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.     

The Relationship and Expression of miR-451a,miR-25-3p and PTEN in Early Peritoneal Endometriotic Lesions and Their Modulation In Vitro

Background: miR-451a can function as a tumor suppresser and has been shown to be elevated in both endometriotic lesion tissue and serum from women with endometriosis. To further explore the role of miR-451a in the pathophysiology of endometriosis, specifically, further evaluating its association with the tumor suppressor, phosphatase and tensin homolog (PTEN), we examined their expression in individual endometriotic lesion tissue to gain insight into their relationship and further explore if miR-451a regulates PTEN expression. Methods: A total of 55 red, peritoneal endometriotic lesions and matched eutopic endometrial specimens were obtained from 46 patients with endometriosis. miR-451a, miR-25-3p and PTEN mRNA levels were assessed by qRT-PCR and reported for each matched eutopic and ectopic sample. To evaluate miR-451a and miR-25-3p expression of miR-25-3p and PTEN, respectively, 12Z cells (endometriotic epithelial cell line) were transfected and miR-25-3p expression was assessed by qRT-PCR, while PTEN protein expression was assessed by Western blotting. Results: PTEN and miR-25-3p expression exhibited an inverse relationship, as did miR-25-3p and miR-451a in individual lesions. Over-expression of miR-451a in 12Z cells resulted in down-regulation of miR-25-3p, while up-regulation of miR-25-3p resulted in down-regulation of PTEN protein expression. Conclusions: By assessing individual endometriotic lesion expression, we discovered an inverse relationship between miR-451a, miR-25-3p and PTEN, while in vitro cell transfection studies suggest that miR-451a may regulate PTEN expression via modulating miR-25-3p.     

Discovery and Characterization of a Baeyer-Villiger Monooxygenase Using Sequence Similarity Network Analysis

Baeyer-Villiger monooxygenases (BVMOs) are important flavin-dependent enzymes which perform oxygen insertion reactions leading to valuable products. As reported in many studies, BVMOs are usually unstable during application, preventing a wider usage in biocatalysis. Here, we discovered a novel NADPH-dependent BVMO which originates from Halopolyspora algeriensis using sequence similarity networks (SSNs). The enzyme is stable at temperatures between 10 °C to 30 °C up to five days after the purification, and yields the normal ester product. In this study, the substrate scope was investigated for a broad range of aliphatic ketones and the enzyme was biochemically characterized to identify optimum reaction conditions. The best substrate (86 % conversion) was 2-dodecanone using purified enzyme. This novel BVMO could potentially be applied as part of an enzymatic cascade or in bioprocesses which utilize aliphatic alkanes as feedstock.     

ChemoGraph: Interactive Visual Exploration of the Chemical Space

Exploratory analysis of the chemical space is an important task in the field of cheminformatics. For example, in drug discovery research, chemists investigate sets of thousands of chemical compounds in order to identify novel yet structurally similar synthetic compounds to replace natural products. Manually exploring the chemical space inhabited by all possible molecules and chemical compounds is impractical, and therefore presents a challenge. To fill this gap, we present ChemoGraph, a novel visual analytics technique for interactively exploring related chemicals. In ChemoGraph, we formalize a chemical space as a hypergraph and apply novel machine learning models to compute related chemical compounds. It uses a database to find related compounds from a known space and a machine learning model to generate new ones, which helps enlarge the known space. Moreover, ChemoGraph highlights interactive features that support users in viewing, comparing, and organizing computationally identified related chemicals. With a drug discovery usage scenario and initial expert feedback from a case study, we demonstrate the usefulness of ChemoGraph.     

Strain Anisotropy Driven Spontaneous Formation of Nanoscrolls from 2D Janus Layers

2D Janus transition metal dichalcogenides (TMDs) have attracted attention due to their emergent properties arising from broken mirror symmetry and self-driven polarization fields. While it has been proposed that their vdW superlattices hold the key to achieving superior properties in piezoelectricity and photovoltaic, available synthesis has ultimately limited their realization. Here, the first packed vdW nanoscrolls made from Janus TMDs through a simple one-drop solution technique are reported. The results, including ab initio simulations, show that the Bohr radius difference between the top sulfur and the bottom selenium atoms within Janus ${\mathrm{M}}_{\mathrm{Se}}^{\mathrm{S}}$ (M = Mo, W) results in a permanent compressive surface strain that acts as a nanoscroll formation catalyst after small liquid interaction. Unlike classical 2D layers, the surface strain in Janus TMDs can be engineered from compressive to tensile by placing larger Bohr radius atoms on top (${\mathrm{M}}_{\mathrm{S}}^{\mathrm{Se}})$to yield inverted C scrolls. Detailed microscopy studies offer the first insights into their morphology and readily formed Moiré lattices. In contrast, spectroscopy and FETs studies establish their excitonic and device properties and highlight significant differences compared to 2D flat Janus TMDs. These results introduce the first polar Janus TMD nanoscrolls and introduce inherent strain-driven scrolling dynamics as a catalyst to create superlattices.     

Strategic cost and sustainability analyses of injection molding and material extrusion additive manufacturing

Economic and environmental costs are assessed for four different plastics manufacturing processes, including cold and hot runner molding as well as stock and upgraded material extrusion three dimensional (3D) printers. A larger stock 3D printer was found to provide a melting capacity of 14.4 ml/h, while a smaller printer with an upgraded extruder had a melting capacity of 36 ml/h. 3D printing at these maximum melting capacities resulted in specific energy consumption (SEC) of 16.5 and 5.28 kWh/kg, respectively, with the latter value being less than 50% of the lowest values reported in the literature. Even so, analysis of these respective processes found them to be only 2.9% and 3.8% efficient relative to their theoretical minimum energy requirements. By comparison, cold and hot runner molding with an all-electric machine had SEC of 1.28 and 0.929 kWh/kg, respectively, with efficiencies of 9.9% and 13.6% relative to the theoretical minima. Breakeven analysis considering the cost and carbon footprint of mold tooling found injection molding was preferable at a production quantity of around 70,000 units. Parametric analysis of model inputs indicates that the breakeven quantities are robust with respect to carbon tax incentives but highly dependent on mold costs, labor costs, and part size. Dimensional and mechanical properties of the molded and 3D printed specimens are also characterized and discussed.