Multiplex genomic recording of enhancer and signal transduction activities in mammalian cells

Measurements of gene expression and signal transduction activity are conventionally performed with methods that require either the destruction or live imaging of a biological sample within the timeframe of interest. Here we demonstrate an alternative paradigm, termed ENGRAM (ENhancer-driven Genomic Recording of transcriptional Activity in Multiplex), in which the activity and dynamics of multiple transcriptional reporters are stably recorded to DNA. ENGRAM is based on the prime editing-mediated insertion of signal- or enhancer-specific barcodes to a genomically encoded recording unit. We show how this strategy can be used to concurrently record the relative activity of at least hundreds of enhancers with high fidelity, sensitivity and reproducibility. Leveraging synthetic enhancers that are responsive to specific signal transduction pathways, we further demonstrate time- and concentration-dependent genomic recording of Wnt, NF-κB, and Tet-On activity. Finally, by coupling ENGRAM to sequential genome editing, we show how serially occurring molecular events can potentially be ordered. Looking forward, we envision that multiplex, ENGRAM-based recording of the strength, duration and order of enhancer and signal transduction activities has broad potential for application in functional genomics, developmental biology and neuroscience.

Multiplex genomic recording of enhancer and signal transduction activity in mammalian cells

Measurements of gene expression and signal transduction activity are conventionally performed with methods that require either the destruction or live imaging of a biological sample within the timeframe of interest. Here we demonstrate an alternative paradigm, termed ENGRAM (ENhancer-driven Genomic Recording of transcriptional Activity in Multiplex), in which the activity and dynamics of multiple transcriptional reporters are stably recorded to DNA. ENGRAM is based on the prime editing-mediated insertion of signal- or enhancer-specific barcodes to a genomically encoded recording unit. We show how this strategy can be used to concurrently genomically record the relative activity of at least hundreds of enhancers with high fidelity, sensitivity and reproducibility. Leveraging synthetic enhancers that are responsive to specific signal transduction pathways, we further demonstrate time- and concentration-dependent genomic recording of Wnt, NF-κB, and Tet-On activity. Finally, by coupling ENGRAM to sequential genome editing, we show how serially occurring molecular events can potentially be ordered. Looking forward, we envision that multiplex, ENGRAM-based recording of the strength, duration and order of enhancer and signal transduction activities has broad potential for application in functional genomics, developmental biology and neuroscience.

Using Underwater Pulse Oximetry in Freediving to Extreme Depths to Study Risk of Hypoxic Blackout and Diving Response Phases

Deep freediving exposes humans to hypoxia and dramatic changes in pressure. The effect of depth on gas exchange may enhance risk of hypoxic blackout (BO) during the last part of the ascent. Our aim was to investigate arterial oxygen saturation (SpO2) and heart rate (HR) in shallow and deep freedives, central variables, which have rarely been studied underwater in deep freediving. Four male elite competitive freedivers volunteered to wear a newly developed underwater pulse oximeter for continuous monitoring of SpO2 and HR during self-initiated training in the sea. Two probes were placed on the temples, connected to a recording unit on the back of the freediver. Divers performed one “shallow” and one “deep” constant weight dive with fins. Plethysmograms were recorded at 30 Hz, and SpO2 and HR were extracted. Mean ± SD depth of shallow dives was 19 ± 3 m, and 73 ± 12 m for deep dives. Duration was 82 ± 36 s in shallow and 150 ± 27 s in deep dives. All divers desaturated more during deeper dives (nadir 55 ± 10%) compared to shallow dives (nadir 80 ± 22%) with a lowest SpO2 of 44% in one deep dive. HR showed a “diving response,” with similar lowest HR of 42 bpm in shallow and deep dives; the lowest value (28 bpm) was observed in one shallow dive. HR increased before dives, followed by a decline, and upon resurfacing a peak after which HR normalized. During deep dives, HR was influenced by the level of exertion across different diving phases; after an initial drop, a second HR decline occurred during the passive “free fall” phase. The underwater pulse oximeter allowed successful SpO2 and HR monitoring in freedives to 82 m depth – deeper than ever recorded before. Divers’ enhanced desaturation during deep dives was likely related to increased exertion and extended duration, but the rapid extreme desaturation to below 50% near surfacing could result from the diminishing pressure, in line with the hypothesis that risk of hypoxic BO may increase during ascent. Recordings also indicated that the diving response is not powerful enough to fully override the exercise-induced tachycardia during active swimming. Pulse oximetry monitoring of essential variables underwater may be an important step to increase freediving safety.

Wireless, miniaturized, semi-implantable electrocorticography microsystem validated in vivo

This paper reports on the design, development, and test of a multi-channel wireless micro-electrocorticography (µECoG) system. The system consists of a semi-implantable, ultra-compact recording unit and an external unit, interfaced through a 2.4 GHz radio frequency data telemetry link with 2 Mbps (partially used) data transfer rate. Encased in a 3D-printed 2.9 cm × 2.9 cm × 2.5 cm cubic package, the semi-implantable recording unit consists of a microelectrode array, a vertically-stacked PCB platform containing off-the-shelf components, and commercially-available small-size 3.7-V, 50 mAh lithium-ion batteries. Two versions of microelectrode array were developed for the recording unit: a rigid 4 × 2 microelectrode array, and a flexible 12 × 6 microelectrode array, 36 of which routed to bonding pads for actual recording. The external unit comprises a transceiver board, a data acquisition board, and a host computer, on which reconstruction of the received signals is performed. After development, assembly, and integration, the system was tested and validated in vivo on anesthetized rats. The system successfully recorded both spontaneous and evoked activities from the brain of the subject.

Experimental and Numerical Investigation of Cutting Characteristics of PA6/PE Nylon Film Subjected to Wedge Indentation Process

In this research work, we aim to evaluate the cutting resistance and deformation of a laminated nylon film subjected to a 42o wedged indentation. One of the problems occurred in the wedge indentation process is the unstable separation and quality of the sheared profile of the worksheet. In order to reveal the effect of cutting parameters on the cutting features, the indentation experiment of 0.16 mm thickness of Polyamide-6/ Polyethylene nylon film (PA6/PE) was conducted; the cutting line force was gotten using a recording unit; the bent-up angle and sheared profile of the worksheet were observed using a high-speed camera. From the experiment results, it was found that the cutting direction was an important factor affected to the bent-up angle and cutting load response of the nylon film. Also, the effect of cutting direction (PA6-PE and PE-PA6) of the nylon film was numerically investigated.