An in vitro platform for study of the human gut microbiome under an oxygen gradient.

The complex, dynamic environment of the human lower gastrointestinal tract is colonized by hundreds of bacterial species that impact health and performance. Ex vivo study of the functional interactions between microbial community members in conditions representative of those in the gut is an ongoing challenge. We have developed an in vitro 40-plex platform that provides an oxygen gradient to support simultaneous maintenance of microaerobic and anaerobic microbes from the gut microbiome that can aid in rapid characterization of microbial interactions and direct comparison of individual microbiome samples. In this report, we demonstrate that the platform more closely maintained the microbial diversity and composition of human donor fecal microbiome samples than strict anaerobic conditions. The oxygen gradient established in the platform allowed the stratification and subsequent sampling of diverse microbial subpopulations that colonize microaerobic and anaerobic micro-environments. With the ability to run forty samples in parallel, the platform has the potential to be used as a rapid screening tool to understand how the gut microbiome responds to environmental perturbations such as toxic compound exposure, dietary changes, or pharmaceutical treatments.

Systematic Approach to Address Early Pandemic's Diagnostic Unmet Needs.

During the first few months of the global Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic, the medical research community had to expeditiously develop, select, and deploy novel diagnostic methods and tools to address the numerous testing challenges presented by the novel virus. Integrating a systematic approach to diagnostic selection with a rapid validation protocol in a clinical setting can shorten the timeline to bring new technologies to practice. In response to the urgent need to provide tools for identifying SARS-CoV-2-positive individuals, we developed a framework for assessing technologies against a set of prioritized performance metrics to guide device selection. We also developed and proposed clinical validation frameworks for the rapid screening of new technologies. The rubric described here represents a versatile approach that can be extended to future technology assessments and can be implemented in preparation for future emerging pathogens.

Geographic source estimation using airborne plant environmental DNA in dust.

Information obtained from the analysis of dust, particularly biological particles such as pollen, plant parts, and fungal spores, has great utility in forensic geolocation. As an alternative to manual microscopic analysis of dust components, we developed a pipeline that utilizes the airborne plant environmental DNA (eDNA) in settled dust to estimate geographic origin. Metabarcoding of settled airborne eDNA was used to identify plant species whose geographic distributions were then derived from occurrence records in the USGS Biodiversity in Service of Our Nation (BISON) database. The distributions for all plant species identified in a sample were used to generate a probabilistic estimate of the sample source. With settled dust collected at four U.S. sites over a 15-month period, we demonstrated positive regional geolocation (within 600 km2 of the collection point) with 47.6% (20 of 42) of the samples analyzed. Attribution accuracy and resolution was dependent on the number of plant species identified in a dust sample, which was greatly affected by the season of collection. In dust samples that yielded a minimum of 20 identified plant species, positive regional attribution was achieved with 66.7% (16 of 24 samples). For broader demonstration, citizen-collected dust samples collected from 31 diverse U.S. sites were analyzed, and trace plant eDNA provided relevant regional attribution information on provenance in 32.2% of samples. This showed that analysis of airborne plant eDNA in settled dust can provide an accurate estimate regional provenance within the U.S., and relevant forensic information, for a substantial fraction of samples analyzed.

The Effect of Hearing-Protection Devices on Auditory Situational Awareness and Listening Effort.

OBJECTIVES: Hearing-protection devices (HPDs) are made available, and often are required, for industrial use as well as military training exercises and operational duties. However, these devices often are disliked, and consequently not worn, in part because they compromise situational awareness through reduced sound detection and localization performance as well as degraded speech intelligibility. In this study, we carried out a series of tests, involving normal-hearing subjects and multiple background-noise conditions, designed to evaluate the performance of four HPDs in terms of their modifications of auditory-detection thresholds, sound-localization accuracy, and speech intelligibility. In addition, we assessed their impact on listening effort to understand how the additional effort required to perceive and process auditory signals while wearing an HPD reduces available cognitive resources for other tasks. DESIGN: Thirteen normal-hearing subjects participated in a protocol, which included auditory tasks designed to measure detection and localization performance, speech intelligibility, and cognitive load. Each participant repeated the battery of tests with unoccluded ears and four hearing protectors, two active (electronic) and two passive. The tasks were performed both in quiet and in background noise. RESULTS: Our findings indicate that, in variable degrees, all of the tested HPDs induce performance degradation on most of the conducted tasks as compared to the open ear. Of particular note in this study is the finding of increased cognitive load or listening effort, as measured by visual reaction time, for some hearing protectors during a dual-task, which added working-memory demands to the speech-intelligibility task. CONCLUSIONS: These results indicate that situational awareness can vary greatly across the spectrum of HPDs, and that listening effort is another aspect of performance that should be considered in future studies. The increased listening effort induced by hearing protectors may lead to earlier cognitive fatigue in noisy environments. Further study is required to characterize how auditory performance is limited by the combination of hearing impairment and the use of HPDs, and how the effects of such limitations can be linked to safe and effective use of hearing protection to maximize job performance.

PERSIA for Direct Fluorescence Measurements of Transcription, Translation, and Enzyme Activity in Cell-Free Systems.

Quantification of biology's central dogma (transcription and translation) is pursued by a variety of methods. Direct, immediate, and ongoing quantification of these events is difficult to achieve. Common practice is to use fluorescent or luminescent proteins to report indirectly on prior cellular events, such as turning on a gene in a genetic circuit. We present an alternative approach, PURExpress-ReAsH-Spinach In-vitro Analysis (PERSIA). PERSIA provides information on the production of RNA and protein during cell-free reactions by employing short RNA and peptide tags. Upon synthesis, these tags yield quantifiable fluorescent signal without interfering with other biochemical events. We demonstrate the applicability of PERSIA in measuring cell-free transcription, translation, and other enzymatic activity in a variety of applications: from sequence-structure-function studies, to genetic code engineering, to testing antiviral drug resistance.

Standardizing Automated DNA Assembly: Best Practices, Metrics, and Protocols Using Robots.

The advancement of synthetic biology requires the ability to create new DNA sequences to produce unique behaviors in biological systems. Automation is increasingly employed to carry out well-established assembly methods of DNA fragments in a multiplexed, high-throughput fashion, allowing many different configurations to be tested simultaneously. However, metrics are required to determine when automation is warranted based on factors such as assembly methodology, protocol details, and number of samples. The goal of our synthetic biology automation work is to develop and test protocols, hardware, and software to investigate and optimize DNA assembly through quantifiable metrics. We performed a parameter analysis of DNA assembly to develop a standardized, highly efficient, and reproducible MoClo protocol, suitable to be used both manually and with liquid-handling robots. We created a key DNA assembly metric (Q-metric) to characterize a given automation method's advantages over conventional manual manipulations with regard to researchers' highest-priority parameters: output, cost, and time. A software tool called Puppeteer was developed to formally capture these metrics, help define the assembly design, and provide human and robotic liquid-handling instructions. Altogether, we contribute to a growing foundation of standardizing practices, metrics, and protocols for automating DNA assembly.