Llama Single Domain Antibodies Specific for the 7 Botulinum Neurotoxin Serotypes as Heptaplex Immunoreagents

There are currently 7 known serotypes of botulinum neurotoxin (BoNT) classified upon non-cross reactivity of neutralizing immunoglobulins. Non-neutralizing immunoglobulins, however, can exhibit cross-reactivities between 2 or more serotypes, particularly mosaic forms, which can hamper the development of highly specific immunoassays, especially if based on polyclonal antisera. Here we employ facile recombinant antibody technology to subtractively select ligands to each of the 7 BoNT serotypes, resulting in populations with very high specificity for their intended serotype.A single llama was immunized with a cocktail of 7 BoNT toxoids to generate a phage display library of single domain antibodies (sdAb, VHH or nanobodies) which were selected on live toxins. Resulting sdAb were capable of detecting both toxin and toxin complex with the best combinations able to detect 100s-10s of pg per 50 microL sample in a liquid bead array. The most sensitive sdAb were combined in a heptaplex assay to identify each of the BoNT serotypes in buffer and milk and to a lesser extent in carrot juice, orange juice and cola. Several anti-A(1) sdAb recognized A2 complex, showing that subtype cross-reactivity within a serotype was evident. Many of our sdAb could act as both captor and tracer for several toxin and toxin complexes suggesting sdAb can be used as architectural probes to indicate BoNT oligomerisation. Six of 14 anti-A clones exhibited inhibition of SNAP-25 cleavage in the neuro-2A assay indicating some sdAb had toxin neutralizing capabilities. Many sdAb were also shown to be refoldable after exposure to high temperatures in contrast to polyclonal antisera, as monitored by circular dichroism.Our panel of molecularly flexible antibodies should not only serve as a good starting point for ruggedizing assays and inhibitors, but enable the intricate architectures of BoNT toxins and complexes to be probed more extensively

Prospective isolation and global gene expression analysis of definitive and visceral endoderm

AbstractIn spite of the therapeutic importance of endoderm derivatives such as the pancreas, liver, lung, and intestine, there are few molecular markers specific for early endoderm. In order to identify endoderm-specific genes as well as to define transcriptional differences between definitive and visceral endoderm, we performed microarray analysis on E8.25 definitive and visceral endoderm. We have developed an early endoderm gene expression signature, and clarified the transcriptional similarities and differences between definitive and visceral endoderm. Additionally, we have developed methods for flow cytometric isolation of definitive and visceral endoderm. These results shed light on the mechanism of endoderm formation and should facilitate investigation of endoderm formation from embryonic stem cells

Thermal properties of halogen-ethane glassy crystals: Effects of orientational disorder and the role of internal molecular degrees of freedom

The thermal conductivity, specific heat, and specific volume of the orientational glass former 1,1,2-trichloro-1,2,2-trifluoroethane (CCl2F-CClF2, F-113) have been measured under equilibrium pressure within the low-temperature range, showing thermodynamic anomalies at ca. 120, 72, and 20 K. The results are discussed together with those pertaining to the structurally related 1,1,2,2-tetrachloro-1,2-difluoroethane (CCl2F-CCl2F, F-112), which also shows anomalies at 130, 90, and 60 K. The rich phase behavior of these compounds can be accounted for by the interplay between several of their degrees of freedom. The arrest of the degrees of freedom corresponding to the internal molecular rotation, responsible for the existence of two energetically distinct isomers, and the overall molecular orientation, source of the characteristic orientational disorder of plastic phases, can explain the anomalies at higher and intermediate temperatures, respectively. The soft-potential model has been used as the framework to describe the thermal properties at low temperatures. We show that the low-temperature anomaly of the compounds corresponds to a secondary relaxation, which can be associated with the appearance of Umklapp processes, i.e., anharmonic phonon-phonon scattering, that dominate thermal transport in that temperature rangeThis work was financially supported in part by the Spanish Ministry of Science and Innovation (Grant Nos. FIS2014-54734-P, FIS2011-23488, and MAT2014-57866- REDT), by the Catalan Government (Grant No. 2014SGR- 0581) and by the Comunidad de Madrid through program NANOFRONTMAG-CM (No. S2013/MIT-2850), as well as by the joint NAS Ukraine and Russian Foundation for Basic Research project “Metastable states of simple condensed systems” (Agreement No. N 7/-2013

Supercooled-liquid and plastic-crystalline state in succinonitrile-glutaronitrile mixtures

We report a thorough characterization of the glassy phases of mixtures of succinonitrile and glutaronitrile via dielectric spectroscopy and differential scanning calorimetry. This system is revealed to be one of the rare examples where both glassy states of matter, a structurally disordered supercooled liquid and an orientationally disordered plastic crystal, can be prepared in the same material. Both disordered states can be easily supercooled, finally arriving at a structural-glass or a glassy-crystal state. Detailed investigations using broadband dielectric spectroscopy enable a comparison of the glassy dynamics in both phases. Just as previously demonstrated for supercooled-liquid and plastic-crystalline ethanol, our experiments reveal very similar relaxational behavior and glass temperatures of both disordered states. Thus the prominent role of orientational degrees of freedom in the glass transition, suggested on the basis of the findings for ethanol, is fully corroborated by the present work. Moreover, the fragilities of both phases are determined and compared for different mixtures. The findings can be well understood within an energy-landscape based explanation of fragility.Comment: 8 pages, 7 figure

Deep vs shallow nature of oxygen vacancies and consequent n -type carrier concentrations in transparent conducting oxides

The source of n -type conductivity in undoped transparent conducting oxides has been a topic of debate for several decades. The point defect of most interest in this respect is the oxygen vacancy, but there are many conflicting reports on the shallow versus deep nature of its related electronic states. Here, using a hybrid quantum mechanical/molecular mechanical embedded cluster approach, we have computed formation and ionization energies of oxygen vacancies in three representative transparent conducting oxides: In 2 O 3 , SnO 2 , and ZnO. We find that, in all three systems, oxygen vacancies form well-localized, compact donors. We demonstrate, however, that such compactness does not preclude the possibility of these states being shallow in nature, by considering the energetic balance between the vacancy binding electrons that are in localized orbitals or in effective-mass-like diffuse orbitals. Our results show that, thermodynamically, oxygen vacancies in bulk In 2 O 3 introduce states above the conduction band minimum that contribute significantly to the observed conductivity properties of undoped samples. For ZnO and SnO 2 , the states are deep, and our calculated ionization energies agree well with thermochemical and optical experiments. Our computed equilibrium defect and carrier concentrations, however, demonstrate that these deep states may nevertheless lead to significant intrinsic n -type conductivity under reducing conditions at elevated temperatures. Our study indicates the importance of oxygen vacancies in relation to intrinsic carrier concentrations not only in In 2 O 3 , but also in SnO 2 and ZnO

The AMMA mulid network for aerosol characterization in West Africa

Three ground based portable low power consumption microlidars (MULID) have been built and deployed at three remote sites in Banizoumbou (Niger), Cinzana (Mali) and M'Bour (Senegal) in the framework of the African Monsoon Multidisciplinary Analyses (AMMA) project for the characterization of aerosols optical properties. A description of the instrument and a discussion of the data inversion method, including a careful analysis of measurement uncertainties (systematic and statistical errors) are presented. Some case studies of typical lidar profiles observed over the Banizoumbou site during 2006 are shown and discussed with respect to the AERONET 7-day back-trajectories and the biomass burning emissions from the Combustion Emission database for the AMMA campaign

Manifestation of classical wave delays in a fully quantized model of the scattering of a single photon

We consider a fully quantized model of spontaneous emission, scattering, and absorption, and study propagation of a single photon from an emitting atom to a detector atom both with and without an intervening scatterer. We find an exact quantum analog to the classical complex analytic signal of an electromagnetic wave scattered by a medium of charged oscillators. This quantum signal exhibits classical phase delays. We define a time of detection which, in the appropriate limits, exactly matches the predictions of a classically defined delay for light propagating through a medium of charged oscillators. The fully quantized model provides a simple, unambiguous, and causal interpretation of delays that seemingly imply speeds greater than c in the region of anomalous dispersion.Comment: 18 pages, 4 figures, revised for clarity, typos corrrecte

Radiocarbon evidence for annual growth rings in a deep sea octocoral (Primnoa resedaeformis)

The deep-sea gorgonian octocoral Primnoa resedaeformis is distributed throughout the Atlantic and Pacific Oceans at depths of 65-3200 m. It has a two-part skeleton of calcite and gorgonin. Towards the inside of the axial skeleton gorgonin and calcite are deposited in concentric growth rings, similar to tree rings. Colonies were collected from the Northeast Channel (northwest Atlantic Ocean, southwest of Nova Scotia, Canada) from depths of 250-475 m. Radiocarbon was measured in individual rings isolated from sections of each colony, after dissolution of calcite. Each {Delta}{sup 14}C measurement was paired with a ring age determined by three amateur ring counters. The precision of ring counts averaged better than {+-} 2 years. Accurate reconstruction of 20th century bomb-radiocarbon shows that (1) the growth rings are formed annually, (2) the gorgonin is derived from surface particulate organic matter (POM) and (3) useful environmental data are recorded in the organic endoskeletons of deep-sea octocorals. These results support the use of Primnoa resedaeformis as a long-term, high resolution monitor of surface ocean conditions, particularly in temperate and boreal environments where proxy data are lacking