Cancer is one of the leading causes of premature death, and, as such, it can be prevented by developing strategies for early and accurate diagnosis. Cancer diagnostics has evolved from the macroscopic detection of malignant tissues to the fine analysis of tumor biomarkers using personalized medicine approaches. Recently, various nanomaterials have been introduced into the molecular diagnostics of cancer. This has resulted in a number of tumor biomarkers that have been detected in vitro and in vivo using nanodevices and corresponding imaging techniques. Atomically precise ligand-protected noble metal quantum nanoclusters represent an interesting class of nanomaterials with a great potential for the detection of tumor biomarkers. They are characterized by high biocompatibility, low toxicity, and suitability for controlled functionalization with moieties specifically recognizing tumor biomarkers. Their non-linear optical properties are of particular importance as they enable the visualization of nanocluster-labeled tumor biomarkers using non-linear optical techniques such as two-photon-excited fluorescence and second harmonic generation. This article reviews liganded nanoclusters among the different nanomaterials used for molecular cancer diagnosis and the relevance of this new class of nanomaterials as non-linear optical probe and contrast agents.

During mitosis, Bub1 kinase phosphorylates histone H2A-T120 to promote centromere sister chromatid cohesion through recruitment of shugoshin (Sgo) proteins. The regulation and dynamics of H2A-T120 phosphorylation are poorly understood. Using quantitative phosphoproteomics we show that Bub1 is autophosphorylated at numerous sites. We confirm mitosis-specific autophosphorylation of a several residues and show that Bub1 activation is primed in interphase but fully achieved only in mitosis. Mutation of a single autophosphorylation site T589 alters kinetochore turnover of Bub1 and results in uniform H2A-T120 phosphorylation and Sgo recruitment along chromosome arms. Consequently, improper sister chromatid resolution and chromosome segregation errors are observed. Kinetochore tethering of Bub1-T589A refocuses H2A-T120 phosphorylation and Sgo1 to centromeres. Recruitment of the Bub1-Bub3-BubR1 axis to kinetochores has recently been extensively studied. Our data provide novel insight into the regulation and kinetochore residency of Bub1 and indicate that its localization is dynamic and tightly controlled through feedback autophosphorylation.

Atomically precise, ligand-protected gold nanoclusters (AuNCs) attract considerable attention as contrast agents in the biosensing field. However, the control of their optical properties and functionalization of surface ligands remain challenging. Here we report a strategy to tailor AuNCs for the precise detection of protein carbonylation—a causal biomarker of ageing. We produce Au₁₅SG₁₃ (SG for glutathione) with atomic precision and functionalize it with a thiolated aminooxy moiety to impart protein carbonyl-binding properties. Mass spectrometry and molecular modelling reveal the key structural features of Au₁₅SG₁₂-Aminooxy and its reactivity towards carbonyls. Finally, we demonstrate that Au₁₅SG₁₂-Aminooxy detects protein carbonylation in gel-based 1D electrophoresis by one- and two-photon excited fluorescence. Importantly, to our knowledge, this is the first application of an AuNC that detects a post-translational modification as a nonlinear optical probe. The significance of post-translational modifications in life sciences may open avenues for the use of Au₁₅SG₁₃ and other nanoclusters as contrast agents with tailored surface functionalization and optical properties.

Monopolar spindle 1 (Mps1) is a conserved apical kinase in the spindle assembly checkpoint (SAC) that ensures accurate segregation of chromosomes during mitosis. Mps1 undergoes extensive auto- and transphosphorylation, but the regulatory and functional consequences of these modifications remain unclear. Recent findings highlight the importance of intermolecular interactions between the N-terminal extension (NTE) of Mps1 and the Hec1 subunit of the NDC80 complex, which control Mps1 localization at kinetochores and activation of the SAC. Whether the NTE regulates other mitotic functions of Mps1 remains unknown. Here, we report that phosphorylation within the NTE contributes to Mps1 activation through relief of catalytic autoinhibition that is mediated by the NTE itself. Moreover, we find that this regulatory NTE function is independent of its role in Mps1 kinetochore recruitment. We demonstrate that the NTE autoinhibitory mechanism impinges most strongly on Mps1-dependent SAC functions and propose that Mps1 activation likely occurs sequentially through dimerization of a "prone-to-autophosphorylate" Mps1 conformer followed by autophosphorylation of the NTE prior to maximal kinase activation segment trans-autophosphorylation. Our observations underline the importance of autoregulated Mps1 activity in generation and maintenance of a robust SAC in human cells.

Oxidative stress and oxidative protein damage occur in various biological processes and diseases. The carbonyl group on amino acid side chains is the most widely used protein oxidation biomarker. Carbonyl groups are commonly detected indirectly through their reaction with 2,4-dinitrophenylhydrazine (DNPH) and subsequent labeling with an anti-DNP antibody. However, the DNPH immunoblotting method lacks protocol standardization, exhibits technical bias, and has low reliability. To overcome these shortcomings, we have developed a new blotting method in which the carbonyl group reacts with the biotin-aminooxy probe to form a chemically stable oxime bond. The reaction speed and the extent of the carbonyl group derivatization are increased by adding a p-phenylenediamine (pPDA) catalyst under neutral pH conditions. These improvements are crucial since they ensure that the carbonyl derivatization reaction reaches a plateau within hours and increases the sensitivity and robustness of protein carbonyl detection. Furthermore, derivatization under pH-neutral conditions facilitates a good SDS-PAGE protein migration pattern, avoids protein loss by acidic precipitation, and is directly compatible with protein immunoprecipitation. This work describes the new Oxime blot method and demonstrates its use in detecting protein carbonylation in complex matrices from diverse biological samples.