 312 of the NMSA, NOAA has the authority to recover monetary damages for injury, destruction, or loss of Sanctuary resources, and to use the recovered monies to restore injured or lost sanctuary resources within the FKNMS. The restoration monitoring program tracks patterns of biological recovery, determines the success of restoration measures, and assesses the resiliency to environmental and anthropogenic disturbances of the site over time. To evaluate restoration success, reference habitats adjacent to the restoration site are concurrently monitored to compare the condition of restored reef areas with “natural” coral reef areas unimpacted by the vessel grounding or other injury. Restoration of the site was completed on July 22, 2002, and thus far two monitoring events have occurred; one in the Fall of 2004, and one in the Summer/Fall of 2006. The monitoring has consisted of: assessment of the structural stability of restoration modules and comparison of the coral recruitment conditions of the modules and reference sites. Corals are divided into Gorgonians, Milleporans, and Scleractinians and (except where noted) recruits are defined as follows: Gorgonians—maximum size (height) 150 mm at first monitoring event, 270 mm at second; Milleporans—maximum size (height) 65 mm at first event, 125 mm at second; Scleractinians—maximum size (greatest diameter) 50 mm at second event (only one species was size-classed at first event, at smaller size). Recruit densities at the restored and reference areas for each event are compared, as are size-class frequency distributions. For the Scleractinians, number and percentage of recruits by species, as well as several common biodiversity indices are provided. Finally, a qualitative comparison of recruit substrate settlement preference is indicated. Generally, results indicate that restored areas are converging on reference areas, based on almost all parameters examined, with one noted exception. Further monitoring is planned and the trends are anticipated to continue; close attention will be paid to the indicated anomaly. (PDF contains 63 pages.)

M/V CONNECTED Coral Reef Restoration Monitoring Report, Monitoring Events 2004-2005. Florida Keys National Marine Sanctuary Monroe County, Florida

This document presents the results of the monitoring of a repaired coral reef injured by the M/V Connected vessel grounding incident of March 27, 2001. This grounding occurred in Florida state waters within the boundaries of the Florida Keys National Marine Sanctuary (FKNMS). The National Oceanic and Atmospheric Administration (NOAA) and the Board of Trustees of the Internal Improvement Trust Fund of the State of Florida, (“State of Florida” or “state”) are the co-trustees for the natural resources within the FKNMS and, thus, are responsible for mediating the restoration of the damaged marine resources and monitoring the outcome of the restoration actions. The restoration monitoring program tracks patterns of biological recovery, determines the success of restoration measures, and assesses the resiliency to environmental and anthropogenic disturbances of the site over time. The monitoring program at the Connected site was to have included an assessment of the structural stability of installed restoration modules and biological condition of reattached corals performed on the following schedule: immediately (i.e., baseline), 1, 3, and 6 years after restoration and following a catastrophic event. Restoration of this site was completed on July 20, 2001. Due to unavoidable delays in the settlement of the case, the “baseline” monitoring event for this site occurred in July 2004. The catastrophic monitoring event occurred on August 31, 2004, some 2 ½ weeks after the passage of Hurricane Charley which passed nearby, almost directly over the Dry Tortugas. In September 2005, the year one monitoring event occurred shortly after the passage of Hurricane Katrina, some 70 km to the NW. This report presents the results of all three monitoring events. (PDF contains 37 pages.)

M/V JACQUELYN L Coral Reef Restoration Monitoring Report, Monitoring Events 2004-2005. Florida Keys National Marine Sanctuary Monroe County, Florida

This document presents the results of the monitoring of a repaired coral reef injured by the M/V Jacquelyn L vessel grounding incident of July 7, 1991. This grounding occurred in Florida state waters within the boundaries of the Florida Keys National Marine Sanctuary (FKNMS). The National Oceanic and Atmospheric Administration (NOAA) and the Board of Trustees of the Internal Improvement Trust Fund of the State of Florida, (“State of Florida” or “state”) are the co-trustees for the natural resources within the FKNMS and, thus, are responsible for mediating the restoration of the damaged marine resources and monitoring the outcome of the restoration actions. The restoration monitoring program tracks patterns of biological recovery, determines the success of restoration measures, and assesses the resiliency to environmental and anthropogenic disturbances of the site over time. The monitoring program at the Jacquelyn L site was to have included an assessment of the structural stability of installed restoration modules and biological condition of reattached corals performed on the following schedule: immediately (i.e., baseline), 1, 3, and 6 years after restoration and following a catastrophic event. Restoration of this site was completed on July 20, 2000. Due to unavoidable delays in the settlement of the case, the “baseline” monitoring event for this site occurred in July 2004. The catastrophic monitoring event occurred on August 31, 2004, some 2 ½ weeks after the passage of Hurricane Charley which passed nearby, almost directly over the Dry Tortugas. In September 2005, the year one monitoring event occurred shortly after the passage of Hurricane Katrina, some 70 km to the NW. This report presents the results of all three monitoring events. (PDF contains 31 pages.)

Construção, clonagem e expressão do fragmento B da toxina diftérica de Corynebacterium diphtheriae (cepa PW- 8) em Mycobacterium bovis BCG sub-cepa Moreau

A vacina anti-diftérica de uso corrente no Brasil (DTP), embora de alta eficácia na prevenção da difteria, está associada com episódios de toxicidade e reatogenicidade no recipiente vacinal, resultantes de proteínas residuais derivadas do processo de produção ou detoxificação. Estratégias para o desenvolvimento de vacinas menos reatogênicas e ao mesmo tempo mais eficazes e economicamente viáveis contra a difteria têm sido alvo de intensa investigação. A alternativa proposta por nosso grupo é a utilização da vacina contra a tuberculose (Mycobacterium bovis BCG sub-cepa Moreau), como vetor do gene que codifica o fragmento B da toxina diftérica (dtb) de 58,3 kDa. Neste trabalho o dtb foi clonado no vetor micobacteriano bifuncional (pUS977) de expressão citoplasmática e os clones recombinantes (pUS977dtbPW8), após a transformação do BCG, foram testados com relação a expressão do DTB em BCG e quanto a antigenicidade frente a anticorpos policlonais anti-toxóide diftérico por Immunobloting. A integridade do gene dtb e a identidade das sequências de DNA da construção plasmidial pUS977dtbPW8 foram confirmadas por sequenciamento de DNA e análise de similaridade. A imunogenicidade do BCGr pUS977dtbPW8 expressando o DTB foi investigada em camundongos BALB/c, os resultados obtidos revelaram uma soroconversão específica (IgG). A infectividade e atividade microbicida do BCGr pUS977dtbPW8 no ambiente intracelular foi avaliada através da infecção de linhagens de células de monócitos humano (THP-1), os dados obtidos indicaram que houve sobrevivência intracelular em até 12 dias. Nesse contexto, esplenócitos dos camundongos imunizados com 30 e 60 dias foram extraídos, mostrando que o BCGr pUS977dtbPW8 persistiu até 60 dias na ausência de pressão seletiva e a viabilidade celular não sofreu alteração significativa durante o período testado. Por outro lado, o BCGr pUS977dtbPW8, quando submetido a seis sub-cultivos consecutivos in vitro não apresentou diferença significativa na capacidade de expressar o DTB, demonstrando portanto a persistência da estabilidade funcional da linhagem recombinante. A estabilidade estrutural da construção pUS977dtbPW8 também foi avaliada por PCR confirmando a presença do gene dtb em colônias do BCGr pUS977dtbPW8 . Adicionalmente, foi possível avaliar preliminarmente in vitro a capacidade soroneutralizante dos soros de camundongos imunizados com BCGr pUS977dtbPW8 após 30 e 60 dias em células VERO. A ação citotóxica da toxina diftérica entre as diluições de 1/4 e 1/16 foram neutralizadas com o pool de soros imunes com 60 dias. Finalmente, em nosso estudo foi possível avaliar o potencial da vacina BCG como vetor de expressão de um antígeno de Corynebacterium diphtheriae in vitro e in vivo.

Effect of the interplay between protein and surface on the properties of adsorbed protein layers.

Although protein adsorption to surface is a common phenomenon, investigation of the process is challenging due to the complexity of the interplay between external factors, protein and surface properties. Therefore experimental approaches have to measure the properties of adsorbed protein layers with high accuracy in order to achieve a comprehensive description of the process. To this end, we used a combination of two biosensing techniques, dual polarization interferometry and quartz crystal microbalance with dissipation. From this, we are able to extract surface coverage values, layer structural parameters, water content and viscoelastic properties to examine the properties of protein layers formed at the liquid/solid interface. Layer parameters were examined upon adsorption of proteins of varying size and structural properties, on surfaces with opposite polarity. We show that "soft" proteins such as unfolded α-synuclein and high molecular weight albumin are highly influenced by the surface polarity, as they form a highly diffuse and hydrated layer on the hydrophilic silica surface as opposed to the denser, less hydrated layer formed on a hydrophobic methylated surface. These layer properties are a result of different orientations and packing of the proteins. By contrast, lysozyme is barely influenced by the surface polarity due to its intrinsic structural stability. Interestingly, we show that for a similar molecular weight, the unfolded α-synuclein forms a layer with the highest percentage of solvation not related to surface coverage but resulting from the highest water content trapped within the protein. Together, these data reveal a trend in layer properties highlighting the importance of the interplay between protein and surface for the design of biomaterials.

Effect of the interplay between protein and surface on the properties of adsorbed protein layers

Although protein adsorption to surface is a common phenomenon, investigation of the process is challenging due to the complexity of the interplay between external factors, protein and surface properties. Therefore experimental approaches have to measure the properties of adsorbed protein layers with high accuracy in order to achieve a comprehensive description of the process. To this end, we used a combination of two biosensing techniques, dual polarization interferometry and quartz crystal microbalance with dissipation. From this, we are able to extract surface coverage values, layer structural parameters, water content and viscoelastic properties to examine the properties of protein layers formed at the liquid/solid interface. Layer parameters were examined upon adsorption of proteins of varying size and structural properties, on surfaces with opposite polarity. We show that "soft" proteins such as unfolded α-synuclein and high molecular weight albumin are highly influenced by the surface polarity, as they form a highly diffuse and hydrated layer on the hydrophilic silica surface as opposed to the denser, less hydrated layer formed on a hydrophobic methylated surface. These layer properties are a result of different orientations and packing of the proteins. By contrast, lysozyme is barely influenced by the surface polarity due to its intrinsic structural stability. Interestingly, we show that for a similar molecular weight, the unfolded α-synuclein forms a layer with the highest percentage of solvation not related to surface coverage but resulting from the highest water content trapped within the protein. Together, these data reveal a trend in layer properties highlighting the importance of the interplay between protein and surface for the design of biomaterials. © 2014 The Authors.

First-principles LDA plus U and GGA plus U study of neptunium dioxide

We have performed a systematic first-principles investigation to calculate the electronic structures, mechanical properties, and phonon-dispersion curves of NpO2. The local-density approximation+U and the generalized gradient approximation+U formalisms have been used to account for the strong on-site Coulomb repulsion among the localized Np 5f electrons. By choosing the Hubbard U parameter around 4 eV, the orbital occupancy characters of Np 5f and O 2p are in good agreement with recent experiments [A. Seibert, T. Gouder, and F. Huber, J. Nucl. Mater. 389, 470 (2009)]. Comparing to our previous study of ThO2, we note that stronger covalency exists in NpO2 due to the more localization behavior of 5f electrons of Np in line with the localization-delocalization trend exhibited by the actinides series.