Involvement of phosphorylated Apis mellifera CREB in gating a honeybee's behavioral response to an external stimulus.

The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees(Apis mellifera)we recently demonstrated a particular high abundance of the phosphorylated honeybee CREB homolog (pAmCREB) in the central brain and in a subpopulation of mushroom body neurons. We hypothesize that these high pAmCREB levels are related to learning and memory formation. Here, we tested this hypothesis by analyzing brain pAmCREB levels in classically conditioned bees and bees experiencing unpaired presentations of conditioned stimulus (CS) and unconditioned stimulus (US). We demonstrate that both behavioral protocols display differences in memory formation but do not alter the level of pAmCREB in bee brains directly after training. Nevertheless, we report that bees responding to the CS during unpaired stimulus presentations exhibit higher levels of pAmCREB than nonresponding bees. In addition, Trichostatin A, a histone deacetylase inhibitor that is thought to enhance histone acetylation by CREB-binding protein, increases the bees' CS responsiveness. We conclude that pAmCREB is involved in gating a bee's behavioral response driven by an external stimulus.

Two inhibitors of the ubiquitin proteasome system enhance long-term memory formation upon olfactory conditioning in the honeybee (Apis mellifera).

In honeybees (Apis mellifera), the proteasome inhibitor Z-Leu-Leu-Leu-CHO (MG132) enhances long-term memory (LTM) formation. Studies in vertebrates using different inhibitors of the proteasome demonstrate the opposite, namely an inhibition of memory formation. The reason for this contradiction remains unclear. MG132 is an inhibitor of the proteasome, but also blocks other proteases. Accordingly, one possible explanation might be that other proteases affected by MG132 are responsible for the enhancement of LTM formation. We test this hypothesis by comparing the effect of MG132 and the more specific proteasome inhibitor clasto-lactacystin beta-lactone (ß-lactone). We show that these two inhibitors block the activity of the proteasome in honeybee brains to a similar extent, do not affect the animals' survival but do enhance LTM retention upon olfactory conditioning. Thus, the enhancement of LTM formation is not due to MG132-specific side effects, but to inhibition of a protease targeted by MG132 and ß-lactone, i.e. the proteasome.

Comparison of middle- and bottom-up mass spectrometry in forced degradation studies of bevacizumab and infliximab.

Monoclonal antibodies (mAbs) used as therapeutics need comprehensive characterization for appropriate quality assurance. For analysis, cost-effective methods are of high importance, especially when it comes to biosimilar development which is based on extended physicochemical characterization. The use of forced degradation to study the occurrence of modifications for analysis is well established in drug development and may be used for the evaluation of critical quality attributes (CQAs). For mAb analysis different procedures of liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses are commonly applied. In this study the middle-up approach is compared to the more expensive bottom-up analysis in a forced oxidation biosimilar comparability study. Bevacizumab and infliximab as well as biosimilar candidates for the two mAbs were forcefully oxidized by H2O2 for 24, 48 and 72 h. For bottom-up, the reduced and alkylated trypsin or Lys-C digested samples were analysed by LC-MS with quadrupole time-of-flight mass analyser (LC-QTOF-MS) to detect susceptible residues. By middle-up analysis several species of every subunit (Fc/2, light chain and Fd') were detected which differed in the number of oxidations. For the most abundant species, results from middle-up were in line with results from bottom-up analysis, confirming the strength of middle-up analysis. However, for less abundant species of some subunits, results differed between the two approaches. In both mAbs, the Fc was extensively oxidized. In infliximab, additional extensive oxidation was found in the Fab. Assignment to specific amino acid residues was finally possible using the results from bottom-up analyses. Interestingly, the C-terminal cysteine of the light chain was partially found triply oxidized in both mAbs. The comparison of susceptibility to oxidation showed high similarity between the reference products and their biosimilar candidates. It is suggested that the findings of middle-up experiments should be complemented by bottom-up analysis to confirm the assignments of the localization of modifications. Once the consistency of results has been established, middle-up analyses are sufficient in extended forced degradation biosimilar studies.

A disruptive clickable antibody design for the generation of antibody-drug conjugates.

Background: Antibody-drug conjugates are cancer therapeutics that combine specificity and toxicity. A highly cytotoxic drug is covalently attached to an antibody that directs it to cancer cells. The conjugation of the drug-linker to the antibody is a key point in research and development as well as in industrial production. The consensus is to conjugate the drug to a surface-exposed part of the antibody to ensure maximum conjugation efficiency. However, the hydrophobic nature of the majority of drugs used in antibody-drug conjugates leads to an increased hydrophobicity of the generated antibody-drug conjugates, resulting in higher liver clearance and decreased stability. Methods: In contrast, we describe a non-conventional approach in which the drug is conjugated in a buried part of the antibody. To achieve this, a ready-to-click antibody design was created in which an azido-based non-canonical amino acid is introduced within the Fab cavity during antibody synthesis using nonsense suppression technology. The Fab cavity was preferred over the Fc cavity to circumvent issues related to cleavage of the IgG1 lower hinge region in the tumor microenvironment. Results: This antibody design significantly increased the hydrophilicity of the generated antibody-drug conjugates compared to the current best-in-class designs based on non-canonical amino acids, while conjugation efficiency and functionality were maintained. The robustness of this native shielding effect and the versatility of this approach were also investigated. Conclusions: This pioneer design may become a starting point for the improvement of antibody-drug conjugates and an option to consider for protecting drugs and linkers from unspecific interactions.

Identification, localization and function of glutamate-gated chloride channel receptors in the honeybee brain.

Glutamate-gated chloride channels (GluCls) are members of the cys-loop ligand-gated ion channel superfamily whose presence has been reported in a variety of invertebrate tissues. In the honeybee, a single gene, amel_glucl, encoding a GluClα subunit, was found in the genome but both the pattern of expression of this gene in the bee brain and its functional role remained unknown. Here we localised the expression sites of the honeybee GluClα subunit at the mRNA and protein levels. To characterise the functional role of GluCls in the honeybee brain, we studied their implication in olfactory learning and memory by means of RNA interference (RNAi) against the GluClα subunit. We found that the GluClα subunit is expressed in the muscles, the antennae and the brain of honeybees. Expression of the GluClα protein was necessary for the retrieval of olfactory memories; more specifically, injection of dsRNA or siRNA resulted in a decrease in retention performances ∼24 h after injection. Knockdown of GluClα subunits impaired neither olfaction nor sucrose sensitivity, and did not affect the capacity to associate odor and sucrose. Our data provide the first evidence for the involvement of glutamate-gated chloride channels in olfactory memory in an invertebrate.

Forced Degradation Testing as Complementary Tool for Biosimilarity Assessment.

Oxidation of monoclonal antibodies (mAbs) can impact their efficacy and may therefore represent critical quality attributes (CQA) that require evaluation. To complement classical CQA, bevacizumab and infliximab were subjected to oxidative stress by H2O2 for 24, 48, or 72 h to probe their oxidation susceptibility. For investigation, a middle-up approach was used utilizing liquid chromatography hyphenated with mass spectrometry (LC-QTOF-MS). In both mAbs, the Fc/2 subunit was completely oxidized. Additional oxidations were found in the light chain (LC) and in the Fd' subunit of infliximab, but not in bevacizumab. By direct comparison of methionine positions, the oxidized residues in infliximab were assigned to M55 in LC and M18 in Fd'. The forced oxidation approach was further exploited for comparison of respective biosimilar products. Both for bevacizumab and infliximab, comparison of posttranslational modification profiles demonstrated high similarity of the unstressed reference product (RP) and the biosimilar (BS). However, for bevacizumab, comparison after forced oxidation revealed a higher susceptibility of the BS compared to the RP. It may thus be considered a useful tool for biopharmaceutical engineering, biosimilarity assessment, as well as for quality control of protein drugs.

Differences in long-term memory stability and AmCREB level between forward and backward conditioned honeybees (Apis mellifera).

In classical conditioning a predictive relationship between a neutral stimulus (conditioned stimulus; CS) and a meaningful stimulus (unconditioned stimulus; US) is learned when the CS precedes the US. In backward conditioning the sequence of the stimuli is reversed. In this situation animals might learn that the CS signals the end or the absence of the US. In honeybees 30 min and 24 h following backward conditioning a memory for the excitatory and inhibitory properties of the CS could be retrieved, but it remains unclear whether a late long-term memory is formed that can be retrieved 72 h following backward conditioning. Here we examine this question by studying late long-term memory formation in forward and backward conditioning of the proboscis extension response (PER). We report a difference in the stability of memory formed upon forward and backward conditioning with the same number of conditioning trials. We demonstrate a transcription-dependent memory 72 h after forward conditioning but do not observe a 72 h memory after backward conditioning. Moreover we find that protein degradation is differentially involved in memory formation following these two conditioning protocols. We report differences in the level of a transcription factor, the cAMP response element binding protein (CREB) known to induce transcription underlying long-term memory formation, following forward and backward conditioning. Our results suggest that these alterations in CREB levels might be regulated by the proteasome. We propose that the differences observed are due to the sequence of stimulus presentation between forward and backward conditioning and not to differences in the strength of the association of both stimuli.