This study has demonstrated that genomewide NIPT for fetal chromosomal abnormalities can provide high resolution, sensitive, and specific detection of a wide range of subchromosomal and whole chromosomal abnormalities that were previously only detectable by invasive karyotype analysis. In some instances, this NIPT also provided additional clarification about the origin of genetic material that had not been identified by invasive karyotype analysis.

In real time: Thrombin activation in vivo can be imaged in real time with ratiometric activatable cell penetrating peptides (RACPPs). RACPPs are designed to combine 1) dual‐emission ratioing, 2) far red to infrared wavelengths for in vivo mammalian imaging, and 3) cleavage‐dependent spatial localization. The most advanced RACPP uses norleucine (Nle)‐TPRSFL as a linker that increases sensitivity to thrombin by about 90‐fold (see figure).

The measurement of trypsin activity directly in blood is important for the development of novel diagnostics and for biomedical research. Presently, most degradative enzyme assays require sample preparation, making them time consuming, costly, and less accurate. We recently demonstrated a simple and rapid electrophoretic assay for the measurement of trypsin activity directly in whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target enzyme. This fragment is then rapidly separated from whole blood by electrophoresis and quantified with a fluorescent detector. In this study, we demonstrate that polyanionic poly-L-glutamic acid-doped polyacrylamide gels can focus the fluorescent cleavage product and markedly improve the LODs of the assay. A LOD of 2 pg in 6 microL (0.3 ng/mL) in whole human blood was achieved after a 1-h reaction of enzyme and substrate followed by 10 min of electrophoresis. This is 50- to 200-fold better than the estimated reference levels for trypsin (15-60 ng/mL) in blood. This straightforward technique now allows for the rapid measurement of clinically relevant levels of trypsin activity in microliter volumes of whole blood, providing a useful tool for the development of novel point-of-care diagnostics.

In biomedical research and clinical diagnostics, it is a major challenge to measure disease-related degradative enzyme activity directly in whole blood. Present techniques for assaying degradative enzyme activity require sample preparation, which makes the assays time-consuming and costly. This study now describes a simple and rapid electrophoretic method that allows detection of degradative enzyme activity directly in whole blood using charge-changing fluorescent peptide substrates. Charge-changing substrates eliminate the need for sample preparation by producing positively charged cleavage fragments that can be readily separated from the oppositely charged fluorescent substrate and blood components by electrophoresis. Two peptide substrates have been developed for pancreatic alpha-chymotrypsin and trypsin. For the first substrate, a detection limit of 3 ng for both alpha-chymotrypsin and trypsin was achieved in whole rat blood using a 4% agarose gel. This substrate had minimal cross-reactivity with the trypsin-like proteases thrombin, plasmin, and kallikrein. For the second substrate (trypsin-specific), a detection limit of about 10-20 pg was achieved using thinner higher resolution 20 and 25% polyacrylamide gels. Thus, the new charge changing peptide substrates enable a simple electrophoretic assay format for the measurement of degradative enzyme activity, which is an important step toward the development of novel point-of-care diagnostics.

Extracellular proteases including thrombin are involved in numerous biological processes and play major roles in a variety of human diseases. The spatial and temporal patterns of activation of proteases in vivo control their biological role in diseases and amenability to therapeutic targeting. Previously we developed activatable cell-penetrating peptides (ACPPs) to monitor matrix metalloproteinase (MMP) and elastase activity in tumors. Later ACPPs detect thrombin activation in atherosclerosis and brain injury. We have now modified the thrombin ACPP in two independent ways, 1) to provide a FRET-dependent emission ratiometric readout and 2) to accelerate the kinetics of cleavage by thrombin. Emission ratioing improves kinetic detection of enzyme activity, because it reflects the ratio of cleaved versus uncleaved probe but cancels out total probe concentration, illumination intensity, detection sensitivity, and tissue thickness. Because pharmacokinetic washout of the uncleaved probe is not necessary, yet the cleavage converts a diffusible substrate into an immobilized product, thrombin activity can be imaged in real time with good spatial resolution. Meanwhile, placement of norleucine-threonine (Nle-Thr) at the P4-P3 substrate positions accelerates the kinetics of thrombin cleavage by 1-2 orders of magnitude, while preserving selectivity against related proteases. The new ratiometric ACPPs detect localized thrombin activation in rapidly forming blood clots minutes after probe injection, and the signal is inhibited by thrombin specific inhibitors.Thrombin is a serine protease and a key regulator of blood coagulation. It is responsible for the proteolytic cleavage and activation of multiple coagulation factors including Factor V,