Exosomes have been proposed as vehicles for microRNA (miRNA) -based intercellular communication and a source of miRNA biomarkers in bodily fluids. Although exosome preparations contain miRNAs, a quantitative analysis of their abundance and stoichiometry is lacking. In the course of studying cancer-associated extracellular miRNAs in patient blood samples, we found that exosome fractions contained a small minority of the miRNA content of plasma. This low yield prompted us to perform a more quantitative assessment of the relationship between miRNAs and exosomes using a stoichiometric approach. We quantified both the number of exosomes and the number of miRNA molecules in replicate samples that were isolated from five diverse sources (i.e., plasma, seminal fluid, dendritic cells, mast cells, and ovarian cancer cells). Regardless of the source, on average, there was far less than one molecule of a given miRNA per exosome, even for the most abundant miRNAs in exosome preparations (mean ± SD across six exosome sources: 0.00825 ± 0.02 miRNA molecules/exosome). Thus, if miRNAs were distributed homogenously across the exosome population, on average, over 100 exosomes would need to be examined to observe one copy of a given abundant miRNA. This stoichiometry of miRNAs and exosomes suggests that most individual exosomes in standard preparations do not carry biologically significant numbers of miRNAs and are, therefore, individually unlikely to be functional as vehicles for miRNA-based communication. We propose revised models to reconcile the exosome-mediated, miRNA-based intercellular communication hypothesis with the observed stoichiometry of miRNAs associated with exosomes.microvesicle | circulating

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Conventional techniques for detecting rare DNA sequences require many cycles of PCR amplification for high sensitivity and specificity, potentially introducing significant biases and errors. While amplification-free methods exist, they rarely achieve the ability to detect single molecules, and their ability to discriminate between single-nucleotide variants is often dictated by the specificity limits of hybridization thermodynamics. Here we show that a direct detection approach using single-molecule kinetic fingerprinting can surpass the thermodynamic discrimination limit by 3 orders of magnitude, with a dynamic range of up to 5 orders of magnitude with optional super-resolution analysis. This approach detects mutations as subtle as the drug-resistance-conferring cancer mutation EGFR T790M (a single C → T substitution) with an estimated specificity of 99.99999%, surpassing even the leading PCR-based methods and enabling detection of 1 mutant molecule in a background of at least 1 million wild-type molecules. This level of specificity revealed rare, heat-induced cytosine deamination events that introduce false positives in PCR-based detection, but which can be overcome in our approach through milder thermal denaturation and enzymatic removal of damaged nucleobases.

Current cell-free DNA (cfDNA) next generation sequencing (NGS) precision oncology workflows are typically limited to targeted and/or disease-specific applications. In advanced cancer, disease burden and cfDNA tumor content are often elevated, yielding unique precision oncology opportunities. We sought to demonstrate the utility of a pan-cancer, rapid, inexpensive, whole genome NGS of cfDNA approach (PRINCe) as a precision oncology screening strategy via ultra-low coverage (~0.01x) tumor content determination through genome-wide copy number alteration (CNA) profiling. We applied PRINCe to a retrospective cohort of 124 cfDNA samples from 100 patients with advanced cancers, including 76 men with metastatic castration-resistant prostate cancer (mCRPC), enabling cfDNA tumor content approximation and actionable focal CNA detection, while facilitating concordance analyses between cfDNA and tissue-based NGS profiles and assessment of cfDNA alteration associations with mCRPC treatment outcomes. Therapeutically relevant focal CNAs were present in 42 (34%) cfDNA samples, including 36 of 93 (39%) mCRPC patient samples harboring AR amplification. PRINCe identified pre-treatment cfDNA CNA profiles facilitating disease monitoring. Combining PRINCe with routine targeted NGS of cfDNA enabled mutation and CNA assessment with coverages tuned to cfDNA tumor content. In mCRPC, genome-wide PRINCe cfDNA and matched tissue CNA profiles showed high concordance (median Pearson correlation = 0.87), and PRINCe detectable AR amplifications predicted reduced time on therapy, independent of therapy type (Kaplan-Meier log-rank test, chi-square = 24.9, p < 0.0001). Our screening approach enables robust, broadly applicable cfDNA-based precision oncology for patients with advanced cancer through scalable identification of therapeutically relevant CNAs and pre-/post-treatment genomic profiles, enabling cfDNA- or tissue-based precision oncology workflow optimization.

ADAM12 has been implicated in cell-cell interactions in myogenesis and cancer, but the structure of the mature form of ADAM12 is not known, and its localization on the cell surface has been questioned. In this report, we show that full-length ADAM12 is N-glycosylated in the endoplasmic reticulum (ER) and proteolytically processed in the trans-Golgi network to an ϳ90-kDa form. The ϳ90-kDa form, which lacks the prodomain, was the predominant form present at the cell surface. Replacement of Leu 73 in the putative ␣-helical region in the prodomain with proline resulted in retention of ADAM12 in the ER and a complete lack of its processing. However, deletion of the entire pro-and metalloprotease domains did not affect the processing and trafficking of ADAM12. In contrast, replacement of the cytoplasmic domain of ADAM12 with that of ADAM9 or adding a c-Myc tag at the C terminus led to a significant increase in transport of the protein to the cell surface. These results suggest that the cytoplasmic domain of ADAM12 plays an important role in regulating ADAM12 exit from the ER. We conclude that properly folded mouse ADAM12, after passing a rate-limiting step of exit from the ER, is processed in the secretory pathway and reaches the cell surface, where it can mediate adhesionmediated signaling.ADAMs, a family of proteins containing a disintegrin and metalloprotease domain, play important roles in many biological processes involving cell-surface proteolysis and cell-cell or cell-matrix interactions (1-3). ADAMs have been implicated in many vital functions during development (4 -7) and in the pathogenesis of cancer (8 -10), rheumatoid arthritis (11), Alzheimer's disease (12), and inflammatory responses (9). With an exception of the ADAMTS subfamily (ADAMs with thrombospondin motifs) and alternative splice variants of several family members, ADAMs are type I transmembrane proteins expressed in all animal organisms from worm to human. A typical ADAM protein contains an N-terminal secretion signal and pro-, metalloprotease, disintegrin-like, cysteine-rich, epidermal growth factor-like, transmembrane, and cytoplasmic domains.The metalloprotease domains of many, but not all, ADAMs contain a consensus sequence (HEXXHXXGXXH) for the active site of zinc-dependent metalloproteases (13). These ADAMs are predicted to be active proteases involved in shedding of the ectodomains of membrane proteins, which is critical for cellsurface remodeling, regulating growth factor availability, and modulating the capacity of cells to respond to extracellular stimuli (14, 15). At least six members of the ADAM family have been demonstrated to have proteolytic activity. ADAM17 (tumor necrosis factor-␣-converting enzyme) releases soluble tumor necrosis factor-␣ from its membrane precursor (16,17). It also cleaves the tumor necrosis factor-␣ receptor, transforming growth factor-␣, and L-selectin (6); interleukin-1 receptor type II (18); amyloid precursor protein (12); ErbB4/HER4, a member of the epidermal growth factor receptor family (19); and Notch (...

ADAM 12, a member of the ADAM (protein containing a disintegrin and metalloprotease) family of metalloprotease-disintegrins, has been implicated in the differentiation and fusion of skeletal myoblasts, and its expression is dramatically up-regulated in many cancer cells. While the extracellular portion of ADAM 12 contains an active metalloprotease and a cell-adhesion domain, the function of the cytoplasmic portion is much less clear. In this paper, we show that the cytoplasmic tail of ADAM 12 mediates interactions with the non-receptor protein tyrosine kinase Src. The interaction is direct, specific, and involves the N-terminal proline-rich region in the cytoplasmic tail of ADAM 12 and the Src homology 3 (SH3) domain of Src. ADAM 12 and Src co-immunoprecipitate from transfected C2C12 cells, suggesting that the two proteins form a complex in vivo. Co-expression of Src and ADAM 12, but not ADAM 9, in C2C12 cells results in activation of the recombinant Src. Moreover, endogenous ADAM 12 associates with and activates endogenous Src in differentiating C2C12 cells. These results indicate that ADAM 12 may mediate adhesion-induced signalling during myoblast differentiation.

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The study aimed to investigate the reliability and validity of the Chinese version of the eating attitudes test (EAT-26) among female adolescents and young adults in Mainland China. This scale was administered to 396 female eating disorder patients and 406 noneating disorder healthy controls, in addition 35 healthy controls completed a retest after a 4-week intervals. Tests for reliability, convergent validity and receiver operating characteristic analysis were performed to detect the psychometric properties. The EAT-26 demonstrated good internal consistency (Cronbach's alpha = 0.822-0.922), test-retest reliability (interclass correlation coefficient = 0.817) and convergent validity(r = 0.450-0.750). The receiver operating characteristic analysis showed that the cut-off 14 for anorexia nervosa and 15 for bulimia nervosa represented good compromises with approximate sensitivity (0.66-0.68) and specificity (0.85-0.86). Our findings provided evidence that the Chinese version of the EAT-26 was a psychometrically reliable and valid self-rating instrument for identifying people suffering from an eating disorder in Mainland China. A clinical cut-off range between 14 and 15 could be used, but caution should be exercised because of the low sensitivity of the tool. Copyright © 2017 John Wiley & Sons, Ltd and Eating Disorders Association.