Detection of somatic mutations and copy number alterations in circulating tumour DNA in patients with primary breast cancer by next-generation sequencing: an in-depth analysis of the Neocent trial to evaluate the efficacy of neoadjuvant therapy

Breast cancer is one of the most common malignant tumours in women worldwide. With the continuous advancement of new technologies, more accurate and earlier detection and diagnosis of cancer have become possible. This thesis explored the use circulating tumour DNA (ctDNA) in patients with breast cancer. In the Neocent study, paired tumour whole exome sequence data was available before and after 21 weeks neoadjuvant therapy and bioinformatics approaches were used to compare exome profiles. Shallow whole genome sequencing (sWGS) was also used to explore somatic copy number alterations (SCNA) in ctDNA from in these patients. A related study in patients with metastatic breast cancer (MBC) evaluated NGS analysis of specific mutations using the Oncomine breast cancer cfDNA assay and results were compared with detection of SCNA by sWGS.  Results from two bioinformatics workflows, Sequenza and SuperFreq, In Neocent tumour exome data showed that SuperFreq analysis was more reflective of focal gene-specific amplification than Sequenza analysis as determined by ddPCR analysis. The results of sWGS showed that unfortunately Neocent baseline plasma had limited or no detectable ctDNA at diagnosis. In the MBC cohort (the CTCM cohort study), results showed that ReproSeq was more sensitive than Oncomine in revealing SCNA. However, Oncomine was more sensitive in detecting ctDNA overall. Finally, in a pilot study comparison of Oncomine ctDNA profiles and treatment outcomes in the ALERT cohort demonstrated the utility of ctDNA testing in monitoring treatment response.  In conclusion, results presented in this thesis demonstrate the potential of ctDNA analysis to monitor breast cancer patients at the time of neoadjuvant therapy and in metastatic disease to inform clinical decision-making.</p

Assessment of Quantification Precision of Histone Post-Translational Modifications by Using an Ion Trap and down To 50 000 Cells as Starting Material

Histone post-translational modifications (PTMs) are fundamental players of chromatin regulation, as they contribute to editing histone chemical properties and recruiting proteins for gene transcription and DNA repair. Mass spectrometry (MS)-based proteomics is currently the most widely adopted strategy for high-throughput quantification of hundreds of histone PTMs. Samples such as primary tissues, complex model systems, and biofluids are hard to retrieve in large quantities. Because of this, it is critical to know whether the amount of sample available would lead to an exhaustive analysis if subjected to MS. In this work, we assessed the reproducibility in quantification of histone PTMs using a wide range of starting material, that is, from 5 000 000 to 50 000 cells. We performed the experiment using four different cell lines, that is, HeLa, 293T, human embryonic stem cells (hESCs), and myoblasts, and we quantified a list of 205 histone peptides using ion trap MS and our in-house software. Results highlighted that the relative abundance of some histone PTMs deviated as little as just 4% when comparing high starting material with histone samples extracted from 50 000 cells, for example, H3K9me2 (40% average abundance). Low abundance PTMs such as H3K4me2 (<3% average abundance) showed higher variability, but still ∼34%. This indicates that most PTMs, and especially abundant ones, are quantified with high precision starting from low cell counts. This study will help scientists to decide whether specific experiments are feasible and to plan how much sample should be reserved for histone analysis using MS

Oscillatory Enzyme Dynamics Revealed by Two-Dimensional Infrared Spectroscopy

Enzymes move on a variety of length and time scales. While much is known about large structural fluctuations that impact binding of the substrates and release of products, little is known about faster motions of enzymes and how these motions may influence enzyme-catalyzed reactions. This Letter reports frequency fluctuations of the azide anion bound to the active site of formate dehydrogenase measured via 2D IR spectroscopy. These measurements reveal an underdamped oscillatory component to the frequency–frequency correlation function when the azide is bound to the NAD⁺ ternary complex. This oscillation disappears when the reduced cofactor is added, indicating that the oscillating contributions most likely come from the charged nicotinamide ring. These oscillatory motions may be relevant to donor–acceptor distance sampling of the catalyzed hydride transfer and therefore may give future insights into the dynamic behavior involved in enzyme catalysis

Additional file 1: of Effectiveness of transcutaneous electrical nerve stimulation for the treatment of migraine: a meta-analysis of randomized controlled trials

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Different Timing Features in Brain Processing of Core and Moral Disgust Pictures: An Event-Related Potentials Study

<div><p>Disgust, an emotion motivating withdrawal from offensive stimuli, protects us from the risk of biological pathogens and sociomoral violations. Homogeneity of its two types, namely, core and moral disgust has been under intensive debate. To examine the dynamic relationship between them, we recorded event-related potentials (ERPs) for core disgust, moral disgust and neutral pictures while participants performed a modified oddball task. ERP analysis revealed that N1 and P2 amplitudes were largest for the core disgust pictures, indicating automatic processing of the core disgust-evoking pictures. N2 amplitudes were higher for pictures evoking moral disgust relative to core disgust and neutral pictures, reflecting a violation of social norms. The core disgust pictures elicited larger P3 and late positive potential (LPP) amplitudes in comparison with the moral disgust pictures which, in turn, elicited larger P3 and LPP amplitudes when compared to the neutral pictures. Taken together, these findings indicated that core and moral disgust pictures elicited different neural activities at various stages of information processing, which provided supporting evidence for the heterogeneity of disgust.</p></div

The schematic illustration of the experimental procedure.

<p>Core disgust: vomit. Moral disgust: a bad antisocial action, a person stamping on a disabled beggar. Picture presentation was terminated by a key pressing or when 1000 ms was elapsed.</p

Stimulus-locked grand average ERP waveforms.

<p>Grand average ERP waveforms recorded from Fz, FCz, Cz, CPz and Pz in response to core disgust (black lines), moral disgust (red lines) and neutral (blue lines) pictures.</p

Topographical maps of voltage amplitudes of N1, P2, N2, P3 and LPP across three conditions.

<p>Topographical maps of voltage amplitudes of N1, P2, N2, P3 and LPP across three conditions.</p

Results of ANOVA for the amplitudes of N1, P2, N2, P3 and LPP components.

<p>Results of ANOVA for the amplitudes of N1, P2, N2, P3 and LPP components.</p

The reaction times and accuracies for three types of stimuli (<i>M±SD</i>).

<p>The reaction times and accuracies for three types of stimuli (<i>M±SD</i>).</p