Hadaean to Palaeoarchaean stagnant-lid tectonics revealed by zircon magnetism.

Plate tectonics is a fundamental factor in the sustained habitability of Earth, but its time of onset is unknown, with ages ranging from the Hadaean to Proterozoic eons1-3. Plate motion is a key diagnostic to distinguish between plate and stagnant-lid tectonics, but palaeomagnetic tests have been thwarted because the planet's oldest extant rocks have been metamorphosed and/or deformed4. Herein, we report palaeointensity data from Hadaean-age to Mesoarchaean-age single detrital zircons bearing primary magnetite inclusions from the Barberton Greenstone Belt of South Africa5. These reveal a pattern of palaeointensities from the Eoarchaean (about 3.9 billion years ago (Ga)) to Mesoarchaean (about 3.3 Ga) eras that is nearly identical to that defined by primary magnetizations from the Jack Hills (JH; Western Australia)6,7, further demonstrating the recording fidelity of select detrital zircons. Moreover, palaeofield values are nearly constant between about 3.9 Ga and about 3.4 Ga. This indicates unvarying latitudes, an observation distinct from plate tectonics of the past 600 million years (Myr) but predicted by stagnant-lid convection. If life originated by the Eoarchaean8, and persisted to the occurrence of stromatolites half a billion years later9, it did so when Earth was in a stagnant-lid regime, without plate-tectonics-driven geochemical cycling.

Electronic transport through single-molecule oligophenyl-diethynyl junctions with direct gold-carbon bonds formed at low temperature.

We report on the first systematic transport study of alkynyl-ended oligophenyl-diethynyl (OPA) single-molecule junctions with direct Au-C anchoring scheme at low temperature using the mechanically controlled break junction technique. Through quantitative statistical analysis of opening traces, conductance histograms and density functional theory studies, we identified different types of junctions, classified by their conductance and stretching behavior, for OPA molecules between Au electrodes with two to four phenyl rings. We performed inelastic electron tunneling spectroscopy and observed the excitation of Au-C vibrational modes confirming the existence of Au-C bonds at low temperature and compared the stability of molecule junctions upon mechanical stretching. Our findings reveal the huge potential for future functional molecule transport studies at low temperature using alkynyl endgroups.

Interplay between Magnetoresistance and Kondo Resonance in Radical Single-Molecule Junctions.

We report transport measurements on tunable single-molecule junctions of the organic perchlorotrityl radical molecule, contacted with gold electrodes at low temperature. The current-voltage characteristics of a subset of junctions shows zero-bias anomalies due to the Kondo effect and in addition elevated magnetoresistance (MR). Junctions without Kondo resonance reveal a much stronger MR. Furthermore, we show that the amplitude of the MR can be tuned by mechanically stretching the junction. On the basis of these findings, we attribute the high MR to an interference effect involving spin-dependent scattering at the metal-molecule interface and assign the Kondo effect to the unpaired spin located in the center of the molecule in asymmetric junctions.

Paleomagnetism indicates that primary magnetite in zircon records a strong Hadean geodynamo.

Determining the age of the geomagnetic field is of paramount importance for understanding the evolution of the planet because the field shields the atmosphere from erosion by the solar wind. The absence or presence of the geomagnetic field also provides a unique gauge of early core conditions. Evidence for a geomagnetic field 4.2 billion-year (Gy) old, just a few hundred million years after the lunar-forming giant impact, has come from paleomagnetic analyses of zircons of the Jack Hills (Western Australia). Herein, we provide new paleomagnetic and electron microscope analyses that attest to the presence of a primary magnetic remanence carried by magnetite in these zircons and new geochemical data indicating that select Hadean zircons have escaped magnetic resetting since their formation. New paleointensity and Pb-Pb radiometric age data from additional zircons meeting robust selection criteria provide further evidence for the fidelity of the magnetic record and suggest a period of high geomagnetic field strength at 4.1 to 4.0 billion years ago (Ga) that may represent efficient convection related to chemical precipitation in Earth's Hadean liquid iron core.

Structural and Hydrogeological Controls on Hydrocarbon and Brine Migration into Drinking Water Aquifers in Southern New York.

Environmental concerns regarding the potential for drinking water contamination in shallow aquifers have accompanied unconventional energy development in the northern Appalachian Basin. These activities have also raised several critical questions about the hydrogeological parameters that control the naturally occurring presence and migration of hydrocarbon gases in shallow aquifers within petroliferous basins. To interrogate these factors, we analyzed the noble gas, dissolved ion, and hydrocarbon gas (molecular and isotopic composition) geochemistry of 98 groundwater samples from south-central New York. All samples were collected ≫1km from unconventional drilling activities and sample locations were intentionally targeted based on their proximity to various types of documented fault systems. In agreement with studies from other petroliferous basins, our results show significant correlations between elevated levels of radiogenic [4 He], thermogenic [CH4 ], and dissolved ions (e.g., Cl, Br, Sr, Ba). In combination, our data suggest that faults have facilitated the transport of exogenous hydrocarbon-rich brines from Devonian source rocks into overlying Upper Devonian aquifer lithologies over geologic time. These data conflict with previous reports, which conclude that hydrodynamic focusing regulates the occurrence of methane and salt in shallow aquifers and leads to elevated levels of these species in restricted flow zones within valley bottoms. Instead, our data suggest that faults in Paleozoic rocks play a fundamental role in gas and brine transport from depth, regulate the distribution of their occurrence in shallow aquifers, and influence the geochemistry of shallow groundwater in this petroliferous basin.

Purification of clinical-grade disulfide stabilized antibody fragment variable--Pseudomonas exotoxin conjugate (dsFv-PE38) expressed in Escherichia coli.

Immunotoxins are rationally designed cancer targeting and killing agents. Disulfide stabilized antibody Fv portion-toxin conjugates (dsFv-toxin) are third generation immunotoxins containing only the antibody fragment variable portions and a toxin fused to the V(H) or V(L). Pseudomonas exotoxin fragment (PE-38) is a commonly used toxin in immunotoxin clinical trials. dsFv-toxin purification was previously published, but the recovery was not satisfactory. This report describes the development of a cGMP production process of the dsFv-toxin that incorporated a novel purification method. The method has been successfully applied to the clinical manufacturing of two dsFv-PE38 immunotoxins, MR1-1 targeting EGFRvIII and HA22 targeting CD22. The two subunits, V(L) and V(H) PE-38 were expressed separately in Escherichia coli using recombinant technology. Following cell lysis, inclusion bodies were isolated from the biomass harvested from fermentation in animal source component-free media. The dsFv-toxin was formed after denaturation and refolding, and subsequently purified to homogeneity through ammonium sulfate precipitation, hydrophobic interaction and ion-exchange chromatography steps. It was shown, in a direct comparison experiment using MR1-1 as model protein, that the recovery from the new purification method was improved three times over that from previously published method. The improved recovery was also demonstrated during the clinical production of two dsFv-PE38 immunotoxins-MR1-1 and HA22.

Comparison of GD2 binding capture ELISA assays for anti-GD2-antibodies using GD2-coated plates and a GD2-expressing cell-based ELISA.

Two assay methods for quantification of the disialoganglioside (GD2)-specific binding activities of anti-GD2 monoclonal antibodies and antibody immunofusion proteins, such as ch14.18 and hu14.18-IL2, were developed. The methods differed in the use of either microtiter plates coated with purified GD2 or plates seeded with GD2-expressing cell lines to bind the anti-GD2 molecules. The bound antibodies were subsequently detected using the reactivity of the antibodies to an HRP-labeled anti-IgG Fc or antibodies recognizing the conjugate IL-2 part of the Hu 14.18IL-2 fusion protein. The bound HRP was detected using reagents such as orthophenylene diamine, 2, 2'-azinobis [3-ethylbenzothiazoline-6-sulfonic acid] or tetramethylbenzidine. The capture ELISA using GD2-coated plates was developed earlier in assay development and used to demonstrate assay specificity and to compare lot-to-lot consistency and stability of ch14.18, and Hu14.18 IL-2 in clinical development. During this study, we found a number of issues related to plate-to-plate variability, GD2 lot variability, and variations due to GD2 storage stability, etc., that frequently lead to assay failure in plates coated with purified GD2. The cell-based ELISA (CbELISA) using the GD2 expressing melanoma cell line, M21/P6, was developed as an alternative to the GD2-coated plate ELISA. The results on the comparability of the capture ELISA on GD2-coated plates and the cell-based assay show that both assays give comparable results. However, the cell-based assay is more consistent and reproducible. Subsequently, the anti-GD2 capture ELISA using the GD2-coated plate was replaced with the CbELISA for product lot release testing and stability assessment.

Evaluation of IRES-mediated, cell-type-specific cytotoxicity of poliovirus using a colorimetric cell proliferation assay.

PVS-RIPO is a recombinant oncolytic poliovirus designed for clinical application to target CD155 expressing malignant gliomas and other malignant diseases. PVS-RIPO does not replicate in healthy neurons and is therefore non-pathogenic in rodent and non-human primate models of poliomyelitis. A tetrazolium salt dye-based cellular assay was developed and qualified to define the cytotoxicity of virus preparations on susceptible cells and to explore the target cell specificity of PVS-RIPO. In this assay, PVS-RIPO inhibited proliferation of U87-MG astrocytoma cells in a dose-dependent manner. However, HEK293 cells were much less susceptible to cell killing by PVS-RIPO. In contrast, the Sabin type 1 live attenuated poliovirus vaccine strain (PV(1)S) was cytotoxic to both HEK293 and U87-MG cells. The correlation between expression of CD155 and cytotoxicity was also explored using six different cell lines. There was little or no expression of CD155 and PVS-RIPO-induced cytotoxicity in Jurkat and Daudi cells. HEK293 was the only cell line tested that showed CD155 expression and resistance to PVS-RIPO cytotoxicity. The results indicate that differential cytotoxicity measured by the colorimetric assay can be used to evaluate the cytotoxicity and cell-type specificity of recombinant strains of poliovirus and to demonstrate lot to lot consistency during the manufacture of viruses intended for clinical use.

Development of a quantitative cell-based ELISA, for a humanized anti-IL-2/IL-15 receptor beta antibody (HuMikbeta(1)), and correlation with functional activity using an antigen-transfected murine cell line.

The HuMikbeta(1), a humanized IgG1 monoclonal antibody directed toward the IL-2/IL-15 receptor beta-chain (CD122), inhibits the actions of the inflammatory cytokine IL-15, and may be useful for immunotherapy of an array of autoimmune disorders as well as diseases associated with the retrovirus human T-cell lymphotrophic virus 1 (HTLV-1). In order to facilitate the production of material for clinical investigation, we developed a cell-based ELISA (CbELISA) for measuring the binding activity, as a potential biological activity marker, of the HuMikbeta(1) monoclonal antibody to a transfected 32D mouse cell line (32Dbeta) expressing IL-2Rbeta antigen on the cell surface. There is specific binding of HuMikbeta(1) to the transfected cell line, titrating out in the concentration range of 1-1,000 ng/ml. Under identical conditions, there was no binding of HuMikbeta(1) to the parent cell line 32D. Satisfactory binding curves with HuMikbeta(1) were obtained with 32Dbeta cells grown between 3 and 19 passages in culture and at seed densities of 2 x 10(5)-4 x 10(6) cells/well. The binding was specific for Mikbeta antibodies recognizing the IL-2/IL-15 receptor beta subunit as demonstrated by binding of HuMikbeta(1), Mikbeta(2) and Mikbeta(3) antibodies, and lack of binding of irrelevant humanized and chimeric antibodies and isotype-matched human IgG1 to the 32Dbeta cell. Also, the human IgG1 and irrelevant humanized and chimeric antibodies did not interfere with the HuMikbeta(1) binding. The assay could detect changes in binding activity of HuMikbeta(1) antibody under stressful conditions (heat and low pH) and the results paralleled the effect of stress on the physicochemical characteristics. More importantly, the binding activity shows an apparent correlation to inhibition of IL-15-induced proliferation of 32Dbeta cells with HuMikbeta(1). In conclusion, the cell-based ELISA method represents a simple, reproducible accurate quantitative assay for monitoring HuMikbeta(1) activity and could be used as a potency marker assay for monitoring the lot-lot consistency and functional stability of HuMikbeta(1) product.

Characterization and analysis of thermal denaturation of antibodies by size exclusion high-performance liquid chromatography with quadruple detection.

Size exclusion chromatography (SEC) coupled with online light scattering, viscometry, refractometry, and UV-visible spectroscopy provides a very powerful tool for studying protein size, shape, and aggregation. This technique can be used to determine the molecular weight of the component peaks independent of the retention times in the SEC column and simultaneously measure the hydrodynamic radius and polydispersity of the protein. We applied this technology by coupling an Agilent Chemstation high-performance liquid chromatography system with a diode array UV-visible detector and a Viscotek 300 EZ Pro triple detector (combination of a light scattering detector, refractometer, and differential pressure viscometer) to characterize and compare the molecular properties of a number of monoclonal antibodies. Our studies reveal that different monoclonal immunoglobulin Gs (IgGs) and chimeric IgGs show slightly different retention times and therefore different molecular weights in gel filtration analysis. However, when they are analyzed by light scattering, refractometry, and viscometry, different IgGs have comparable molecular weight, molecular homogeneity (polydispersity), and size. Gel filtration coupled with UV or refractive index detection suggests that antibodies purified and formulated for preclinical and clinical development are more than 95% monomer with little or no detectable soluble aggregates. Light scattering measurements showed the presence of trace amounts of soluble aggregate in all the IgG preparations. The different IgG molecules showed different susceptibility to heat and pH. One of the murine antibodies was considerably less stable than the others at 55 degrees C. The application of this powerful technology for the characterization of monoclonal antibodies of therapeutic potential is discussed.

Evaluation of the compatibility of a second generation recombinant anthrax vaccine with aluminum-containing adjuvants.

Recombinant protective antigen (rPA) is the active pharmaceutical ingredient in a second generation anthrax vaccine undergoing pre-clinical evaluation. This rPA vaccine differs from the currently licensed vaccine, anthrax vaccine adsorbed (AVA), in that the sole component is a recombinant form of protective antigen (PA). Unlike AVA the rPA vaccine contains no lethal factor (LF) or edema factor (EF), components of the two bipartite toxins, nor many other Bacillus anthracis-related contaminating proteins that are present in AVA. The proposed clinical protocol involves adsorption of the rPA to an aluminum-based adjuvant. The adsorptive characteristics of rPA and two aluminum-containing adjuvants were examined in a physiological buffer with and without EDTA. Based on the pI of rPA (pI=5.6) and the zero charge point of aluminum hydroxide adjuvant (11.5) and aluminum phosphate adjuvant (4.5), it was predicted and demonstrated that rPA bound in a more efficient manner to aluminum hydroxide adjuvant than to aluminum phosphate adjuvant in the physiological buffer. Binding of the rPA to the aluminum hydroxide adjuvant was decreased by increasing amounts of phosphate in the buffer. The adsorptive capacity for rPA onto aluminum hydroxide adjuvant in the physiological buffer and in water were calculated to be 0.46 mg rPA/mg aluminum in DPBS and 0.73 mg rPA/mg aluminum in water. This study also demonstrated that upon desorption from the aluminum hydroxide adjuvant the rPA was physically intact and free of detectable aggregates. Further, the eluted material was biologically active in an in vitro cytotoxicity assay. Desorption was only possible after an overnight incubation of 2-8 degrees C and not after a room temperature incubation reflecting increased contact with the aluminum hydroxide adjuvant over time. These data suggest that the interaction between rPA and aluminum hydroxide adjuvant is predominantly electrostatic in character.

Development of a quantitative antigen-specific cell-based ELISA for the 7G7/B6 monoclonal antibody directed toward IL-2Ralpha.

Interleukin-2 receptor alpha (IL-2Ralpha, CD25) has been identified as a valuable target for immunotherapy. The 7G7/B6 monoclonal antibody, a mouse IgG2a kappa, recognizes an epitope of the IL-2Ralpha peptide, other than that identified by anti-Tac. This antibody is currently being explored for potential therapeutic and diagnostic applications. Here, we show a cell-based enzyme-linked immunosorbent assay (CbELISA) method for quantitative measurement of the binding activity of the 7G7/B6 antibody to the Kit-225-iG3 cell line expressing IL-2Ralpha antigen on the cell surface. The cell- and antigen-specificity of the assay was established using specific cell lines and irrelevant control antibodies. Satisfactory binding curves were demonstrated with Kit-225-iG3 cells grown between 3 and 25 passages in culture and at seed densities of 2 x 10(5)-4 x 10(6) cells/well. The assay shows reproducible dose-response curves in the concentration range of 10-1000 ng/ml. The assay validation data presented here indicate that this CbELISA assay is quantitative, reproducible, robust, precise, and can be used to test the biological activity, lot to lot comparison, and stability of 7G7/B6 monoclonal antibody.