SignificanceThe recent advances in cancer immunotherapy motivated us to investigate the clonal structure of the T cell receptor repertoire in breast tumors, normal breast, and blood in the same individuals. We found quantitatively distinct clonal structures in all three tissues, which enabled us to predict whether tissue is normal or tumor solely by comparing the repertoire of the tissue with blood. T cell receptor sequences shared between patients’ tumors are rare and, in general, do not appear to be specific to the cancer.

Genomic characterization of circulating tumor cells (CTCs) may prove useful as a surrogate for conventional tissue biopsies. This is particularly important as studies have shown different mutational profiles between CTCs and ctDNA in some tumor subtypes. However, isolating rare CTCs from whole blood has significant hurdles. Very limited DNA quantities often can’t meet NGS requirements without whole genome amplification (WGA). Moreover, white blood cells (WBC) germline contamination may confound CTC somatic mutation analyses. Thus, a good CTC enrichment platform with an efficient WGA and NGS workflow are needed. Here, Vortex label-free CTC enrichment platform was used to capture CTCs. DNA extraction was optimized, WGA evaluated and targeted NGS tested. We used metastatic colorectal cancer (CRC) as the clinical target, HCT116 as the corresponding cell line, GenomePlex® and REPLI-g as the WGA methods, GeneRead DNAseq Human CRC Panel as the 38 gene panel. The workflow was further validated on metastatic CRC patient samples, assaying both tumor and CTCs. WBCs from the same patients were included to eliminate germline contaminations. The described workflow performed well on samples with sufficient DNA, but showed bias for rare cells with limited DNA input. REPLI-g provided an unbiased amplification on fresh rare cells, enabling an accurate variant calling using the targeted NGS. Somatic variants were detected in patient CTCs and not found in age matched healthy donors. This demonstrates the feasibility of a simple workflow for clinically relevant monitoring of tumor genetics in real time and over the course of a patient’s therapy using CTCs.

Cepstral- and spectral-based measures that have been previously studied in dysphonia characterized by breathiness and roughness are effective in distinguishing strained dysphonia from normal voice quality. The utility of these acoustic measures is supported by their moderate-to-high relationship with perceptually rated strain severity.

Mononuclear non-heme Fe­(II)- and α-ketoglutarate-dependent oxygenases (FeDOs) catalyze a site-selective C–H hydroxylation. Variants of these enzymes in which a conserved Asp/Glu residue in the Fe­(II)-binding facial triad is replaced by Ala/Gly can, in some cases, bind various anionic ligands and catalyze non-native chlorination and bromination reactions. In this study, we explore the binding of different anions to an FeDO facial triad variant, SadX, and the effects of that binding on HO• vs X• rebound. We establish not only that chloride and bromide enable non-native halogenation reactions but also that all anions investigated, including azide, cyanate, formate, and fluoride, significantly accelerate and influence the site selectivity of SadX hydroxylation catalysis. Azide and cyanate also lead to the formation of products resulting from N3 •, NCO•, and OCN• rebound. While fluoride rebound is not observed, the rate acceleration provided by this ligand leads us to calculate barriers for HO• and F• rebound from a putative Fe­(III)­(OH)­(F) intermediate. These calculations suggest that the lack of fluorination is due to the relative barriers of the HO• and F• rebound transition states rather than an inaccessible barrier for F• rebound. Together, these results improve our understanding of the FeDO facial triad variant tolerance of different anionic ligands, their ability to promote rebound involving these ligands, and inherent rebound preferences relative to HO• that will aid efforts to develop non-native catalysis using these enzymes.