DNA assembly with error correction on a droplet digital microfluidics platform.

BACKGROUND: Custom synthesized DNA is in high demand for synthetic biology applications. However, current technologies to produce these sequences using assembly from DNA oligonucleotides are costly and labor-intensive. The automation and reduced sample volumes afforded by microfluidic technologies could significantly decrease materials and labor costs associated with DNA synthesis. The purpose of this study was to develop a gene assembly protocol utilizing a digital microfluidic device. Toward this goal, we adapted bench-scale oligonucleotide assembly methods followed by enzymatic error correction to the Mondrian™ digital microfluidic platform. RESULTS: We optimized Gibson assembly, polymerase chain reaction (PCR), and enzymatic error correction reactions in a single protocol to assemble 12 oligonucleotides into a 339-bp double- stranded DNA sequence encoding part of the human influenza virus hemagglutinin (HA) gene. The reactions were scaled down to 0.6-1.2 µL. Initial microfluidic assembly methods were successful and had an error frequency of approximately 4 errors/kb with errors originating from the original oligonucleotide synthesis. Relative to conventional benchtop procedures, PCR optimization required additional amounts of MgCl2, Phusion polymerase, and PEG 8000 to achieve amplification of the assembly and error correction products. After one round of error correction, error frequency was reduced to an average of 1.8 errors kb- 1. CONCLUSION: We demonstrated that DNA assembly from oligonucleotides and error correction could be completely automated on a digital microfluidic (DMF) platform. The results demonstrate that enzymatic reactions in droplets show a strong dependence on surface interactions, and successful on-chip implementation required supplementation with surfactants, molecular crowding agents, and an excess of enzyme. Enzymatic error correction of assembled fragments improved sequence fidelity by 2-fold, which was a significant improvement but somewhat lower than expected compared to bench-top assays, suggesting an additional capacity for optimization.

A comparison of physical activity, physical fitness levels, BMI and blood pressure of adults with intellectual disability, who do and do not take part in Special Olympics Ireland programmes: Results from the SOPHIE study.

People with an intellectual disability are less physically active, live more sedentary lives, have lower fitness levels and are more likely to be overweight or obese than the general population. No evidence exists on the impact of participation in Special Olympics Ireland (SOI) on physical activity and physical fitness levels. Adults with intellectual disabilities (16-64 years) were recruited from services and SOI clubs. Physical measures included waist circumference, height, weight, blood pressure, heart rate and 6-min walking test. Self-report questionnaires gathered data on physical activity levels. Actigraph (GT3X) accelerometers were used to gain an objective measure of physical activity. SOI participants accumulated more moderate to vigorous physical activity per day, had higher fitness levels and more positive health profile scores than those not taking part in SOI. SOI has the potential to make a positive difference to people's physical health and subsequently their overall health and well-being.

An examination of the nutritional intake and anthropometric status of individuals with intellectual disabilities: Results from the SOPHIE study.

The prevalence of obesity appears greater in people with intellectual disabilities than those in the general population. This study aimed to examine the nutritional intake and anthropometric status of individuals with intellectual disabilities. Participants aged 16-64 years were recruited from intellectual disability service provider organizations ( n = 131). Data were collected using questionnaires; 4-day food dairies and weight, height and waist circumference measurements. Participants' mean body mass index (BMI) was 29.4 kg/m2 ± 6.1, 2.4% were underweight, 22.6% were normal weight, 28.2% were overweight and 46.8% were obese. Having a diagnosis of Down syndrome ( p = 0.03) was associated with increasing BMI. Increasing waist circumference was associated with increasing severity of ID ( p = 0.04). The mean-reported energy intake was 1890 kcal/day. Mean energy intakes from sugar, fat and saturated fat were above recommendations and few participants met micronutrient recommended daily amounts. This study highlights the alarming prevalence of overweight and obesity and poor diet quality of individuals with intellectual disabilities.

People with intellectual disability and their families' perspectives of Special Olympics Ireland: Qualitative findings from the SOPHIE study.

Previous research has highlighted that while involvement in Special Olympics can have benefits for athletes and their families, there can also be many barriers to participation. This qualitative study, which was part of a large mixed-method study, examined the experiences and perspectives of people with intellectual disability, their families and staff who work with them, about Special Olympics Ireland (SOI). A total of 47 participants (15 athletes, 6 non-athletes, 18 family members and 8 staff members) participated in focus group and individual interviews. Supplemental data, gathered as part of the larger study extracted from open-ended survey questions completed by 97 family members also informed this element. Findings revealed four main themes: impact of participation on athletes, impact of involvement on families, barriers to participation and how to enhance participation rates. Involvement in Special Olympics impacted positively on the quality of life of athletes and families. Enhanced availability of user-friendly information and service accessibility were important drivers identified for enhancing participation rates in Special Olympics.

Caffeine overdose resulting in severe rhabdomyolysis and acute renal failure.

Despite the fact that caffeine is the most commonly used stimulant in modern society, cases of caffeine overdose are relatively rare, with fatalities reported from doses of 10 g and higher (Nord J Psychiatry. 2006;60:97-106). Large doses produce symptoms associated with stimulation of the cardiovascular, central nervous, and gastrointestinal symptoms (Associates of the California Poison Control Center, Poisoning and Drug Overdose, pp. 142-143. 5th Ed). We present the first reported case of a man with delayed presentation to the emergency department after ingesting 24 g of caffeine in a suicide attempt who suffered complications of severe rhabdomyolysis and acute renal failure requiring subsequent hemodialysis.

Door-to-balloon times from freestanding emergency departments meet ST-segment elevation myocardial infarction reperfusion guidelines.

BACKGROUND: Freestanding emergency departments (FEDs) introduce a challenge to physicians who care for the patient with an ST-segment elevation myocardial infarction (STEMI) because treatment is highly time dependent. FEDs have no percutaneous coronary intervention (PCI) capabilities, which necessitates transfer to a PCI-capable facility or fibrinolysis. STUDY OBJECTIVE: Our aim was to determine the proportion of STEMI patients who arrived to an FED and were subsequently transferred for PCI and met the door-to-balloon reperfusion guidelines of 90 min. METHODS: This was a dual-center retrospective cohort review of all patients 18 years and older who were diagnosed with an STEMI and presented to the main hospital-affiliated FEDs. Electronic medical records and emergency medical services documentation were reviewed for all cases since the opening of the FEDs in July 2007 and August 2009, respectively. Key time points were abstracted and statistical evaluation was performed using Fisher's exact test. RESULTS: A total of 47 patients met inclusion criteria. Median door-to-transport time was 34 min (interquartile range [IQR] 15 min). Median transport time from the FEDs to the main hospital catheterization laboratory was 21 min (IQR 5 min). Median arrival at the catheterization laboratory-to-balloon time was 25 min (IQR 13 min). Median total door-to-balloon time was 83 min (IQR 10.5 min), with 78.7% meeting the American Heart Association's recommended guidelines of ≤ 90 min. CONCLUSION: STEMI patients initially seen at two FEDs achieved door-to-balloon time goals of < 90 min.

Spontaneous septic arthritis in a patient without trauma, coinfection, or immunosuppression.

Septic arthritis is a rare infection, most often affecting the knee and hip [1]. Infections are often secondary to joint repair or replacement surgery, systemic infection, or intravenous recreational drug use [1,2].Diabetes, rheumatoid arthritis, hepatic dysfunction, and immunosuppression are common risk factors [1,2]. Although septic arthritis can occur spontaneously, such occurrences are rare. We report a case of a previously healthy 54-year-old woman with no known risk factors presenting to a freestanding emergency department with 5 days of shoulder pain.

Emergency Department Point-of-care Ultrasound Identification of Suspected Lemierre's Syndrome: A Case Report.

INTRODUCTION: Lemierre's syndrome is septic thrombophlebitis of the internal jugular vein, most commonly associated with head and neck infections. While central catheters are associated with venous thromboembolism and catheter-associated bacterial infections, cases of Lemierre's syndrome caused by central catheters are extraordinarily rare. CASE REPORT: We detail a case of Lemierre's syndrome resulting from a peripherally inserted central catheter in a pregnant female patient. Diagnosis of this rare and potentially life-threatening disease process was expedited using point-of-care ultrasound. CONCLUSION: Diagnosis of rare but potentially life- or limb-threatening pathologies is paramount to the successful practice of emergency medicine. Identifying these rare disease processes requires a high index of suspicion and a work-up strategy that includes consideration of medical history in combination with lab and imaging findings to determine appropriate interventions.

Digital assay for rapid electronic quantification of clinical pathogens using DNA nanoballs.

Fast and accurate detection of nucleic acids is key for pathogen identification. Methods for DNA detection generally rely on fluorescent or colorimetric readout. The development of label-free assays decreases costs and test complexity. We present a novel method combining a one-pot isothermal generation of DNA nanoballs with their detection by electrical impedance. We modified loop-mediated isothermal amplification by using compaction oligonucleotides that self-assemble the amplified target into nanoballs. Next, we use capillary-driven flow to passively pass these nanoballs through a microfluidic impedance cytometer, thus enabling a fully compact system with no moving parts. The movement of individual nanoballs is detected by a change in impedance providing a quantized readout. This approach is flexible for the detection of DNA/RNA of numerous targets (severe acute respiratory syndrome coronavirus 2, HIV, ß-lactamase gene, etc.), and we anticipate that its integration into a standalone device would provide an inexpensive (<$5), sensitive (10 target copies), and rapid test (<1 hour).

Glucagon-like peptide-1 receptor agonism improves metabolic, biochemical, and histopathological indices of nonalcoholic steatohepatitis in mice.

These preclinical studies aimed to 1) increase our understanding the dietary induction of nonalcoholic steatohepatitis (NASH), and, 2) further explore the utility and mechanisms of glucagon-like peptide-1 receptor (GLP-1R) agonism in NASH. We compared the effects of a high trans-fat (HTF) or high lard fat (HLF) diet on key facets of nonalcoholic fatty liver disease (NAFLD)/NASH in Lep(ob)/Lep(ob) and C57BL6J (B6) mice. Although HLF-fed mice experienced overall greater gains in weight and adiposity, the addition of trans-fat better mirrored pathophysiological features of NASH (e.g., hepatomegaly, hepatic lipid, and fibrosis). Administration of AC3174, an exenatide analog, and GLP-1R agonist to Lep(ob)/Lep(ob) and B6 ameliorated hepatic endpoints in both dietary models. Next, we assessed whether AC3174-mediated improvements in diet-induced NASH were solely due to weight loss in HTF-fed mice. AC3174-treatment significantly reduced body weight (8.3%), liver mass (14.2%), liver lipid (12.9%), plasma alanine aminotransferase, and triglycerides, whereas a calorie-restricted, weight-matched group demonstrated only modest nonsignificant reductions in liver mass (9%) and liver lipid (5.1%) relative to controls. Treatment of GLP-1R-deficient (GLP-1RKO) mice with AC3174 had no effect on body weight, adiposity, liver or plasma indices pointing to the GLP-1R-dependence of AC3174's effects. Interestingly, the role of endogenous GLP-1Rs in NASH merits further exploration as the GLP-1RKO model was protected from the deleterious hepatic effects of HTF. Our pharmacological data further support the clinical evaluation of the utility of GLP-1R agonists for treatment of NASH.

An assessment of workplace programmes designed to control inhalation risks using respiratory protective equipment.

OBJECTIVES: Few studies have assessed respiratory protective equipment (RPE) failures at the organizational level despite evidence to suggest that compliance with good practice may be low. The aim of this study was to develop an understanding of what current RPE programmes look like across industry and how this compares with good practice. METHODS: Twenty cross-industry site visits were conducted with companies that had RPE programmes in place. Visits involved management interviews to explore current RPE systems and procedures and the decision making underpinning these. Observations of RPE operatives were included followed by short interviews to discuss the behaviours observed. Post-site assessments jointly undertaken by an RPE scientist and psychologist produced ratings for each site on six critical aspects of RPE programmes (knowledge/awareness, selection, use, training/information, supervision, and storage/cleaning/maintenance). Overall ratings for theoretical competence (i.e. management knowledge of RPE) and practical control (i.e. actual RPE practice on the shop floor) were also given. Qualitative analysis was performed on all interview data. RESULTS: The performance of RPE programmes varied across industry. Fewer than half the companies visited were considered to have an acceptable level of theoretical competence and practical control. Four distinct groups emerged from the 20 sites studied, ranging from Learners (low theoretical competence and practical control--four sites), Developers (acceptable theoretical competence and low practical control--five sites), and Fortuitous (low theoretical competence and acceptable practical control--two sites), to Proficient (acceptable theoretical competence and practical control--nine sites). None of the companies visited were achieving optimal control through the use of RPE. Widespread inadequacies were found with programme implementation, particularly training, supervision, and maintenance. CONCLUSIONS: Our taxonomy based on the four groups (Learners, Developers, Fortuitous, and Proficient) provided a useful expert-informed tool for explaining the variation in performance of RPE programmes across industry. Although further research and development are required, this taxonomy offers a useful starting point for the development of practical tools that may assist managers in making the much-needed improvements to all facets of programme implementation, particularly training, supervision, and maintenance.

The impact of two freestanding emergency departments on a tertiary care center.

BACKGROUND: Freestanding emergency departments (FEDs) have become increasingly popular as the need for emergency care continues to grow. OBJECTIVE: To analyze the impact of two FEDs on a local tertiary care center's patient volume and admission rates. METHODS: A retrospective analysis examined monthly volume and admission rates for the main ED and two FEDs located 9.6 and 12 miles away. Main ED census records were divided into three distinct time frames: period A (control) was January 2007 through June 2007. Period B was July 2007 through July 2009 when one FED was open. Period C was August 2009 through June 2010 when both FEDs were open. A two-factor analysis of variance was used to analyze admission rates while adjusting for monthly variation. RESULTS: The mean monthly patient volume for the main ED was 4709 for period A, but dropped significantly (p<0.01) to 4447 for period B, and again dropped significantly (p<0.01) to 4242 during period C. The volume for all facilities increased throughout the study period. A combined monthly volume increase to 5642 occurred in Period B, and increased to 6808 in Period C. The adjusted mean admission rate at the main ED for period A was 0.221, which dropped somewhat, though not significantly (p=0.3505) to 0.213 for period B, and then significantly (p<0.01) to 0.189 for period C. CONCLUSION: Opening two FEDs decreased the volume and admission rates for the main ED and increased the overall ED volume for the health care system.

The Use of Whole Blood in Rural Trauma Leads to Decreased Resource Utilization.

BACKGROUND: Nearly 40% of trauma deaths result from uncontrolled hemorrhage. Most of these deaths occur within 24 hours, highlighting the importance of early resuscitation. Balanced component resuscitation has been shown to improve outcomes in hemorrhagic shock. However, hemostatic properties may then be decreased, leading to inadequate coagulopathy treatment or higher transfusion requirements. Data comparing the efficacy of component vs. whole blood (WB) resuscitation in early trauma is poor, particularly in the rural population. This study investigates WB use and resource utilization at a rural Level 1 trauma center. METHODS: A prospective cohort study with historical controls (HC) was performed using patients over age 17 presenting as the highest priority trauma. Two units of WB were available to patients with signs of hemorrhagic shock, with subsequent transfusions via massive transfusion protocol or thromboelastography guidance. Component utilization, time to hemorrhage control, complications, and transfer times were examined. RESULTS: Forty patients received WB vs. 153 HC. WB patients had lower complication rates (35% vs. 55.6%; P = .02), and a significant reduction in pRBC utilization in the emergency department (0 vs. 2; P < .0001) and throughout admission (2.0 vs. 4.0; P = .0003). All patients had prolonged transport times given the rural setting (1.42 hours HC vs. 2.03 hours WB; P = .002). DISCUSSION: Unlike most urban WB studies, this study occurred in a rural area with extended transportation times, when WB is inaccessible for patients. Despite this delay, WB patients demonstrated lower component utilization and complication rates. Further research is needed to characterize the impact of early WB access.

Droplet-based pyrosequencing using digital microfluidics.

The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned on a printed-circuit board was used to control the formation, transportation, merging, mixing, and splitting of submicroliter-sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, deoxyribonucleotide triphosphates (dNTPs), and DNA templates. The DNA templates were anchored to magnetic beads which enabled them to be thoroughly washed between nucleotide additions. Reagents and protocols were optimized to maximize signal over background, linearity of response, cycle efficiency, and wash efficiency. As an initial demonstration of feasibility, a portion of a 229 bp Candida parapsilosis template was sequenced using both a de novo protocol and a resequencing protocol. The resequencing protocol generated over 60 bp of sequence with 100% sequence accuracy based on raw pyrogram levels. Excellent linearity was observed for all of the homopolymers (two, three, or four nucleotides) contained in the C. parapsilosis sequence. With improvements in microfluidic design it is expected that longer reads, higher throughput, and improved process integration (i.e., "sample-to-sequence" capability) could eventually be achieved using this low-cost platform.

Development of a multicentre automated model to reduce planning variability in radiotherapy of prostate cancer.

BACKGROUND AND PURPOSE: Inter-institutional studies highlighted correlation between consistent radiotherapy quality and improved overall patient survival. In treatment planning automation has the potential to address differences due to user-experience and training, promoting standardisation. The aim of this study was to evaluate implementation and clinical effect of a multicentre collaboratively-developed automated planning model for Intensity-Modulated Radiation Therapy/Volumetric-Modulated Arc Therapy of prostate. The model was built using a variety of public institutions' clinical plans, incorporating different contouring and dose protocols, aiming at minimising their variation. METHODS AND MATERIALS: A model using 110 clinically approved and treated prostate plans provided by different radiotherapy centres was built with RapidPlan (RP), for use on intact and post-prostatectomy prostate cases. The model was validated, distributed and introduced into clinical practice in all institutions. To investigate its impact a total of 126 patients, originally manually inverse planned (OP), were replanned using RP without additional planner manual intervention. Target and organ-at-risk (OAR) metrics were statistically compared between original and automated plans. RESULTS: For all centres combined and individually, RP provided plans comparable or superior to OP for all dose metrics. Statistically significant reductions with RP were found in bladder (V40Gy) and rectal (V50Gy) low doses (within 2.3% and 3.4% for combined and 4% and 10% individually). No clinically significant changes were seen for the PTV, independently of seminal vesicle inclusion. CONCLUSION: This project showed it is feasible to develop, share and implement RP models created with plans from different institutions treated with a variety of techniques and dose protocols, with the potential of improving treatment planning results and/or efficiency despite the original variability.

Porous AlGaN-Based Ultraviolet Distributed Bragg Reflectors.

Utilising dislocation-related vertical etching channels in gallium nitride, we have previously demonstrated a simple electrochemical etching (ECE) process that can create layered porous GaN structures to form distributed Bragg reflectors for visible light at wafer scale. Here, we apply the same ECE process to realise AlGaN-based ultraviolet distributed Bragg reflectors (DBRs). These are of interest because they could provide a pathway to non-absorbing UV reflectors to enhance the performance of UV LEDs, which currently have extremely low efficiency. We have demonstrated porous AlGaN-based UV DBRs with a peak reflectance of 89% at 324 nm. The uniformity of these devices is currently low, as the as-grown material has a high density of V-pits and these alter the etching process. However, our results indicate that if the material growth is optimised, the ECE process will be useful for the fabrication of UV reflectors.

Scalable Device for Automated Microbial Electroporation in a Digital Microfluidic Platform.

Electrowetting-on-dielectric (EWD) digital microfluidic laboratory-on-a-chip platforms demonstrate excellent performance in automating labor-intensive protocols. When coupled with an on-chip electroporation capability, these systems hold promise for streamlining cumbersome processes such as multiplex automated genome engineering (MAGE). We integrated a single Ti:Au electroporation electrode into an otherwise standard parallel-plate EWD geometry to enable high-efficiency transformation of Escherichia coli with reporter plasmid DNA in a 200 nL droplet. Test devices exhibited robust operation with more than 10 transformation experiments performed per device without cross-contamination or failure. Despite intrinsic electric-field nonuniformity present in the EP/EWD device, the peak on-chip transformation efficiency was measured to be 8.6 ± 1.0 × 108 cfu·µg-1 for an average applied electric field strength of 2.25 ± 0.50 kV·mm-1. Cell survival and transformation fractions at this electroporation pulse strength were found to be 1.5 ± 0.3 and 2.3 ± 0.1%, respectively. Our work expands the EWD toolkit to include on-chip microbial electroporation and opens the possibility of scaling advanced genome engineering methods, like MAGE, into the submicroliter regime.