Priming agents transiently reduce the clearance of cell-free DNA to improve liquid biopsies.

Liquid biopsies enable early detection and monitoring of diseases such as cancer, but their sensitivity remains limited by the scarcity of analytes such as cell-free DNA (cfDNA) in blood. Improvements to sensitivity have primarily relied on enhancing sequencing technology ex vivo. We sought to transiently augment the level of circulating tumor DNA (ctDNA) in a blood draw by attenuating its clearance in vivo. We report two intravenous priming agents given 1 to 2 hours before a blood draw to recover more ctDNA. Our priming agents consist of nanoparticles that act on the cells responsible for cfDNA clearance and DNA-binding antibodies that protect cfDNA. In tumor-bearing mice, they greatly increase the recovery of ctDNA and improve the sensitivity for detecting small tumors.

Inhalable point-of-care urinary diagnostic platform.

Although low-dose computed tomography screening improves lung cancer survival in at-risk groups, inequality remains in lung cancer diagnosis due to limited access to and high costs of medical imaging infrastructure. We designed a needleless and imaging-free platform, termed PATROL (point-of-care aerosolizable nanosensors with tumor-responsive oligonucleotide barcodes), to reduce resource disparities for early detection of lung cancer. PATROL formulates a set of DNA-barcoded, activity-based nanosensors (ABNs) into an inhalable format. Lung cancer-associated proteases selectively cleave the ABNs, releasing synthetic DNA reporters that are eventually excreted via the urine. The urinary signatures of barcoded nanosensors are quantified within 20 min at room temperature using a multiplexable paper-based lateral flow assay. PATROL detects early-stage tumors in an autochthonous lung adenocarcinoma mouse model with high sensitivity and specificity. Tailoring the library of ABNs may enable not only the modular PATROL platform to lower the resource threshold for lung cancer early detection tools but also the rapid detection of chronic pulmonary disorders and infections.

An intravenous DNA-binding priming agent protects cell-free DNA and improves the sensitivity of liquid biopsies.

Blood-based, or "liquid," biopsies enable minimally invasive diagnostics but have limits on sensitivity due to scarce cell-free DNA (cfDNA). Improvements to sensitivity have primarily relied on enhancing sequencing technology ex vivo . Here, we sought to augment the level of circulating tumor DNA (ctDNA) detected in a blood draw by attenuating the clearance of cfDNA in vivo . We report a first-in-class intravenous DNA-binding priming agent given 2 hours prior to a blood draw to recover more cfDNA. The DNA-binding antibody minimizes nuclease digestion and organ uptake of cfDNA, decreasing its clearance at 1 hour by over 150-fold. To improve plasma persistence and limit potential immune interactions, we abrogated its Fc-effector function. We found that it protects GC-rich sequences and DNase-hypersensitive sites, which are ordinarily underrepresented in cfDNA. In tumor-bearing mice, priming improved tumor DNA recovery by 19-fold and sensitivity for detecting cancer from 6% to 84%. These results suggest a novel method to enhance the sensitivity of existing DNA-based cancer testing using blood biopsies.

A nanoparticle priming agent reduces cellular uptake of cell-free DNA and enhances the sensitivity of liquid biopsies.

Liquid biopsies are enabling minimally invasive monitoring and molecular profiling of diseases across medicine, but their sensitivity remains limited by the scarcity of cell-free DNA (cfDNA) in blood. Here, we report an intravenous priming agent that is given prior to a blood draw to increase the abundance of cfDNA in circulation. Our priming agent consists of nanoparticles that act on the cells responsible for cfDNA clearance to slow down cfDNA uptake. In tumor-bearing mice, this agent increases the recovery of circulating tumor DNA (ctDNA) by up to 60-fold and improves the sensitivity of a ctDNA diagnostic assay from 0% to 75% at low tumor burden. We envision that this priming approach will significantly improve the performance of liquid biopsies across a wide range of clinical applications in oncology and beyond.

Direct capture of neutralized RBD enables rapid point-of-care assessment of SARS-CoV-2 neutralizing antibody titer.

Neutralizing antibody (NAb) titer is a key biomarker of protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but point-of-care methods for assessing NAb titer are not widely available. Here, we present a lateral flow assay that captures SARS-CoV-2 receptor-binding domain (RBD) that has been neutralized from binding angiotensin-converting enzyme 2 (ACE2). Quantification of neutralized RBD in this assay correlates with NAb titer from vaccinated and convalescent patients. This methodology demonstrated superior performance in assessing NAb titer compared with either measurement of total anti-spike immunoglobulin G titer or quantification of the absolute reduction in binding between ACE2 and RBD. Our testing platform has the potential for mass deployment to aid in determining at population scale the degree of protective immunity individuals may have following SARS-CoV-2 vaccination or infection and can enable simple at-home assessment of NAb titer.

Protease Activity Analysis: A Toolkit for Analyzing Enzyme Activity Data.

Analyzing the activity of proteases and their substrates is critical to defining the biological functions of these enzymes and to designing new diagnostics and therapeutics that target protease dysregulation in disease. While a wide range of databases and algorithms have been created to better predict protease cleavage sites, there is a dearth of computational tools to automate analysis of in vitro and in vivo protease assays. This necessitates individual researchers to develop their own analytical pipelines, resulting in a lack of standardization across the field. To facilitate protease research, here we present Protease Activity Analysis (PAA), a toolkit for the preprocessing, visualization, machine learning analysis, and querying of protease activity data sets. PAA leverages a Python-based object-oriented implementation that provides a modular framework for streamlined analysis across three major components. First, PAA provides a facile framework to query data sets of synthetic peptide substrates and their cleavage susceptibilities across a diverse set of proteases. To complement the database functionality, PAA also includes tools for the automated analysis and visualization of user-input enzyme-substrate activity measurements generated through in vitro screens against synthetic peptide substrates. Finally, PAA supports a set of modular machine learning functions to analyze in vivo protease activity signatures that are generated by activity-based sensors. Overall, PAA offers the protease community a breadth of computational tools to streamline research, taking a step toward standardizing data analysis across the field and in chemical biology and biochemistry at large.

Urinary detection of lung cancer in mice via noninvasive pulmonary protease profiling.

Lung cancer is the leading cause of cancer-related death, and patients most commonly present with incurable advanced-stage disease. U.S. national guidelines recommend screening for high-risk patients with low-dose computed tomography, but this approach has limitations including high false-positive rates. Activity-based nanosensors can detect dysregulated proteases in vivo and release a reporter to provide a urinary readout of disease activity. Here, we demonstrate the translational potential of activity-based nanosensors for lung cancer by coupling nanosensor multiplexing with intrapulmonary delivery and machine learning to detect localized disease in two immunocompetent genetically engineered mouse models. The design of our multiplexed panel of sensors was informed by comparative transcriptomic analysis of human and mouse lung adenocarcinoma datasets and in vitro cleavage assays with recombinant candidate proteases. Intrapulmonary administration of the nanosensors to a Kras- and Trp53-mutant lung adenocarcinoma mouse model confirmed the role of metalloproteases in lung cancer and enabled accurate detection of localized disease, with 100% specificity and 81% sensitivity. Furthermore, this approach generalized to an alternative autochthonous model of lung adenocarcinoma, where it detected cancer with 100% specificity and 95% sensitivity and was not confounded by lipopolysaccharide-driven lung inflammation. These results encourage the clinical development of activity-based nanosensors for the detection of lung cancer.

Procaine Inhibits Osteo/Odontogenesis through Wnt/ß-Catenin Inactivation.

INTRODUCTION: Periodontitis is a complex pathology characterized by the loss of alveolar bone. The causes and the mechanisms that promote this bone resorption still remain unknown. The knowledge of the critical regulators involved in the alteration of alveolar bone homeostasis is of great importance for developing molecular therapies. Procaine is an anesthetic drug with demethylant properties, mainly used by dentists in oral surgeries. The inhibitor role of Wnt signaling of procaine was described in vitro in colon cancer cells. METHODS: In this work we evaluated the role of procaine (1 uM) in osteo/odontogenesis of rat bone marrow mesenchymal stem cells. Similarly, the mechanisms whereby procaine achieves these effects were also studied. RESULTS: Procaine administration led to a drastic decrease of calcium content, alkaline phosphatase activity, alizarin red staining and an increase in the expression of Matrix Gla Protein. With respect to osteo/odontogenic markers, procaine decreased early and mature osteo/odontogenic markers. In parallel, procaine inhibited canonical Wnt/ß-catenin pathway, observing a loss of nuclear ß-catenin, a decrease in Lrp5 and Frizzled 3, a significant increase of sclerostin and Gsk3ß and an increase of phosphorylated ß-catenin. The combination of osteo/odontogenic stimuli and Lithium Chloride decreased mRNA expression of Gsk3ß, recovered by Procaine. Furthermore it was proved that Procaine alone dose dependently increases the expression of Gsk3ß and ß-catenin phosphorylation. These effects of procaine were also observed on mature osteoblast. Interestingly, at this concentration of procaine no demethylant effects were observed. CONCLUSIONS: Our results demonstrated that procaine administration drastically reduced the mineralization and osteo/odontogenesis of bone marrow mesenchymal stem cells inhibiting Wnt/ß-catenin pathway through the increase of Gsk3ß expression and ß-catenin phosphorylation.