A non-enzymatic test for SARS-CoV-2 RNA using DNA nanoswitches.

The emergence of a highly contagious novel coronavirus in 2019 led to an unprecedented need for large scale diagnostic testing. The associated challenges including reagent shortages, cost, deployment delays, and turnaround time have all highlighted the need for an alternative suite of low-cost tests. Here, we demonstrate a diagnostic test for SARS-CoV-2 RNA that provides direct detection of viral RNA and eliminates the need for costly enzymes. We employ DNA nanoswitches that respond to segments of the viral RNA by a change in shape that is readable by gel electrophoresis. A new multi-targeting approach samples 120 different viral regions to improve the limit of detection and provide robust detection of viral variants. We apply our approach to a cohort of clinical samples, positively identifying a subset of samples with high viral loads. Since our method directly detects multiple regions of viral RNA without amplification, it eliminates the risk of amplicon contamination and renders the method less susceptible to false positives. This new tool can benefit the COVID-19 pandemic and future emerging outbreaks, providing a third option between amplification-based RNA detection and protein antigen detection. Ultimately, we believe this tool can be adapted both for low-resource onsite testing as well as for monitoring viral loads in recovering patients.

Fluorometric Determination of DNA Nanostructure Biostability.

The analysis and improvement of DNA nanostructure biostability is one of the keys areas of progress needed in DNA nanotechnology applications. Here, we present a plate-compatible fluorometric assay for measuring DNA nanostructure biostability using the common intercalator ethidium bromide. We demonstrate the assay by testing the biostability of duplex DNA, a double crossover DNA motif, and a DNA origami nanostructure against different nucleases and in fetal bovine serum. This method scales well to measure a large number of samples using a plate reader and can complement existing methods for assessing and developing robust DNA nanostructures.

High-throughput single-molecule quantification of individual base stacking energies in nucleic acids.

Base stacking interactions between adjacent bases in DNA and RNA are important for many biological processes and in biotechnology applications. Previous work has estimated stacking energies between pairs of bases, but contributions of individual bases has remained unknown. Here, we use a Centrifuge Force Microscope for high-throughput single molecule experiments to measure stacking energies between adjacent bases. We found stacking energies strongest between purines (G|A at -2.3 ± 0.2 kcal/mol) and weakest between pyrimidines (C|T at -0.5 ± 0.1 kcal/mol). Hybrid stacking with phosphorylated, methylated, and RNA nucleotides had no measurable effect, but a fluorophore modification reduced stacking energy. We experimentally show that base stacking can influence stability of a DNA nanostructure, modulate kinetics of enzymatic ligation, and assess accuracy of force fields in molecular dynamics simulations. Our results provide insights into fundamental DNA interactions that are critical in biology and can inform design in biotechnology applications.

Male Youth Ice Hockey Concussion Incidence in a USA Hockey Membership-Adjusted Population: A Peak in 2011 and the Impact of Major Rule Changes.

OBJECTIVE: To investigate the incidence of youth ice hockey-related concussions preceding and following the implementation of new body-checking and head contact rules by USA hockey in 2011. We hypothesized a decrease in concussions after the rule change. DESIGN: Retrospective analysis. SETTING: United States emergency department (ED) data queried in the National Electronic InjurySurveillance System (NEISS). PATIENTS: National Electronic Injury Surveillance System reported male youth (≤18 years) ice hockey concussion cases from January 1, 2002, to December 31, 2016. In total, 848 players were diagnosed with concussion, representing a national estimate of 17 374 cases. INDEPENDENT VARIABLES: Time, specifically years. MAIN OUTCOME MEASURES: Incidences and incidence rates (measured per 10 000 person-years) of male youth ice hockey concussions. Annual trends were analyzed using descriptive and linear or polynomial regression analysis. RESULTS: The national estimate of youth ice hockey-related concussions seen in US emergency departments (EDs) increased significantly from 656 in 2007 to 2042 in 2011 (P < 0.01). During the same period, their respective incidence increased significantly from 21.8 to 66.8 per 10 000, before dropping through 2016 (P < 0.05). After 2011, concussions decreased from 1965 in 2012 to 1292 in 2016 (P = 0.055). The gap in concussion incidence between the 11 to 12 and 13 to 14 divisions widened after 2011 (before 2011: 41 vs 49 per 10 000 person-years [P = 0.80]; after 2011: 45 and 89, respectively [P < 0.01]). CONCLUSIONS: US EDs experienced a significant increase in youth ice hockey concussion visits from 2007 to 2011. After the 2011 rule changes, concussion visits decreased significantly from 2012 to 2016.

The Use of Cangrelor Infusions After Endovascular Aortic Repair With Prophylactic Lumbar Drain Placement.

Perioperative lumbar drains commonly are placed for spinal cord protection in patients undergoing endovascular aortic repair. However, the logistics of postoperative neuraxial drain removal is challenging in the presence of systemic antithrombotic therapy. This retrospective case series describes the novel use of cangrelor infusions in this high-risk setting. All lumbar drains were placed preoperatively, and descriptive data were collected including cangrelor infusion duration, time to lumbar drain removal after the infusion discontinuation, clinical course, and overall patient outcomes. There were no neurologic complications associated with lumbar drain insertion or removal, and median time to lumbar drain removal was 150 minutes after cangrelor infusion discontinuation. While further study is needed to validate its efficacy and safety, this case series highlights the promise of cangrelor infusions for systemic antithrombotic therapy in the cardiovascular/surgical intensive care unit.

Rapid, Affordable, and Scalable SARS-CoV-2 Detection From Saliva.

Here we present an inexpensive, rapid, and robust reverse-transcription loop-mediated isothermal amplification (RT-LAMP)-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection method that is easily scalable, enabling point-of-care facilities and clinical labs to determine results from patients' saliva directly in 30 minutes for less than $2 per reaction. The method uses a novel combination of widely available reagents that can be prepared in bulk, plated, and frozen and remain stable until samples are received. This innovation dramatically reduces preparation time, enabling high-throughput automation and testing with time to results (including setup) in less than 1 hour for 96 patient samples simultaneously when using a 384-well format. By using a dual reporter (phenol red pH indicator for end-point detection and SYTO-9 fluorescent dye for real time), the assay also provides internal validation of results and redundancy in the event of an instrument malfunction.

Molecular Characterization of Increased Amplicon Lengths in SARS-CoV-2 Reverse Transcription Loop-Mediated Isothermal Amplification Assays.

Loop-mediated isothermal amplification (LAMP) is a power tool for the amplification of specific RNA and DNA targets. Much like PCR, LAMP requires primers that surround a target amplification region and generates exponential product through a unique highly specific daisy-chain single-temperature amplification reaction. However, until recently, attempts to amplify targets of greater than 200 base pairs (bp) have been mostly unsuccessful and limited to short amplicon targets of less than 150 bp. Although short amplicons have the benefit of a rapid detection (<40 min), they do not allow for the prediction of RNA integrity based on RNA length and possible intactness. In this study, 8 primer sets were developed using 2 LAMP primer-specific software packages against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid gene with insert lengths ranging from 262 to 945 bp in order to amplify and infer the integrity of viral RNA. Because these amplification lengths are greater than the current methods that use an insert length of 130 or less, they require a longer incubation, modified primer and temperature strategies, and the addition of specific adjuncts to prevent nonspecific amplification. This proof of concept study resulted in successful reverse transcription LAMP reactions for amplicon targets of 262, 687, 693, and 945 bp using a clinical nasopharyngeal NP sample, purified SARS-CoV-2 RNA, and crude lysate containing inactivated virus.

Loop-Mediated Isothermal Amplification Detection of SARS-CoV-2 and Myriad Other Applications.

As the second year of the COVID-19 pandemic begins, it remains clear that a massive increase in the ability to test for SARS-CoV-2 infections in a myriad of settings is critical to controlling the pandemic and to preparing for future outbreaks. The current gold standard for molecular diagnostics is the polymerase chain reaction (PCR), but the extraordinary and unmet demand for testing in a variety of environments means that both complementary and supplementary testing solutions are still needed. This review highlights the role that loop-mediated isothermal amplification (LAMP) has had in filling this global testing need, providing a faster and easier means of testing, and what it can do for future applications, pathogens, and the preparation for future outbreaks. This review describes the current state of the art for research of LAMP-based SARS-CoV-2 testing, as well as its implications for other pathogens and testing. The authors represent the global LAMP (gLAMP) Consortium, an international research collective, which has regularly met to share their experiences on LAMP deployment and best practices; sections are devoted to all aspects of LAMP testing, including preanalytic sample processing, target amplification, and amplicon detection, then the hardware and software required for deployment are discussed, and finally, a summary of the current regulatory landscape is provided. Included as well are a series of first-person accounts of LAMP method development and deployment. The final discussion section provides the reader with a distillation of the most validated testing methods and their paths to implementation. This review also aims to provide practical information and insight for a range of audiences: for a research audience, to help accelerate research through sharing of best practices; for an implementation audience, to help get testing up and running quickly; and for a public health, clinical, and policy audience, to help convey the breadth of the effect that LAMP methods have to offer.

Sequence-selective purification of biological RNAs using DNA nanoswitches.

Nucleic acid purification is a critical aspect of biomedical research and a multibillion-dollar industry. Here we establish sequence-selective RNA capture, release, and isolation using conformationally responsive DNA nanoswitches. We validate purification of specific RNAs ranging in size from 22 to 401 nt with up to 75% recovery and 99.98% purity in a benchtop process with minimal expense and equipment. Our method compared favorably with bead-based extraction of an endogenous microRNA from cellular total RNA, and can be programmed for multiplexed purification of multiple individual RNA targets from one sample. Coupling our approach with downstream LC/MS, we analyzed RNA modifications in 5.8S ribosomal RNA, and found 2'-O-methylguanosine, 2'-O-methyluridine, and pseudouridine in a ratio of ~1:7:22. The simplicity, low cost, and low sample requirements of our method make it suitable for easy adoption, and the versatility of the approach provides opportunities to expand the strategy to other biomolecules.

Wi-Fi Live-Streaming Centrifuge Force Microscope for Benchtop Single-Molecule Experiments.

The ability to apply controlled forces to individual molecules has been revolutionary in shaping our understanding of biophysics in areas as diverse as dynamic bond strength, biological motor operation, and DNA replication. However, the methodology to perform single-molecule experiments remains relatively inaccessible because of cost and complexity. In 2010, we introduced the centrifuge force microscope (CFM) as a platform for accessible and high-throughput single-molecule experimentation. The CFM consists of a rotating microscope with which prescribed centrifugal forces can be applied to microsphere-tethered biomolecules. In this work, we develop and demonstrate a next-generation Wi-Fi CFM that offers unprecedented ease of use and flexibility in design. The modular CFM unit fits within a standard benchtop centrifuge and connects by Wi-Fi to an external computer for live control and streaming at near gigabit speeds. The use of commercial wireless hardware allows for flexibility in programming and provides a streamlined upgrade path as Wi-Fi technology advances. To facilitate ease of use, detailed build and setup instructions, as well as LabVIEW-based control software and MATLAB-based analysis software, are provided. We demonstrate the instrument's performance by analysis of force-dependent dissociation of short DNA duplexes of 7, 8, and 9 bp. We showcase the sensitivity of the approach by resolving distinct dissociation kinetic rates for a 7 bp duplex in which one G-C basepair is mutated to an A-T basepair.

Characterizing Hand Infections in an Underserved Population: The Role of Diabetic Status in Antibiotic Choice and Infection Location.

Introduction Patients with diabetes mellitus (DM) in underserved communities are at greater risk for hand infections. We aimed to describe the features of hand infections presenting to an urban hospital via laboratories, microbiology, and antibiotic choice with respect to diabetic status. Materials and Methods Patients presenting with any hand infection were reviewed and stratified by DM status and infection location. Labs, culture results, antibiotic regimens, and significant predictors of laboratories or infection location were analyzed. Results Fifty-three patients were included: DM ( n = 24), no-DM ( n = 24), and unknown status ( n = 5). Culture rates were comparable between all groups. Mean erythrocyte sedimentation rate (ESR) was significantly higher in DM (76.19 vs. 51.33); mean white blood cell count (WBC) and C-reactive protein (CRP) were comparable. Diabetics had higher odds of increased ESR (odds ratio [OR] = 1.03). Diabetics received vancomycin/piperacillin/tazobactam (VAN/PTZ) significantly more often (52% vs. 8%). Providers treated DM with VAN/PTZ or any VAN-containing regimen more often than with any other regimen. Proximal infections had significantly higher mean CRP (136.9 vs. 50.5) and WBC (5.19 vs. 3.9) and higher CRP (OR = 1.02). Conclusion This study highlights the need for systematic criteria to better risk- stratify patients for appropriate antibiotic treatment. It may not be appropriate to treat both groups differently, as overly aggressive antibiotic selection may contribute to drug-resistance development.

Second Generation Nanoporous Silicon Nitride Membranes for High Toxin Clearance and Small Format Hemodialysis.

Conventional hemodialysis (HD) uses floor-standing instruments and bulky dialysis cartridges containing ≈2 m2 of 10 micrometer thick, tortuous-path membranes. Portable and wearable HD systems can improve outcomes for patients with end-stage renal disease by facilitating more frequent, longer dialysis at home, providing more physiological toxin clearance. Developing devices with these benefits requires highly efficient membranes to clear clinically relevant toxins in small formats. Here, the ability of ultrathin (<100 nm) silicon-nitride-based membranes to reduce the membrane area required to clear toxins by orders of magnitude is shown. Advanced fabrication methods are introduced that produce nanoporous silicon nitride membranes (NPN-O) that are two times stronger than the original nanoporous nitride materials (NPN) and feature pore sizes appropriate for middle-weight serum toxin removal. Single-pass benchtop studies with NPN-O (1.4 mm2 ) demonstrate the extraordinary clearance potential of these membranes (105 mL min-1 m-2 ), and their intrinsic hemocompatibility. Results of benchtop studies with nanomembranes, and 4 h dialysis of uremic rats, indicate that NPN-O can reduce the membrane area required for hemodialysis by two orders of magnitude, suggesting the performance and robustness needed to enable small-format hemodialysis, a milestone in the development of small-format hemodialysis systems.

Knee dislocation with popliteal artery disruption: A nationwide analysis from 2005 to 2013.

OBJECTIVE: Few have compared short-term outcomes following knee dislocations with or without concomitant popliteal artery disruption (PAD). METHODS: The Nationwide Inpatient Sample was used to identify 2175 patients admitted for knee dislocation from 2005 to 2013 (concomitant PAD: n = 210/9.7%; without: n = 1965/90.3%). RESULTS: Patients with PAD were younger, more often male, Black and Hispanic, and with Medicaid (all p ≤ 0.013). PADs were associated with 11.0-times higher odds of increased LOS (95%CI, 6.6-18.4) and 2.8-times higher odds of experiencing any complication (95%CI, 2.03-3.92). Female sex was a protective factor against increased LOS, (OR = 0.65; 95%CI, 0.48-0.88). CONCLUSION: High suspicion index should be maintained for concomitant vascular injuries following knee dislocations.

Total shoulder arthroplasty in octogenarians: Is there a higher risk of adverse outcomes?

This study used the National Surgical Quality Improvement Program to evaluate octogenarians who underwent total shoulder arthroplasty (TSA). Specifically, we evaluated: (1) patient demographics; (2) perioperative factors; and (3) 30-day postoperative complications. Compared to controls, the octogenarians had more females, white patients, lower BMIs, fewer smokers, less functionally independent, higher ASA scores, shorter operative times, and longer LOS. Octogenarians had greater odds for developing any (OR = 2.05; 95%CI, 1.70-2.46), any major (OR = 2.28; 95%CI, 1.66-3.13), and any minor (OR = 1.99; 95%CI, 1.63-2.45) complications. Perioperative risk management strategies for elective TSA in the elderly may help mitigate the increased perioperative risks associated with age.

Shoulder Lesion in a 69 Year Old Woman.

Milwaukee Shoulder Syndrome (MSS) is a painful progressive arthropathy in which hydroxyapatite crystal deposition in synovial tissue induces lysosomal release of collagenase and neutral proteases. These enzymes are destructive to periarticular tissue, including the synovium, articular cartilage, rotator cuff muscles, and the intrasynovial cortical bone. MSS predominantly occurring in women (90%) over the age of 70 years of age with a clinical history marked by recurrent joint effusions and pain, which classically worsens at night. Our patient is a 69-year-old woman who presented with progressive shoulder pain, most prominent at night, with limited range of motion and swelling; intermittent discharge; and intermittent neck pain that radiated to her right upper extremity. Her medical history was notable for invasive carcinoma of the right breast treated with mastectomy and radiation. She was also treated with radiation therapy for right shoulder pain and a lucent right shoulder lesion presumed to be metastatic breast cancer. The remainder of her medical history consists of hypertension, diabetes mellitus, hyperlipidemia, and uneventful bilateral total knee arthroplasties. At presentation, she denied constitutional symptoms. Based on the patient's history and physical exam the differential diagnosis included primary and metastatic malignancy, radiation induced sarcoma and necrosis, infection, Charcot disease, and crystal arthropathies. Physical exam, laboratory findings, and imaging studies led us to the diagnosis of MSS.

The effect of pelvic motion on spinopelvic parameters.

BACKGROUND CONTEXT: To date, many studies have examined how pelvic position affects the spinal curvature and spinopelvic parameters. However, these studies focus on a static relationship, comparing pelvis and spine in a relaxed or baseline position only. Indeed, the spinopelvic connection is dynamic, as subjects can easily be taught to rotate their pelvis anteriorly or posteriorly on the femoral head, all while maintaining an erect posture. Therefore, for a true understanding of pelvic influence on the spinal column, it is necessary to examine spinopelvic parameters in multiple pelvic positions within the same subject. PURPOSE: The objective of this study was to examine the dynamic effect of pelvic motion on the spine and associated radiographic parameters. STUDY DESIGN: This is a single-center, cross-sectional study of 50 healthy, asymptomatic volunteers. PATIENT SAMPLE: Subjects were recruited and screened based on the following criteria: between 18 and 79 years of age; no known spinal, pelvic, or lower extremity pain lasting for >48 hours; no history of spinal, pelvic, or lower extremity dysfunction requiring medical care; no radiographic evidence of spinal or pelvic abnormality, scoliosis deformity, or other associated spinal pathologies; not currently pregnant and with no possibility of being pregnant; and a body mass index of <30. 64. The subjects were screened and 14 were excluded for a total of 50 subjects. OUTCOME MEASURES: The outcome measures included thoracic kyphosis (TK), lumbar lordosis (LL), sagittal vertical axis (SVA), pelvic tilt (PT), sacral slope (SS), and pelvic incidence (PI). MATERIALS AND METHODS: This study was funded by a Small Exploratory Research Grant from the Scoliosis Research Society. Each subject was instructed and observed to stand in three different positions: pelvic resting, anterior pelvic rotation, and posterior pelvic rotation. Lateral standing radiographs were taken in each position and each image was examined by an orthopedic spine surgeon who digitally measured the TK, LL, SVA, PT, SS, and PI. The data were then statistically examined to determine the affect of pelvic position on each parameter. RESULTS: Subjects demonstrated a measurable, statistically significant change in each parameter with pelvic rotation. There was a clear pattern of change for LL, PT, and SS with the anterior and posterior pelvic rotations. A change in LL demonstrated a strong correlation with changes in all measured parameters with pelvic rotation. CONCLUSIONS: In asymptomatic subjects, pelvic motion affects the position of the spinal column and resultant spinopelvic parameters. The results of this study demonstrate that one can intentionally change the position of the pelvis and the adjacent spinal column in space. Knowledge of this relationship is important to the understanding of sagittal balance and could influence the treatment of patients with spinal deformity.

Mind the Gap. A systematic review to identify usability and safety challenges and practices during electronic health record implementation.

OBJECTIVE: Decisions made during electronic health record (EHR) implementations profoundly affect usability and safety. This study aims to identify gaps between the current literature and key stakeholders' perceptions of usability and safety practices and the challenges encountered during the implementation of EHRs. MATERIALS AND METHODS: Two approaches were used: a literature review and interviews with key stakeholders. We performed a systematic review of the literature to identify usability and safety challenges and best practices during implementation. A total of 55 articles were reviewed through searches of PubMed, Web of Science and Scopus. We used a qualitative approach to identify key stakeholders' perceptions; semi-structured interviews were conducted with a diverse set of health IT stakeholders to understand their current practices and challenges related to usability during implementation. We used a grounded theory approach: data were coded, sorted, and emerging themes were identified. Conclusions from both sources of data were compared to identify areas of misalignment. RESULTS: We identified six emerging themes from the literature and stakeholder interviews: cost and resources, risk assessment, governance and consensus building, customization, clinical workflow and usability testing, and training. Across these themes, there were misalignments between the literature and stakeholder perspectives, indicating major gaps. DISCUSSION: Major gaps identified from each of six emerging themes are discussed as critical areas for future research, opportunities for new stakeholder initiatives, and opportunities to better disseminate resources to improve the implementation of EHRs. CONCLUSION: Our analysis identified practices and challenges across six different emerging themes, illustrated important gaps, and results suggest critical areas for future research and dissemination to improve EHR implementation.

Nanoporous membrane robustness / stability in small form factor microfluidic filtration system.

The development of wearable hemodialysis (HD) devices that replace center-based HD holds the promise to improve both outcomes and quality-of-life for patients with end-stage-renal disease (ERD). A prerequisite for these devices is the development of highly efficient membranes that can achieve high toxin clearance in small footprints. The ultrathin nanoporous membrane material developed by our group is orders of magnitude more permeable than conventional HD membranes. We report on our progress making a prototype wearable dialysis unit. First, we present data from benchtop studies confirming that clinical levels of urea clearance can be obtained in a small animal model with low blood flow rates. Second, we report on efforts to improve the mechanical robustness of high membrane area dialysis devices.