New realm of precision multiplexing enabled by massively-parallel single molecule UltraPCR.

PCR has been a reliable and inexpensive method for nucleic acid detection in the past several decades. In particular, multiplex PCR is a powerful tool to analyze many biomarkers in the same reaction, thus maximizing detection sensitivity and reducing sample usage. However, balancing the amplification kinetics between amplicons and distinguishing them can be challenging, diminishing the broad adoption of high order multiplex PCR panels. Here, we present a new paradigm in PCR amplification and multiplexed detection using UltraPCR. UltraPCR utilizes a simple centrifugation workflow to split a PCR reaction into ∼34 million partitions, forming an optically clear pellet of spatially separated reaction compartments in a PCR tube. After in situ thermocycling, light sheet scanning is used to produce a 3D reconstruction of the fluorescent positive compartments within the pellet. At typical sample DNA concentrations, the magnitude of partitions offered by UltraPCR dictate that the vast majority of target molecules occupy a compartment uniquely. This single molecule realm allows for isolated amplification events, thereby eliminating competition between different targets and generating unambiguous optical signals for detection. Using a 4-color optical setup, we demonstrate that we can incorporate 10 different fluorescent dyes in the same UltraPCR reaction. We further push multiplexing to an unprecedented level by combinatorial labeling with fluorescent dyes - referred to as "comboplex" technology. Using the same 4-color optical setup, we developed a 22-target comboplex panel that can detect all targets simultaneously at high precision. Collectively, UltraPCR has the potential to push PCR applications beyond what is currently available, enabling a new class of precision genomics assays.

Bilateral Gluteal Fasciocutaneous Advancement Flaps With and Without Tie-Over Sutures in Treatment of Chronic Pilonidal Disease: A Prospective Case Series.

PURPOSE: Flap procedures following pilonidal excision have high recurrence and dehiscence rates. We present a cosmetic, outpatient technique to reconstruction via bilateral gluteal fasciocutaneous advancement flaps with and without tie-over sutures. METHODS: This is a prospective case series of 51 patients (40 males and 11 females). Following elliptical excision of pilonidal disease, gluteal fasciocutaneous advancement flaps were elevated circumferentially using blunt, discontinuous dissection, and a multilayered closure was performed. The resulting scar was midline. Thirty-five patients (68.6%) also had two full-thickness, compressing sutures tied over rolled up gauze. RESULTS: Patients had a mean age of 28.2 and body mass index of 26.8. Eight (15.9%) were smokers and 11 (21.6%) were obese. At a mean follow-up of 38.7 months, there were no recurrences and 19 (37.3%) patients had wound dehiscence. There was no significant difference in dehiscence between patients with and without tie-over sutures (31.4% vs 50%, P = 0.20). There was no significant difference in dehiscence between smokers and non-smokers, (62.5% vs 41.9%, P = 0.47), or between obese and non-obese patients (36.4% vs 46.3%, P = 0.51). Obese patients with tie-over sutures had significantly less dehiscence compared to obese patients without tie-over sutures (14% vs 75%, P = 0.03). CONCLUSION: Bilateral gluteal fasciocutaneous advancement flap with consideration of tie-over sutures is an outpatient treatment for chronic pilonidal disease with resultant midline scar and with no recurrence in our series.

Next-Generation Digital Polymerase Chain Reaction: High-Dynamic-Range Single-Molecule DNA Counting via Ultrapartitioning.

Digital PCR (dPCR) was first conceived for single-molecule quantitation. However, current dPCR systems often require DNA templates to share partitions due to limited partitioning capacities. Here, we introduce UltraPCR, a next-generation dPCR system where DNA counting is performed in a single-molecule regimen through a 6-log dynamic range using a swift and parallelized workflow. Each UltraPCR reaction is divided into >30 million partitions without microfluidics to achieve single template occupancy. Combined with a unique emulsion chemistry, partitions are optically clear, enabling the use of a three-dimensional imaging technique to rapidly detect DNA-positive partitions. Single-molecule occupancy also allows for more straightforward multiplex assay development due to the absence of partition-specific competition. As a proof of concept, we developed a 222-plex UltraPCR assay and demonstrated its potential use as a rapid, low-cost screening assay for noninvasive prenatal testing for as low as 4% trisomy fraction samples with high precision, accuracy, and reproducibility.

Calculated cell-specific intracellular hydrogen peroxide concentration: Relevance in cancer cell susceptibility during ascorbate therapy.

The high extracellular hydrogen peroxide (H2O2) concentrations generated during pharmacological ascorbate (P-AscH-) therapy has been shown to exhibit a high flux into susceptible cancer cells leading to a decrease in clonogenic survival. It is hypothesized that the intracellular H2O2 concentration for susceptibility is independent of cell type and that the variation observed in dosing is associated with differences in the cell-specific overall steady-state intracellular H2O2 concentration values. The steady-state variation in intracellular H2O2 concentration is coupled to a number of cellular specific transport and reaction factors including catalase activity and membrane permeability. Here a lumped-parameter mathematical modeling approach, assuming a catalase-dominant peroxide removal mechanism, is used to calculate intracellular H2O2 concentration for several cell lines. Experimental measurements of critical parameters pertaining to the model are obtained. The cell lines investigated are normal pancreatic cells, H6c7, the pancreatic cancer cell line, MIA PaCa-2 and the glioblastoma cell lines, LN-229, T98G, and U-87; all which vary in susceptibility. The intracellular H2O2 concentration estimates are correlated with the clonogenic surviving fraction for each cell line, in-vitro. The results showed that, despite the fact that the experimental parameters including catalase concentration and plasma membrane permeability demonstrated significant variability across cell lines, the calculated steady-state intracellular to extracellular H2O2 concentration ratio did not vary significantly across cell lines. Thus, the calculated intracellular H2O2 concentration is not unique in characterizing susceptibility. These results imply that, although intracellular H2O2 concentration plays a key role in cellular susceptibility to P-AscH- adjuvant therapy, its overall contribution in a unifying mechanism across cell types is complex.