High-throughput droplet digital PCR system for absolute quantitation of DNA copy number

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ~2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100,000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.     

Improved nursing home end-stage renal disease patient participation in physical therapy with onsite,more frequent dialysis

Introduction

For end-stage renal disease (ESRD) patients residing in skilled nursing facilities (SNFs), the logistics and physical exhaustion of life-saving hemodialysis therapy often conflict with rehabilitation goals. Integration of dialysis care with rehabilitation programs in a scalable and cost-efficient manner has been a significant challenge. SNF-resident ESRD patients receiving onsite, more frequent hemodialysis (MFD) have reported rapid post-dialysis recovery. We examined whether such patients have improved Physical Therapy (PT) participation.

Methods

We conducted a retrospective electronic medical records review of SNF-resident PT participation rates within a multistate provider of SNF rehabilitation care from January 1, 2022 to June 1, 2022. We compared three groups: ESRD patients receiving onsite MFD (Onsite-MFD), ESRD patients receiving offsite, conventional 3×/week dialysis (Offsite-Conventional-HD), and the general non-ESRD SNF rehabilitation population (Non-ESRD). We evaluated physical therapy participation rates based on a predefined metric of missed or shortened (<15 min) therapy days. Baseline demographics and functional status were assessed.

Findings

Ninety-two Onsite-MFD had 2084 PT sessions scheduled, 12,916 Non-ESRD had 225,496 PT sessions scheduled, and 562 Offsite-Conventional-HD had 9082 PT sessions scheduled. In mixed model logistic regression, Onsite-MFD achieved higher PT participation rates than Offsite-Conventional-HD (odds ratio: 1.8, CI: 1.1–3.0; p < 0.03), and Onsite-MFD achieved equivalent PT participation rates to Non-ESRD (odds ratio: 1.2, CI: 0.3–1.9; p < 0.46). Baseline mean ± SD Charlson Comorbidity score was significantly higher in Onsite-MFD (4.9 ± 2.0) and Offsite-Conventional-HD (4.9 ± 1.8) versus Non-ESRD (2.6 ± 2.0; p < 0.001). Baseline mean self-care and mobility scores were significantly lower in Onsite-MFD versus Non-ESRD or Offsite-Conventional-HD.

Discussion

SNF-resident ESRD patients receiving MFD colocated with rehabilitation had higher PT participation rates than those conventionally dialyzed offsite and equivalent PT participation rates to the non-ESRD SNF-rehabilitation general population, despite being sicker, less independent, and less mobile. We report a scalable program integrating dialysis and rehabilitation care as a potential solution for ESRD patients recovering from acute hospitalization.     

Lysosomes Targeting pH Activable Imaging-Guided Photodynamic Agents

Photodynamic therapy (PDT) is a photochemistry-based medical treatment combining light at a specific wavelength and a photosensitizer (PS) in the presence of oxygen. Application of PDT as a conventional treatment is limited and clearly the approval in clinics of new PS is challenging. The selective accumulation of the PS in the targeted malignant cells is of paramount importance to reduce the side effects that are typical of the current worldwide approved PS. Here we report a new series of aniline- and iodine-substituted BODIPY derivatives ( 1 – 3 ) as promising lysosome-targeting and pH-responsive theranostic PS, which displayed a significant in vitro light-induced cytotoxicity, efficient imaging properties and low dark toxicity (for 2 and 3 ). These compounds were obtained in few reproducible synthetic steps and good yields. Spectroscopic and electrochemical measurements along with computational calculations confirmed the quenching of the emissive properties of the PS, while both fluorescence and ¹O₂ emission were obtained only under acidic conditions inducing amine protonation. The pK_a values and pH-dependent emissive properties of 1 – 3 being established, their cellular uptake and activation in the lysosomal vesicles (pH≈4-5) were confirmed by their co-localization with the commercial LysoTracker deep red and light-induced cytotoxicity (IC₅₀ between 0.16 and 0.06 μM) against HeLa cancer cells.