Improving Adherence to AAP Acute Otitis Media Guidelines in an Academic Pediatrics Practice through a Quality Improvement Project.

Introduction: Acute otitis media (AOM) is the most common reason for antibiotic use in children. The American Academy of Pediatrics (AAP) published its latest AOM guidelines in 2013. A safety-net antibiotic prescription (SNAP) is recommended for some patients based on age, severity, and duration of symptoms. At baseline, 78% of patients diagnosed with AOM in our general pediatrics practice met AAP guidelines, and 20% of eligible patients received a SNAP according to guidelines. We aimed to increase adherence to AAP AOM guidelines in an academic general pediatrics clinic from 78% to 90% by January 2020. Methods: A quality improvement team determined key drivers and developed interventions. Patients included were 6 months to 12 years old with AOM. Encounters were reviewed for adherence to AAP AOM guidelines. During the project, interventions included an ear pain note template, which generated guideline-based recommendations, note template education in clinic orientation sessions, a didactic session on AOM management, and reminders on workstations. Data were analyzed using P-charts. Results: Percent of AOM encounters (n = 1266) adhering to AAP AOM guidelines increased from 78% to 92%. We also reviewed two process measures. First, the use of the ear pain note template increased from 0% to 44%. Second, the percent of AOM encounters where an eligible patient received a SNAP increased from 21% to 78% (encounters n = 421). Conclusion: We demonstrate increased adherence to AAP AOM guidelines, including improved use of SNAPs after introducing a note template with clinical decision support and provider educational sessions.

Becoming Active Bystanders and Advocates: Teaching Medical Students to Respond to Bias in the Clinical Setting.

INTRODUCTION: Incidents of bias and microaggressions are prevalent in the clinical setting and are disproportionately experienced by racial minorities, women, and medical students. These incidents contribute to burnout. Published efforts to address these incidents are growing, but gaps remain regarding the long-term efficacy of these curricular models. We developed and longitudinally evaluated a workshop that taught medical students a framework to respond to incidents of bias or microaggressions. METHODS: In October 2019, 102 Vanderbilt core clerkship medical students participated in an hour-long, interactive, case-based workshop centered around the 3 D's response behavior framework: (1) direct, (2) distract, and (3) delegate. Participants were surveyed before and after the training, and both qualitative and quantitative data were collected. A refresher workshop was offered 8 months later, which added two additional D's: delay and display discomfort. RESULTS: After the workshop, respondents' knowledge of the assessed topics improved significantly, as did their confidence in addressing both personally experienced and witnessed incidents. Respondents initially indicated a high likelihood of using response behaviors to address incidents. The workshop did not consistently modify behavioral responses to experienced or witnessed incidents. Ninety-one percent of respondents agreed the workshop was effective. DISCUSSION: This workshop provided an effective curriculum to sustainably improve participant knowledge and confidence in responding to incidents of bias and microaggressions. This resource can be adopted by educators at other institutions.

Roundup causes embryonic development failure and alters metabolic pathways and gut microbiota functionality in non-target species.

BACKGROUND: Research around the weedkiller Roundup is among the most contentious of the twenty-first century. Scientists have provided inconclusive evidence that the weedkiller causes cancer and other life-threatening diseases, while industry-paid research reports that the weedkiller has no adverse effect on humans or animals. Much of the controversial evidence on Roundup is rooted in the approach used to determine safe use of chemicals, defined by outdated toxicity tests. We apply a system biology approach to the biomedical and ecological model species Daphnia to quantify the impact of glyphosate and of its commercial formula, Roundup, on fitness, genome-wide transcription and gut microbiota, taking full advantage of clonal reproduction in Daphnia. We then apply machine learning-based statistical analysis to identify and prioritize correlations between genome-wide transcriptional and microbiota changes. RESULTS: We demonstrate that chronic exposure to ecologically relevant concentrations of glyphosate and Roundup at the approved regulatory threshold for drinking water in the US induce embryonic developmental failure, induce significant DNA damage (genotoxicity), and interfere with signaling. Furthermore, chronic exposure to the weedkiller alters the gut microbiota functionality and composition interfering with carbon and fat metabolism, as well as homeostasis. Using the "Reactome," we identify conserved pathways across the Tree of Life, which are potential targets for Roundup in other species, including liver metabolism, inflammation pathways, and collagen degradation, responsible for the repair of wounds and tissue remodeling. CONCLUSIONS: Our results show that chronic exposure to concentrations of Roundup and glyphosate at the approved regulatory threshold for drinking water causes embryonic development failure and alteration of key metabolic functions via direct effect on the host molecular processes and indirect effect on the gut microbiota. The ecological model species Daphnia occupies a central position in the food web of aquatic ecosystems, being the preferred food of small vertebrates and invertebrates as well as a grazer of algae and bacteria. The impact of the weedkiller on this keystone species has cascading effects on aquatic food webs, affecting their ability to deliver critical ecosystem services. Video Abstract.

High-Quality Assemblies for Three Invasive Social Wasps from the Vespula Genus.

Social wasps of the genus Vespula have spread to nearly all landmasses worldwide and have become significant pests in their introduced ranges, affecting economies and biodiversity. Comprehensive genome assemblies and annotations for these species are required to develop the next generation of control strategies and monitor existing chemical control. We sequenced and annotated the genomes of the common wasp (Vespula vulgaris), German wasp (Vespula germanica), and the western yellowjacket (Vespula pensylvanica). Our chromosome-level Vespula assemblies each contain 176-179 Mb of total sequence assembled into 25 scaffolds, with 10-200 unanchored scaffolds, and 16,566-18,948 genes. We annotated gene sets relevant to the applied management of invasive wasp populations, including genes associated with spermatogenesis and development, pesticide resistance, olfactory receptors, immunity and venom. These genomes provide evidence for active DNA methylation in Vespidae and tandem duplications of venom genes. Our genomic resources will contribute to the development of next-generation control strategies, and monitoring potential resistance to chemical control.

Increasing the accuracy of nanopore DNA sequencing using a time-varying cross membrane voltage.

Nanopore DNA sequencing is limited by low base-calling accuracy. Improved base-calling accuracy has so far relied on specialized base-calling algorithms, different nanopores and motor enzymes, or biochemical methods to re-read DNA molecules. Two primary error modes hamper sequencing accuracy: enzyme mis-steps and sequences with indistinguishable signals. We vary the driving voltage from 100 to 200 mV, with a frequency of 200 Hz, across a Mycobacterium smegmatis porin A (MspA) nanopore, thus changing how the DNA strand moves through the nanopore. A DNA helicase moves the DNA through the nanopore in discrete steps, and the variable voltage moves the DNA continuously between these steps. The electronic signal produced with variable voltage is used to overcome the primary error modes in base calling. We found that single-passage de novo base-calling accuracy of 62.7 ± 0.5% with a constant driving voltage improves to 79.3 ± 0.3% with a variable driving voltage. The variable-voltage sequencing mode is complementary to other methods to boost the accuracy of nanopore sequencing and could be incorporated into any enzyme-actuated nanopore sequencing device.

A football helmet prototype that reduces linear and rotational acceleration with the addition of an outer shell.

OBJECTIVEAmid the public health controversy surrounding American football, a helmet that can reduce linear and rotational acceleration has the potential to decrease forces transmitted to the brain. The authors hypothesized that a football helmet with an outer shell would reduce both linear and rotational acceleration. The authors' objectives were to 1) determine an optimal material for a shock-absorbing outer shell and 2) examine the ability of an outer shell to reduce linear and/or rotational acceleration.METHODSA laboratory-based investigation was undertaken using an extra-large Riddell Revolution football helmet. Two materials (Dow Corning Dilatant Compound and Sorbothane) were selected for their non-Newtonian properties (changes in viscosity with shear stress) to develop an outer shell. External pads were attached securely to the helmet at 3 locations: the front boss, the side, and the back. The helmet was impacted 5 times per location at 6 m/sec with pneumatic ram testing. Two-sample t-tests were used to evaluate linear/rotational acceleration differences between a helmet with and a helmet without the outer shell.RESULTSSorbothane was superior to the Dow Corning compound in force reduction and recovered from impact without permanent deformation. Of 5 different grades, 70-duro (a unit of hardness measured with a durometer) Sorbothane was found to have the greatest energy dissipation and stiffness, and it was chosen as the optimal outer-shell material. The helmet prototype with the outer shell reduced linear acceleration by 5.8% (from 75.4g to 71.1g; p < 0.001) and 10.8% (from 89.5g to 79.8g; p = 0.033) at the side and front boss locations, respectively, and reduced rotational acceleration by 49.8% (from 9312.8 rad/sec2 to 4671.7 rad/sed2; p < 0.001) at the front boss location.CONCLUSIONSSorbothane (70 duro) was chosen as the optimal outer-shell material. In the outer-shell prototype helmet, the results demonstrated a 5%-10% reduction in linear acceleration at the side and front boss locations, and a 50% reduction in rotational acceleration at the front boss location. Given the paucity of publicly reported helmet-design literature and the importance of rotational acceleration in head injuries, the substantial reduction seen in rotational acceleration with this outer-shell prototype holds the potential for future helmet-design improvements.

Assembly of 913 microbial genomes from metagenomic sequencing of the cow rumen.

The cow rumen is adapted for the breakdown of plant material into energy and nutrients, a task largely performed by enzymes encoded by the rumen microbiome. Here we present 913 draft bacterial and archaeal genomes assembled from over 800 Gb of rumen metagenomic sequence data derived from 43 Scottish cattle, using both metagenomic binning and Hi-C-based proximity-guided assembly. Most of these genomes represent previously unsequenced strains and species. The draft genomes contain over 69,000 proteins predicted to be involved in carbohydrate metabolism, over 90% of which do not have a good match in public databases. Inclusion of the 913 genomes presented here improves metagenomic read classification by sevenfold against our own data, and by fivefold against other publicly available rumen datasets. Thus, our dataset substantially improves the coverage of rumen microbial genomes in the public databases and represents a valuable resource for biomass-degrading enzyme discovery and studies of the rumen microbiome.

Decoding long nanopore sequencing reads of natural DNA.

Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length, which can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides a foundation for nanopore sequencing of long, natural DNA strands.

Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA.

Precise and efficient mapping of epigenetic markers on DNA may become an important clinical tool for prediction and identification of ailments. Methylated CpG sites are involved in gene expression and are biomarkers for diseases such as cancer. Here, we use the engineered biological protein pore Mycobacterium smegmatis porin A (MspA) to detect and map 5-methylcytosine and 5-hydroxymethylcytosine within single strands of DNA. In this unique single-molecule tool, a phi29 DNA polymerase draws ssDNA through the pore in single-nucleotide steps, and the ion current through the pore is recorded. Comparing current levels generated with DNA containing methylated CpG sites to current levels obtained with unmethylated copies of the DNA reveals the precise location of methylated CpG sites. Hydroxymethylation is distinct from methylation and can also be mapped. With a single read, the detection efficiency in a quasirandom DNA strand is 97.5 ± 0.7% for methylation and 97 ± 0.9% for hydroxymethylation.

Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase.

Nanopore technologies are being developed for fast and direct sequencing of single DNA molecules through detection of ionic current modulations as DNA passes through a pore's constriction. Here we demonstrate the ability to resolve changes in current that correspond to a known DNA sequence by combining the high sensitivity of a mutated form of the protein pore Mycobacterium smegmatis porin A (MspA) with phi29 DNA polymerase (DNAP), which controls the rate of DNA translocation through the pore. As phi29 DNAP synthesizes DNA and functions like a motor to pull a single-stranded template through MspA, we observe well-resolved and reproducible ionic current levels with median durations of ∼28 ms and ionic current differences of up to 40 pA. Using six different DNA sequences with readable regions 42-53 nucleotides long, we record current traces that map to the known DNA sequences. With single-nucleotide resolution and DNA translocation control, this system integrates solutions to two long-standing hurdles to nanopore sequencing.

Unsupported planar lipid membranes formed from mycolic acids of Mycobacterium tuberculosis.

The cell wall of mycobacteria includes a thick, robust, and highly impermeable outer membrane made from long-chain mycolic acids. These outer membranes form a primary layer of protection for mycobacteria and directly contribute to the virulence of diseases such as tuberculosis and leprosy. We have formed in vitro planar membranes using pure mycolic acids on circular apertures 20 to 90 µm in diameter. We find these membranes to be long lived and highly resistant to irreversible electroporation, demonstrating their general strength. Insertion of the outer membrane channel MspA into the membranes was observed indicating that the artificial mycolic acid membranes are suitable for controlled studies of the mycobacterial outer membrane and can be used in nanopore DNA translocation experiments.