Synonymous codon usage regulates translation initiation.

Nonoptimal synonymous codons repress gene expression, but the underlying mechanisms are poorly understood. We and others have previously shown that nonoptimal codons slow translation elongation speeds and thereby trigger messenger RNA (mRNA) degradation. Nevertheless, transcript levels are often insufficient to explain protein levels, suggesting additional mechanisms by which codon usage regulates gene expression. Using reporters in human and Drosophila cells, we find that transcript levels account for less than half of the variation in protein abundance due to codon usage. This discrepancy is explained by translational differences whereby nonoptimal codons repress translation initiation. Nonoptimal transcripts are also less bound by the translation initiation factors eIF4E and eIF4G1, providing a mechanistic explanation for their reduced initiation rates. Importantly, translational repression can occur without mRNA decay and deadenylation, and it does not depend on the known nonoptimality sensor, CNOT3. Our results reveal a potent mechanism of regulation by codon usage where nonoptimal codons repress further rounds of translation.

A multiplexed, paired-pooled droplet digital PCR assay for detection of SARS-CoV-2 in saliva.

In response to the SARS-CoV-2 pandemic, we developed a multiplexed, paired-pool droplet digital PCR (MP4) screening assay. Key features of our assay are the use of minimally processed saliva, 8-sample paired pools, and reverse-transcription droplet digital PCR (RT-ddPCR) targeting the SARS-CoV-2 nucleocapsid gene. The limit of detection was determined to be 2 and 12 copies per µl for individual and pooled samples, respectively. Using the MP4 assay, we routinely processed over 1,000 samples a day with a 24-h turnaround time and over the course of 17 months, screened over 250,000 saliva samples. Modeling studies showed that the efficiency of 8-sample pools was reduced with increased viral prevalence and that this could be mitigated by using 4-sample pools. We also present a strategy for, and modeling data supporting, the creation of a third paired pool as an additional strategy to employ under high viral prevalence.

Measuring interprofessional education and collaborative practice competencies: a content validity study of the Jefferson Teamwork Observation Guide®.

Collaborative practice (CP) is integral in meeting the Quadruple Aim of healthcare, with effective team-based practice linked to improving all four components. Evidence of the validity of tools measuring collaborative practice competencies is lacking in educational and practice settings. The Jefferson Teamwork Observation Guide® (JTOG®), a real-time, 360-degree competency-based assessment tool administered via mobile app, provides formative feedback to learners in educational settings and helps practitioners develop and refine team-based behaviors in clinical settings. This study examines content validity evidence in terms of the linkage of JTOG items with the four Interprofessional Education Collaborative (IPEC) core competencies, along with two additional domains of leadership and patient-centeredness. Results provide content validity evidence to support use of the JTOG in interprofessional collaborative practice (IPCP) settings. The Teams and Teamwork competency was linked with every item, which is consistent with JTOG as a measure of teamwork. Aligning with the 2016 IPEC update, the JTOG items are all intercorrelated and together represent coverage across all competency areas. While items were typically linked to multiple competencies, each item only had one primary linkage. Analyses revealed that there is sufficient evidence of content validity relative to the intended IPCP competencies, and the JTOG tool is promising in its role to fill a gap in extant literature to measure collaborative practice behaviors.

A Multi-color Bicistronic Biosensor to Compare the Translation Dynamics of Different Open Reading Frames at Single-molecule Resolution in Live Cells.

Here, we describe how to image and quantitate the translation dynamics of a bicistronic biosensor that we recently created to fairly compare cap-dependent and IRES-mediated translation at single-molecule resolution in live human cells. This technique employs a pair of complementary intrabodies loaded into living cells that co-translationally bind complementary epitopes in the two separate ORFs of the bicistronic biosensor. This causes the biosensor to fluoresce in different colors depending on which ORF/epitopes are translated. Using the biosensor together with high-resolution fluorescence microscopy and single-molecule tracking analysis allows for the quantitative comparison of translation dynamics between the two ORFs at a resolution of tens-of-nanometers in space and sub-seconds in time, which is not possible with more traditional GFP or luciferase reporters. Since both ORFs are on the same biosensor, they experience the same microenvironment, allowing a fair comparison of their relative translational activities. In this protocol, we describe how to get this assay up and running in cultured human cells so that translation dynamics can be studied under both normal and stressful cellular conditions. We also provide a number of useful tips and notes to help express components at appropriate levels inside cells for optimal live cell imaging. Graphical abstract: Steps required for 3-color single-molecule translation imaging and analysis.

Bead Loading Proteins and Nucleic Acids into Adherent Human Cells.

Many live-cell imaging experiments use exogenous particles (e.g., peptides, antibodies, beads) to label or function within cells. However, introducing proteins into a cell across its membrane is difficult. The limited selection of current methods struggles with low efficiency, requires expensive and technically demanding equipment, or functions within narrow parameters. Here, we describe a relatively simple and cost-effective technique for loading DNA, RNA, and proteins into live human cells. Bead loading induces a temporary mechanical disruption to the cell membrane, allowing macromolecules to enter adherent, live mammalian cells. At less than 0.01 USD per experiment, bead loading is the least expensive cell loading method available. Moreover, bead loading does not substantially stress cells or impact their viability or proliferation. This manuscript describes the steps of the bead loading procedure, adaptations, variations, and technical limitations. This methodology is especially suited for live-cell imaging but provides a practical solution for other applications requiring the introduction of proteins, beads, RNA, or plasmids into living, adherent mammalian cells.

Author Correction: Quantifying the dynamics of IRES and cap translation with single-molecule resolution in live cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Quantifying the dynamics of IRES and cap translation with single-molecule resolution in live cells.

Viruses use internal ribosome entry sites (IRES) to hijack host ribosomes and promote cap-independent translation. Although they are well-studied in bulk, the dynamics of IRES-mediated translation remain unexplored at the single-molecule level. Here, we developed a bicistronic biosensor encoding distinct repeat epitopes in two open reading frames (ORFs), one translated from the 5' cap, and the other from the encephalomyocarditis virus IRES. When combined with a pair of complementary probes that bind the epitopes cotranslationally, the biosensor lights up in different colors depending on which ORF is translated. Using the sensor together with single-molecule tracking and computational modeling, we measured the kinetics of cap-dependent versus IRES-mediated translation in living human cells. We show that bursts of IRES translation are shorter and rarer than bursts of cap translation, although the situation reverses upon stress. Collectively, our data support a model for translational regulation primarily driven by transitions between translationally active and inactive RNA states.

Lighting up single-mRNA translation dynamics in living cells.

Over the past five years, technological advances have made it possible to image the translation of single mRNA in the natural context of living cells. With these advances, researchers are beginning to shed light on when, where, and to what degree mRNA are translated with single-molecule precision. These works provide insight into the heterogeneity of translation amongst single transcripts, behavior that is averaged out in complementary bulk assays. In this review, we discuss the rapidly maturing field of live-cell, single-mRNA imaging of translation, beginning with a brief overview of recent technological advances. The remainder of the review focuses on the new biological insights gained from these technologies. We conclude with a discussion of the future of this technology.

Fatal Case of Legionnaires' Disease After Home Exposure to Legionella pneumophila Serogroup 3 - Wisconsin, 2018.

In January 2018, the Wisconsin Department of Health Services, Division of Public Health (DPH), received a report of a culture-confirmed case of Legionnaires' disease. The patient, who was immunocompromised, had died at a local hospital 10 days after being admitted. DPH and an infection preventionist from the hospital investigated to determine the source of the infection and prevent additional cases. Because the case was suspected to be nosocomial, health care facility water samples were tested for Legionella. When these samples were negative, water sources in the patient's home were tested. These tested positive for Legionella pneumophila, and the bacteria remained after an attempt to remediate. The patient and home isolates were identified as L. pneumophila serogroup 3, sequence type 93, by whole-genome multilocus sequence typing. A second resident of the home did not become ill. This case highlights the potential for immunocompromised persons and others at risk for Legionnaires' disease to be exposed to Legionella through home water systems containing the bacteria and demonstrates the difficulty of home remediation. This case also illustrates the role of lower respiratory tract specimens in the identification of less common Legionella infections (e.g., L. pneumophila serogroup 3) and confirmation of the infection source.

A meta-analysis of gender stereotypes and bias in experimental simulations of employment decision making.

Gender bias continues to be a concern in many work settings, leading researchers to identify factors that influence workplace decisions. In this study we examine several of these factors, using an organizing framework of sex distribution within jobs (including male- and female-dominated jobs as well as sex-balanced, or integrated, jobs). We conducted random effects meta-analyses including 136 independent effect sizes from experimental studies (N = 22,348) and examined the effects of decision-maker gender, amount and content of information available to the decision maker, type of evaluation, and motivation to make careful decisions on gender bias in organizational decisions. We also examined study characteristics such as type of participant, publication year, and study design. Our findings revealed that men were preferred for male-dominated jobs (i.e., gender-role congruity bias), whereas no strong preference for either gender was found for female-dominated or integrated jobs. Second, male raters exhibited greater gender-role congruity bias than did female raters for male-dominated jobs. Third, gender-role congruity bias did not consistently decrease when decision makers were provided with additional information about those they were rating, but gender-role congruity bias was reduced when information clearly indicated high competence of those being evaluated. Fourth, gender-role congruity bias did not differ between decisions that required comparisons among ratees and decisions made about individual ratees. Fifth, decision makers who were motivated to make careful decisions tended to exhibit less gender-role congruity bias for male-dominated jobs. Finally, for male-dominated jobs, experienced professionals showed smaller gender-role congruity bias than did undergraduates or working adults.

Impact of antioxidant supplementation on chemotherapeutic toxicity: a systematic review of the evidence from randomized controlled trials.

Much debate has focused on whether antioxidants interfere with the efficacy of cancer chemotherapy. The objective of this study is to systematically review the randomized, controlled clinical trial evidence evaluating the effects of concurrent use of antioxidants with chemotherapy on toxic side effects. We performed a search of literature from 1966-October 2007 using MEDLINE, Cochrane, CinAhl, AMED, AltHealthWatch and EMBASE databases. Randomized, controlled clinical trials reporting antioxidant-based mitigation of chemotherapy toxicity were included in the final tally. Searches were performed following a standardized protocol for systematic reviews. Only 33 of 965 articles considered, including 2,446 subjects, met the inclusion criteria. Antioxidants evaluated were: glutathione (11), melatonin (7), vitamin A (1), an antioxidant mixture (2), N-acetylcysteine (2), vitamin E (5), selenium (2), L-carnitine (1), Co-Q10 (1) and ellagic acid (1). The majority (24) of the 33 studies included reported evidence of decreased toxicities from the concurrent use of antioxidants with chemotherapy. Nine studies reported no difference in toxicities between the 2 groups. Only 1 study (vitamin A) reported a significant increase in toxicity in the antioxidant group. Five studies reported the antioxidant group completed more full doses of chemotherapy or had less-dose reduction than control groups. Statistical power and poor study quality were concerns with some studies. This review provides the first systematically reviewed evidence that antioxidant supplementation during chemotherapy holds potential for reducing dose-limiting toxicities. However, well-designed studies evaluating larger populations of patients given specific antioxidants defined by dose and schedule relative to chemotherapy are warranted.

Impact of antioxidant supplementation on chemotherapeutic efficacy: a systematic review of the evidence from randomized controlled trials.

PURPOSE: Much debate has arisen about whether antioxidant supplementation alters the efficacy of cancer chemotherapy. Some have argued that antioxidants scavenge the reactive oxygen species integral to the activity of certain chemotherapy drugs, thereby diminishing treatment efficacy. Others suggest antioxidants may mitigate toxicity and thus allow for uninterrupted treatment schedules and a reduced need for lowering chemotherapy doses. The objective of this study is to systematically review the literature in order to compile results from randomized trials that evaluate concurrent use of antioxidants with chemotherapy. DESIGN: MEDLINE, Cochrane, CinAhl, AMED, AltHealthWatch and EMBASE databases were searched. Only randomized, controlled clinical trials that reported survival and/or tumor response were included in the final tally. The literature searches were performed in duplicate following a standardized protocol. No meta-analysis was performed due to heterogeneity of tumor types and treatment protocols used in trials that met the inclusion criteria. RESULTS: Of 845 articles considered, 19 trials met the inclusion criteria. Antioxidants evaluated were: glutathione (7), melatonin (4), vitamin A (2), an antioxidant mixture (2), vitamin C (1), N-acetylcysteine (1), vitamin E (1) and ellagic acid (1). Subjects of most studies had advanced or relapsed disease. CONCLUSION: None of the trials reported evidence of significant decreases in efficacy from antioxidant supplementation during chemotherapy. Many of the studies indicated that antioxidant supplementation resulted in either increased survival times, increased tumor responses, or both, as well as fewer toxicities than controls; however, lack of adequate statistical power was a consistent limitation. Large, well-designed studies of antioxidant supplementation concurrent with chemotherapy are warranted.