Mechanism of an animal toxin-antidote system.

Toxin-antidote systems are selfish genetic elements composed of a linked toxin and antidote. The peel-1 zeel-1 toxin-antidote system in C. elegans consists of a transmembrane toxin protein PEEL-1 which acts cell autonomously to kill cells. Here we investigate the molecular mechanism of PEEL-1 toxicity. We find that PEEL-1 requires a small membrane protein, PMPL-1, for toxicity. Together, PEEL-1 and PMPL-1 are sufficient for toxicity in a heterologous system, HEK293T cells, and cause cell swelling and increased cell permeability to monovalent cations. Using purified proteins, we show that PEEL-1 and PMPL-1 allow ion flux through lipid bilayers and generate currents which resemble ion channel gating. Our work suggests that PEEL-1 kills cells by co-opting PMPL-1 and creating a cation channel.

Nanopores map the acid-base properties of a single site in a single DNA molecule.

Nanopores are increasingly powerful tools for single molecule sensing, in particular, for sequencing DNA, RNA and peptides. This success has spurred efforts to sequence non-canonical nucleic acid bases and amino acids. While canonical DNA and RNA bases have pKas far from neutral, certain non-canonical bases, natural RNA modifications, and amino acids are known to have pKas near neutral pHs at which nanopore sequencing is typically performed. Previous reports have suggested that the nanopore signal may be sensitive to the protonation state of an individual moiety. We sequenced ion currents with the MspA nanopore using a single stranded DNA containing a single non-canonical DNA base (Z) at various pH conditions. The Z-base has a near-neutral pKa ∼ 7.8. We find that the measured ion current is remarkably sensitive to the protonation state of the Z-base. We demonstrate how nanopores can be used to localize and determine the pKa of individual moieties along a polymer. More broadly, these experiments provide a path to mapping different protonation sites along polymers and give insight in how to optimize sequencing of polymers that contain moieties with near-neutral pKas.

Enzymatic synthesis and nanopore sequencing of 12-letter supernumerary DNA.

The 4-letter DNA alphabet (A, T, G, C) as found in Nature is an elegant, yet non-exhaustive solution to the problem of storage, transfer, and evolution of biological information. Here, we report on strategies for both writing and reading DNA with expanded alphabets composed of up to 12 letters (A, T, G, C, B, S, P, Z, X, K, J, V). For writing, we devise an enzymatic strategy for inserting a singular, orthogonal xenonucleic acid (XNA) base pair into standard DNA sequences using 2'-deoxy-xenonucleoside triphosphates as substrates. Integrating this strategy with combinatorial oligos generated on a chip, we construct libraries containing single XNA bases for parameterizing kmer basecalling models for commercially available nanopore sequencing. These elementary steps are combined to synthesize and sequence DNA containing 12 letters - the upper limit of what is accessible within the electroneutral, canonical base pairing framework. By introducing low-barrier synthesis and sequencing strategies, this work overcomes previous obstacles paving the way for making expanded alphabets widely accessible.

Observing inhibition of the SARS-CoV-2 helicase at single-nucleotide resolution.

The genome of SARS-CoV-2 encodes for a helicase (nsp13) that is essential for viral replication and highly conserved across related viruses, making it an attractive antiviral target. Here we use nanopore tweezers, a high-resolution single-molecule technique, to gain detailed insight into how nsp13 turns ATP-hydrolysis into directed motion along nucleic acid strands. We measured nsp13 both as it translocates along single-stranded DNA or unwinds double-stranded DNA. Our data reveal nsp13's single-nucleotide steps, translocating at ∼1000 nt/s or unwinding at ∼100 bp/s. Nanopore tweezers' high spatiotemporal resolution enables detailed kinetic analysis of nsp13 motion. As a proof-of-principle for inhibition studies, we observed nsp13's motion in the presence of the ATPase inhibitor ATPγS. We construct a detailed picture of inhibition in which ATPγS has multiple mechanisms of inhibition. The dominant mechanism of inhibition depends on the application of assisting force. This lays the groundwork for future single-molecule inhibition studies with viral helicases.

Pharmacist-Led Discharge Transitions of Care Interventions for Pediatric Patients: A Narrative Review.

Transitions of care (TOC) before, during, and after hospital discharge are an opportune setting to optimize medication management. The quality standards for pediatric care transitions, however, are lacking, leading to reduced health outcomes in children. This narrative review characterizes the pediatric populations that would benefit from focused, TOC interventions. Different types of medication-focused TOC interventions during hospital discharge are described, including medication reconciliation, education, access, and adherence tools. Various TOC intervention delivery models following hospital discharge are also reviewed. The goal of this narrative review is to help pediatric pharmacists and pharmacy leaders better understand TOC interventions and integrate them into the hospital discharge process for children and their caregivers.

Assessing Readability of an 8-Letter Expanded Deoxyribonucleic Acid Alphabet with Nanopores.

Chemists have now synthesized new kinds of DNA that add nucleotides to the four standard nucleotides (guanine, adenine, cytosine, and thymine) found in standard Terran DNA. Such "artificially expanded genetic information systems" are today used in molecular diagnostics; to support directed evolution to create medically useful receptors, ligands, and catalysts; and to explore issues related to the early evolution of life. Further applications are limited by the inability to directly sequence DNA containing nonstandard nucleotides. Nanopore sequencing is well-suited for this purpose, as it does not require enzymatic synthesis, amplification, or nucleotide modification. Here, we take the first steps to realize nanopore sequencing of an 8-letter "hachimoji" expanded DNA alphabet by assessing its nanopore signal range using the MspA (Mycobacterium smegmatis porin A) nanopore. We find that hachimoji DNA exhibits a broader signal range in nanopore sequencing than standard DNA alone and that hachimoji single-base substitutions are distinguishable with high confidence. Because nanopore sequencing relies on a molecular motor to control the motion of DNA, we then assessed the compatibility of the Hel308 motor enzyme with nonstandard nucleotides by tracking the translocation of single Hel308 molecules along hachimoji DNA, monitoring the enzyme kinetics and premature enzyme dissociation from the DNA. We find that Hel308 is compatible with hachimoji DNA but dissociates more frequently when walking over C-glycoside nucleosides, compared to N-glycosides. C-glycocide nucleosides passing a particular site within Hel308 induce a higher likelihood of dissociation. This highlights the need to optimize nanopore sequencing motors to handle different glycosidic bonds. It may also inform designs of future alternative DNA systems that can be sequenced with existing motors and pores.

Risk of Inappropriately Timed Live Vaccination After Pediatric Cardiovascular Surgery.

OBJECTIVE: The American Academy of Pediatrics (AAP), Advisory Committee on Immunization Practices (ACIP), and Centers for Disease Control and Prevention (CDC) recommend delaying live vaccinations up to 11 months after transfusions of certain blood products due to the risk of immunoglobulins decreasing immunization efficacy. Because vaccination schedules recommend live immunizations at 12 months, infants aged 5 to 12 months who undergo cardiac surgery requiring blood products are potentially at risk for improper vaccination. The objective of this study was to identify the risk of inappropriately timed live vaccination in pediatric patients after cardiovascular surgery. METHODS: This single-center, retrospective chart review included 345 patients 5 to 12 months of age who underwent cardiovascular surgery between January 1, 2010, and December 31, 2016. Included patients received packed red blood cells (PRBCs) and/or platelets during the surgical admission and a live vaccine within the first 18 months of life. The primary endpoint was the incidence of live vaccine administration within 7 months of receiving PRBCs and/or platelets. RESULTS: Of the 345 included patients, 67% (n = 230) were inappropriately vaccinated after receiving platelets and/or PRBCs during cardiac surgery. CONCLUSIONS: Infants who undergo cardiac surgery between the ages of 5 and 12 months are at risk for inappropriate live vaccination timing. A clinically significant percentage of pediatric patients who received blood products during a cardiac surgical admission later received live vaccines at times that were inconsistent with AAP, ACIP, and CDC recommendations. Future interventions aimed at educating providers and patients may be warranted.

Inhibition of the SARS-CoV-2 helicase at single-nucleotide resolution.

The genome of SARS-CoV-2 encodes for a helicase called nsp13 that is essential for viral replication and highly conserved across related viruses, making it an attractive antiviral target. Here we use nanopore tweezers, a high-resolution single-molecule technique, to gain detailed insight into how nsp13 turns ATP-hydrolysis into directed motion along nucleic acid strands. We measured nsp13 both as it translocates along single-stranded DNA or unwinds short DNA duplexes. Our data confirm that nsp13 uses the inchworm mechanism to move along the DNA in single-nucleotide steps, translocating at ~1000 nt/s or unwinding at ~100 bp/s. Nanopore tweezers' high spatio-temporal resolution enables observation of the fundamental physical steps taken by nsp13 even as it translocates at speeds in excess of 1000 nucleotides per second enabling detailed kinetic analysis of nsp13 motion. As a proof-of-principle for inhibition studies, we observed nsp13's motion in the presence of the ATPase inhibitor ATPγS. Our data reveals that ATPγS interferes with nsp13's action by affecting several different kinetic processes. The dominant mechanism of inhibition differs depending on the application of assisting force. These advances demonstrate that nanopore tweezers are a powerful method for studying viral helicase mechanism and inhibition.

Prescription Patterns for Pulmonary Vasodilators in the Treatment of Pulmonary Hypertension Associated With Chronic Lung Diseases: Insights From a Clinician Survey.

Background: Pulmonary hypertension is a complication of chronic lung diseases (PH-CLD) associated with significant morbidity and mortality. Management guidelines for PH-CLD emphasize the treatment of the underlying lung disease, but the role of PH-targeted therapy remains controversial. We hypothesized that treatment approaches for PH-CLD would be variable across physicians depending on the type of CLD and the severity of PH. Methods and Results: Between May and July 2020, we conducted an online survey of PH experts asking for their preferred treatment approach in seven hypothetical cases of PH-CLD of varying severity. We assessed agreement amongst clinicians for initial therapy choice using Fleiss' kappa calculations. Over 90% of respondents agreed that they would treat cases of severe PH in the context of mild lung disease with some form of PH-targeted therapy. For cases of severe PH in the context of severe lung disease, over 70% of respondents agreed to use PH-targeted therapy. For mild PH and mild lung disease cases, <50% of respondents chose to start PH-specific therapy. There was overall poor agreement between respondents in the choice to use mono-, double or triple combination therapy with PH-specific agents in all cases. Conclusion: Although management guidelines discourage the routine use of PH-targeted therapies to treat PH-CLD patients, most physicians choose to treat patients with some form of PH-targeted therapy. The choice of therapy and treatment approach are variable and appear to be influenced by the severity of the PH and the underlying lung disease.

Low-dose intrathecal rituximab is a safe and potentially effective treatment for pediatric neuroimmunologic disorders.

Historically, treatment options for refractory neuroimmunologic disorders have been limited. Use of intrathecal rituximab has been described in a few case reports but experience in pediatric patients is limited. Here, we report our experience with intrathecal rituximab in 5 pediatric patients with refractory neuroimmunologic conditions. Patients were identified based on treatment-refractory symptoms despite first and second-line therapies and treated according to a standardized protocol. Although individual outcomes varied, intrathecal rituximab showed a favorable safety profile and was well-tolerated. Three out of five patients showed evidence of a positive clinical response assessed by modified Rankin score or Mitchell-Pike Opsoclonus-Myoclonus score. Findings from this retrospective observational study suggest that intrathecal rituximab is a safe and potentially effective therapy in carefully selected patients with refractory neuroimmunologic disorders despite appropriate first and second-line therapies.

Pulmonary Vein Stenosis and Pulmonary Hypertension Following a Catheter-Based Radiofrequency Ablation for Atrial Fibrillation: A Case Report.

BACKGROUND Pulmonary vein (PV) stenosis is a rare condition characterized by progressive luminal size reduction of one or more pulmonary veins (PVs), which can increase postcapillary pressure resulting in shortness of breath, cough, hemoptysis, and pulmonary hypertension (PH). The diagnosis of PV stenosis requires a high degree of suspicion. PV stenosis is a rare but recognized complication of catheter-based radiofrequency ablation (RFA) for atrial fibrillation (AF). CASE REPORT We present a case of a 78-year-old man who underwent a surgical MAZE procedure followed by catheter-based RFA to treat AF. He subsequently developed shortness of breath, exercise limitation, and PH. The patient was ultimately diagnosed with PV stenosis, which was a sequela of the RFA and the cause of his PH. The patient was treated by stenting of his PV, with improvement in his exercise capacity and PH. Follow-up imaging showed improved pulmonary blood flow and reduced pulmonary pressures. CONCLUSIONS We conclude that PV stenosis should be high in the differential as the cause of dyspnea in patients with PH and a previous history of RFA for AF management. Early recognition and treatment can prevent complete occlusion of the affected PV and lead to an improvement in the patient's symptoms and quality of life.

Temporary Membrane Permeabilization via the Pore-Forming Toxin Lysenin.

Pore-forming toxins are alluring tools for delivering biologically-active, impermeable cargoes to intracellular environments by introducing large conductance pathways into cell membranes. However, the lack of regulation often leads to the dissipation of electrical and chemical gradients, which might significantly affect the viability of cells under scrutiny. To mitigate these problems, we explored the use of lysenin channels to reversibly control the barrier function of natural and artificial lipid membrane systems by controlling the lysenin's transport properties. We employed artificial membranes and electrophysiology measurements in order to identify the influence of labels and media on the lysenin channel's conductance. Two cell culture models: Jurkat cells in suspension and adherent ATDC5 cells were utilized to demonstrate that lysenin channels may provide temporary cytosol access to membrane non-permeant propidium iodide and phalloidin. Permeability and cell viability were assessed by fluorescence spectroscopy and microscopy. Membrane resealing by chitosan or specific media addition proved to be an effective way of maintaining cellular viability. In addition, we loaded non-permeant dyes into liposomes via lysenin channels by controlling their conducting state with multivalent metal cations. The improved control over membrane permeability might prove fruitful for a large variety of biological or biomedical applications that require only temporary, non-destructive access to the inner environment enclosed by natural and artificial membranes.

Diagnosis and Management of Pulmonary Hypertension in the Modern Era: Insights from the 6th World Symposium.

The past 20 years have seen major advances in the diagnosis and management of pulmonary hypertension, a disease associated with significant morbidity and mortality. The 6th World Symposium in Pulmonary Hypertension (WSPH) took place in February 2018 and attempted to consolidate the current knowledge in the field into practical recommendations to help prioritize an action plan to improve patient outcomes and identify future research directions. In this review, we will summarize the highlights of the 6th WSPH proceedings, including revisions to the hemodynamic definitions and classification of the various types of pulmonary hypertension, genetic advances, approaches to risk stratification, and updated treatment algorithms.

Safety of enteral sildenafil in hemodynamically unstable children.

BACKGROUND: Enteral sildenafil may be used in the intensive care unit for treatment of pulmonary arterial hypertension. We aimed to determine if initial enteral sildenafil dosing is safe in children receiving concurrent vasoactive infusions. METHODS: We performed a single-centre retrospective chart review that included patients less than 2 years of age in paediatric and cardiovascular intensive care units at an academic medical centre from 1 January, 2010 to 30 November, 2016. Included patients received concomitant enteral sildenafil and a continuously infused vasoactive agent. Exclusion criteria consisted of mechanical circulatory support, any form of dialysis, or a suspicion of septic shock at the time of sildenafil initiation. We sought to identify patients who developed worsening hemodynamic instability after initiation of enteral sildenafil defined as one or more of the following observations within 24 hours of sildenafil initiation: sildenafil discontinuation, total fluid bolus receipt >10 ml/kg, increased vasoactive support, epinephrine intravenous push administration, and/or the initiation of mechanical circulatory support. RESULTS: Worsening hemodynamic instability was identified in 35% of the 130-patient cohort. Patients younger than 4 months were at increased risk of further hemodynamic instability compared with older patients (56% versus 44%, p = 0.0003) despite receiving lower median doses (1.28 mg/kg/day versus 1.78 mg/kg/day, p = 0.01). CONCLUSIONS: Critically ill children receiving vasoactive infusions may be at increased risk for further hemodynamic instability after initiation of enteral sildenafil, particularly in younger patients. This population may benefit from lower starting enteral sildenafil doses of 0.25 mg/kg/dose or less every 8 hours to avoid further hemodynamic compromise.

Utility of routine methemoglobin laboratory assays in critically ill pediatric subjects receiving inhaled nitric oxide.

PURPOSE: Inhaled nitric oxide (iNO) has been associated with safety risks including reports of methemoglobinemia. While standard of care recommends routine monitoring of methemoglobin in subjects on iNO therapy, the utility of this practice remains unknown. MATERIALS AND METHODS: This retrospective chart review aimed to determine the frequency of methemoglobinemia in pediatric patients receiving iNO. Included subjects were under 18 years of age receiving iNO therapy with at least one methemoglobin concentration measured from 10/18/2014 to 11/18/2016. RESULTS: In total, 1809 methemoglobin concentrations were collected in 247 subjects during the study period. Median age was 0.33 (0.04-0.83) years. The mean methemoglobin concentration was 1.33% (±0.42) while receiving a mean iNO dose of 11.71 ppm (±7.97). Twenty-nine subjects had a total of 131 methemoglobin concentrations analyzed while receiving iNO doses above 20 ppm which were similar to the entire cohort at 1.33% (±0.42); (p = .95). CONCLUSIONS: Pediatric patients receiving iNO at doses below 40 ppm have minimal risk of developing clinically significant methemoglobinemia. Routine, ongoing monitoring of metHb levels in all pediatric subjects receiving iNO therapy at doses <40 ppm without the presence of risk factors predisposing the subject to increased risk of methemoglobinemia is unnecessary and should be avoided.

Establishment of prophylactic enoxaparin dosing recommendations to achieve targeted anti-factor Xa concentrations in children with CHD.

BACKGROUND: Enoxaparin may be used to prevent central venous catheter-related thrombosis in patients with CHD. We aimed to determine whether current enoxaparin dosing regimens effectively achieve anti-factor Xa concentrations within prophylactic goal ranges in this patient population. METHODS: We implemented a formal protocol aimed at reducing central venous catheter-related thrombosis in children with CHD in January, 2016. Standard empiric prophylactic enoxaparin dosing regimens were used - for example, 0.75 mg/kg/dose every 12 hours for patients <2 months of age and 0.5 mg/kg/dose every 12 hours for patients ⩾2 months of age - with anti-factor Xa goal range of 0.25-0.49 IU/ml. Patients <2 years of age who received enoxaparin and had at least one valid steady-state anti-factor Xa measurement between 25 January, 2016 and 31 August, 2016 were retrospectively reviewed. RESULTS: During the study period, 47 patients had 186 anti-factor Xa concentrations measured, of which 20 (11%) were above and 112 (60%) were below the prophylactic goal range. Anti-factor Xa concentrations within the goal range were ultimately achieved in 31 patients. Median dose required to achieve anti-factor Xa concentrations within the prophylactic range was 0.89 mg/kg/dose (25, 75%: 0.75, 1.11) for patients <2 months (n=23 patients) and 0.79 mg/kg/dose (25, 75%: 0.62, 1.11) for patients ⩾2 months (n=8 patients). CONCLUSIONS: Enoxaparin doses required to achieve prophylactic anti-factor Xa concentrations in young children with CHD were consistently higher than the currently recommended prophylactic dosing regimens. Further study is needed to determine whether dose titration to achieve prophylactic anti-factor Xa concentrations is effective in preventing central venous catheter-related thrombosis.

Empirical Vancomycin Dosing in Pediatric Patients with Congenital Heart Disease and the Impact of Cardiopulmonary Bypass on Trough Concentrations.

OBJECTIVES: To determine the optimal vancomycin dosing regimen to achieve empirical goal trough concentrations in pediatric patients with congenital heart disease and to examine the impact of cardiopulmonary bypass on vancomycin dosing requirements. METHODS: Patients younger than 18 years admitted to the pediatric cardiovascular intensive care unit (CVICU) at our institution from October 1, 2012-December 31, 2014, who received at least one dose of vancomycin, were reviewed retrospectively. Included patients had a steady-state vancomycin trough concentration drawn during the study period. The first steady-state vancomycin trough drawn after being initiated on empirical vancomycin therapy was analyzed for each patient. Excluded patients were those who received mechanical circulatory support, any form of renal replacement therapy, or had a serum creatinine result greater than 1.0 mg/dl on the day of vancomycin initiation. RESULTS: Overall, 77 patients met inclusion criteria, of which 57.1% had undergone cardiopulmonary bypass (CPB) before CVICU admission. Median age was 62 days (interquartile range [IQR] 8.3-176 days). Median daily vancomycin dose was 36.25 mg/kg/day (IQR 29-40 mg/kg/day), resulting in a median steady-state trough of 10.0 µg/ml (IQR 6.3-12.9 µg/ml). Therapeutic troughs occurred in 50.6% of patients; supratherapeutic and subtherapeutic concentrations were attained in 18.2% and 31.2% of patients, respectively. A subgroup analysis of patients who were post-CPB revealed that the only additional variable to affect vancomycin trough concentrations was aortic cross-clamp time (median 56 min, IQR 0-123.3 min, p=0.02). CONCLUSIONS: Empirical vancomycin dosing to achieve troughs of 8-15 µg/dl in patients with congenital heart disease without evidence of significant acute kidney injury should be 30 mg/kg/day for neonates, 35-40 mg/kg/day for infants, and 45 mg/kg/day in children, with adjustments required for patients with elevated creatinine or significant aortic cross-clamp time. The receipt and duration of CPB did not affect total daily vancomycin dose requirements.

Optimizing Guideline-Recommended Antibiotic Doses for Pediatric Infective Endocarditis.

The American Heart Association recently published an updated scientific statement on the management of infective endocarditis in childhood. The recommendations included for vancomycin, aminoglycoside, and ß-lactam dosing and monitoring are based primarily on expert opinion and do not consider available evidence for dose optimization based on pharmacokinetic and pharmacodynamic principles in pediatric patients. This is concerning because even when clinically necessary, some practitioners may be hesitant to deviate from guideline-recommended doses. In this perspective, we highlight potential areas for improvement in the statement-recommended doses and summarize evidence supporting antibiotic dosing optimization. The addition of a pediatric clinical pharmacist with expertise in antibiotic dosing to the panel would be beneficial for future updates.

Implementation of a diuretic stewardship program in a pediatric cardiovascular intensive care unit to reduce medication expenditures.

PURPOSE: The implementation of a diuretic stewardship program in a pediatric cardiovascular intensive care unit (ICU) is described. METHODS: This retrospective study compared the use of i.v. chlorothiazide and i.v. ethacrynic acid in pediatric cardiovascular surgery patients before and after implementation of a diuretic stewardship program. All pediatric patients admitted to the pediatric cardiovascular service were included. The cardiovascular surgery service was educated on formal indications for specific diuretic agents, and the diuretic stewardship program was implemented on January 1, 2013. Under the stewardship program, i.v. ethacrynic acid was indicated in patients with a sulfonamide allergy, and i.v. chlorothiazide was considered appropriate in patients receiving maximized i.v. loop diuretic doses. A detailed review of the pharmacy database and medical records was performed for each patient to determine i.v. chlorothiazide and i.v. ethacrynic acid use and expenditures, appropriateness of use, days using a ventilator, and cardiovascular ICU length of stay. RESULTS: After implementation of diuretic stewardship, the use of i.v. chlorothiazide decreased by 74% (531 fewer doses) while i.v. ethacrynic acid use decreased by 92% (47 fewer doses), resulting in a total reduction of $91,398 in expenditures on these diuretics over the six-month study period and an estimated annual saving of over $182,000. The median number of days using a ventilator and the length of ICU stay did not differ significantly during the study period. CONCLUSION: Implementation of a diuretic stewardship program reduced the use of i.v. chlorothiazide and i.v. ethacrynic acid without adversely affecting clinical outcomes such as ventilator days and length of stay in a pediatric cardiovascular ICU.