m-Tyrosine recovery (%) from m-tyrosine treated non-sterile soil with seedlings of lettuce, littleseed canarygrass or bamboo.

<p>Shared letters indicate no significant differences in mean m-tyrosine recovery from the soils of the three assay species as determined by one-way ANOVAs, and post ANOVA Tukey test (P<0.05).</p

m-Tyrosine (µg/g soil) recovery from non-sterile or sterile soil treated with 0, 4.25, 8.5, 17, 34, 68 or 136 µg m-tyrosine/g soil and incubated at 22 or 30°C.

<p>Error bars indicate 1 SE.</p

Schematic depiction of the differences in the availability/recovery of a putative chemical to a competing species from a given amount of chemical produced and released from a plant in <i>in vitro</i> assays as compared to <i>in situ</i> assays that include soil and the associated microbial (bulk and rhizosphere) communities.

<p>Sorption of chemicals onto soil particles, chemical decomposition and/or microbial degradation of chemicals are major mechanisms that influence their ability to accumulate to phytotoxic levels and influence the growth of neighboring target plants <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004700#pone.0004700-Huang1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004700#pone.0004700-Inderjit7" target="_blank">[40]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004700#pone.0004700-Inderjit8" target="_blank">[41]</a>. Sometimes physical sorption of a chemical on soil particles can actually concentrate the chemical to a level that may become physiologically active. Therefore, sorption may affect allelopathy both negatively and positively.</p

Microbial activity as indicated by CO₂ release (µg CO₂ released/g soil/h) of soil treated with 0, 4.25, 8.5, 17, 34, 68 or 136 µg m-tyrosine/g soil, incubated at 22 or 30°C.

<p>Error bars indicate 1 SE. Values in parenthesis indicate % recovery of m-tyrosine in soil.</p

Sequence of events and stimulus conditions in Experiment 1.

<p>As indicated in (A) in each trial the participant had to name the objects shown as cued prime and as probe. The five conditions concerning the relation between probe and prime display are illustrated in (B). Accordingly, the probe could be identical to either the cued (attended) prime, the ignored (unattended) prime, or to their respective mirror-reflected versions. For each condition, priming was measured in terms of the response time reduction relative to the unprimed baseline condition.</p

Results for Experiment 2.

<p>Priming means as a function of attentional allocation (Attended vs Ignored) and view (Same-Name-Different-Exemplar vs Reflected) for each age group in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061041#s3" target="_blank">Experiment 2</a>. Error bars represent standard errors.</p

Results for Experiment 1.

<p>Priming means as a function of attentional allocation (Attended vs Ignored) and view (Identical vs Reflected) for each age group in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061041#s2" target="_blank">Experiment 1</a>. Error bars represent standard errors.</p

Custom-Designed Affinity Capture LC-MS F(ab′)2 Assay for Biotransformation Assessment of Site-Specific Antibody Drug Conjugates

Affinity capture liquid chromatography–mass spectrometry (LC-MS) intact antibody assay has been widely used for direct drug-to-antibody ratio (DAR) and catabolite characterization of antibody-drug conjugates (ADCs). However, the intact mass spectra of new ADCs, which incorporate new types of linkers and payloads other than maytansines and auristatins, are more complex than those examined previously. The current method has showed some limitations in elucidating certain structural modifications. Herein, we report an alternative analytical approach for ADCs, such as THIOMAB antibody-drug conjugates (TDCs), where the linker drugs are site-specifically conjugated in the Fab region. The newly developed affinity capture LC-MS F­(ab′)­2 assay incorporates affinity capture of human IgGs via binding to the Fab region, followed by on-bead IdeS digestion to remove the Fc domain specifically and uniformly. The resulting F­(ab′)­2 (∼100 kDa) fragments contain the key ADC biotransformation information, such as drug-to-antibody ratio and drug metabolism and are more readily analyzed by electrospray ionization LC-MS than the intact ADC (∼150 kDa). The reduced size of analytes results in improved mass spectral sensitivity and resolution. In addition, the reduced and optimized sample preparation time, for example, rapid removal of the Fc fragment by IdeS digestion, minimizes assay artifacts of drug metabolism and skewed DAR profiles that may result from the prolonged incubation times (e.g., overnight enzymatic treatment for Fc deglycosylation). The affinity capture LC-MS F­(ab′)­2 assay provides more detailed and accurate information on ADC biotransformations in vivo, enabling analysis of low-dose, labile, and complex site-specific ADCs with linker-drug conjugated in the Fab region

High-Resolution Accurate-Mass Mass Spectrometry Enabling In-Depth Characterization of <i>in Vivo</i> Biotransformations for Intact Antibody-Drug Conjugates

Antibody-drug conjugates (ADCs) represent a promising class of therapeutics for the targeted delivery of highly potent cytotoxic drugs to tumor cells to improve bioactivity while minimizing side effects. ADCs are composed of both small and large molecules and therefore have complex molecular structures. <i>In vivo</i> biotransformations may further increase the complexity of ADCs, representing a unique challenge for bioanalytical assays. Quadrupole-time-of-flight mass spectrometry (Q-TOF MS) with electrospray ionization has been widely used for characterization of intact ADCs. However, interpretation of ADC biotransformations with small mass changes, for the intact molecule, remains a limitation due to the insufficient mass resolution and accuracy of Q-TOF MS. Here, we have investigated <i>in vivo</i> biotransformations of multiple site-specific THIOMAB antibody-drug conjugates (TDCs), in the intact form, using a high-resolution, accurate-mass (HR/AM) MS approach. Compared with conventional Q-TOF MS, HR/AM Orbitrap MS enabled more comprehensive identification of ADC biotransformations. It was particularly beneficial for characterizing ADC modifications with small mass changes such as partial drug loss and hydrolysis. This strategy has significantly enhanced our capability to elucidate ADC biotransformations and help understand ADC efficacy and safety <i>in vivo</i>

Modulating Antibody–Drug Conjugate Payload Metabolism by Conjugation Site and Linker Modification

Previous investigations on antibody-drug conjugate (ADC) stability have focused on drug release by linker-deconjugation due to the relatively stable payloads such as maytansines. Recent development of ADCs has been focused on exploring technologies to produce homogeneous ADCs and new classes of payloads to expand the mechanisms of action of the delivered drugs. Certain new ADC payloads could undergo metabolism in circulation while attached to antibodies and thus affect ADC stability, pharmacokinetics, and efficacy and toxicity profiles. Herein, we investigate payload stability specifically and seek general guidelines to address payload metabolism and therefore increase the overall ADC stability. Investigation was performed on various payloads with different functionalities (e.g., PNU-159682 analog, tubulysin, cryptophycin, and taxoid) using different conjugation sites (HC-A118C, LC-K149C, and HC-A140C) on THIOMAB antibodies. We were able to reduce metabolism and inactivation of a broad range of payloads of THIOMAB antibody-drug conjugates by employing optimal conjugation sites (LC-K149C and HC-A140C). Additionally, further payload stability was achieved by optimizing the linkers. Coupling relatively stable sites with optimized linkers provided optimal stability and reduction of payloads metabolism in circulation in vivo