Human cardiac ß-myosin powerstroke energetics: Thin filament, Pi displacement, and mutation effects.

The powerstroke of human cardiac ß-myosin is an important stage of the cross-bridge cycle that generates force for muscle contraction. However, the starting structure of this process has never been resolved, and the relative timing of the powerstroke and inorganic phosphate (Pi) release is still controversial. In this study, we generated an atomistic model of myosin on the thin filament and utilized metadynamics simulations to predict the absent starting structure of the powerstroke. We demonstrated that the displacement of Pi from the active site during the powerstroke is likely necessary, reducing the energy barrier of the conformation change. The effects of the presence of the thin filament, the hypertrophic cardiomyopathy mutation R712L, and the binding of mavacamten on the powerstroke process were also investigated.

Longitudinal Follow-Up of Children With HLHS and Association Between Norwood Shunt Type and Long-Term Outcomes: The SVR III Study.

OBJECTIVE: In the SVR trial (Single Ventricle Reconstruction), newborns with hypoplastic left heart syndrome were randomly assigned to receive a modified Blalock-Taussig-Thomas shunt (mBTTS) or a right ventricle-to-pulmonary artery shunt (RVPAS) at Norwood operation. Transplant-free survival was superior in the RVPAS group at 1 year, but no longer differed by treatment group at 6 years; both treatment groups had accumulated important morbidities. In the third follow-up of this cohort (SVRIII [Long-Term Outcomes of Children With Hypoplastic Left Heart Syndrome and the Impact of Norwood Shunt Type]), we measured longitudinal outcomes and their risk factors through 12 years of age. METHODS: Annual medical history was collected through record review and telephone interviews. Cardiac magnetic resonance imaging (CMR), echocardiogram, and cycle ergometry cardiopulmonary exercise tests were performed at 10 through 14 years of age among participants with Fontan physiology. Differences in transplant-free survival and complication rates (eg, arrhythmias or protein-losing enteropathy) were identified through 12 years of age. The primary study outcome was right ventricular ejection fraction (RVEF) by CMR, and primary analyses were according to shunt type received. Multivariable linear and Cox regression models were created for RVEF by CMR and post-Fontan transplant-free survival. RESULTS: Among 549 participants enrolled in SVR, 237 of 313 (76%; 60.7% male) transplant-free survivors (mBTTS, 105 of 147; RVPAS, 129 of 161; both, 3 of 5) participated in SVRIII. RVEF by CMR was similar in the shunt groups (RVPAS, 51±9.6 [n=90], and mBTTS, 52±7.4 [n=75]; P=0.43). The RVPAS and mBTTS groups did not differ in transplant-free survival by 12 years of age (163 of 277 [59%] versus 144 of 267 [54%], respectively; P=0.11), percentage predicted peak Vo2 for age and sex (74±18% [n=91] versus 72±18% [n=84]; P=0.71), or percentage predicted work rate for size and sex (65±20% versus 64±19%; P=0.65). The RVPAS versus mBTTS group had a higher cumulative incidence of protein-losing enteropathy (5% versus 2%; P=0.04) and of catheter interventions (14 versus 10 per 100 patient-years; P=0.01), but had similar rates of other complications. CONCLUSIONS: By 12 years after the Norwood operation, shunt type has minimal association with RVEF, peak Vo2, complication rates, and transplant-free survival. RVEF is preserved among the subgroup of survivors who underwent CMR assessment. Low transplant-free survival, poor exercise performance, and accruing morbidities highlight the need for innovative strategies to improve long-term outcomes in patients with hypoplastic left heart syndrome. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT0245531.

Transition Path Sampling Study of Engineered Enzymes That Catalyze the Morita-Baylis-Hillman Reaction: Why Is Enzyme Design so Difficult?

It is hoped that artificial enzymes designed in laboratories can be efficient alternatives to chemical catalysts that have been used to synthesize organic molecules. However, the design of artificial enzymes is challenging and requires a detailed molecular-level analysis to understand the mechanism they promote in order to design efficient variants. In this study, we computationally investigate the mechanism of proficient Morita-Baylis-Hillman enzymes developed using a combination of computational design and directed evolution. The powerful transition path sampling method coupled with in-depth post-processing analysis has been successfully used to elucidate the different chemical pathways, transition states, protein dynamics, and free energy barriers of reactions catalyzed by such laboratory-optimized enzymes. This research provides an explanation for how different chemical modifications in an enzyme affect its catalytic activity in ways that are not predictable by static design algorithms.

Inverse heavy enzyme isotope effects in methylthioadenosine nucleosidases.

Heavy enzyme isotope effects occur in proteins substituted with 2H-, 13C-, and 15N-enriched amino acids. Mass alterations perturb femtosecond protein motions and have been used to study the linkage between fast motions and transition-state barrier crossing. Heavy enzymes typically show slower rates for their chemical steps. Heavy bacterial methylthioadenosine nucleosidases (MTANs from Helicobactor pylori and Escherichia coli) gave normal isotope effects in steady-state kinetics, with slower rates for the heavy enzymes. However, both enzymes revealed rare inverse isotope effects on their chemical steps, with faster chemical steps in the heavy enzymes. Computational transition-path sampling studies of H. pylori and E. coli MTANs indicated closer enzyme-reactant interactions in the heavy MTANs at times near the transition state, resulting in an improved reaction coordinate geometry. Specific catalytic interactions more favorable for heavy MTANs include improved contacts to the catalytic water nucleophile and to the adenine leaving group. Heavy bacterial MTANs depart from other heavy enzymes as slowed vibrational modes from the heavy isotope substitution caused improved barrier-crossing efficiency. Improved sampling frequency and reactant coordinate distances are highlighted as key factors in MTAN transition-state stabilization.

Reintervention Before Bidirectional Cavopulmonary Shunt and Intermediate Outcomes in Children with Single Ventricle Who Underwent Main Pulmonary Artery Banding.

Unplanned reinterventions following pulmonary artery banding (PAB) in single ventricle patients are common before stage 2 palliation (S2P) but associated risk factors are unknown. We hypothesized that reintervention is more common when PAB is placed at younger age and with a looser band, reflected by lower PAB pressure gradient. Retrospective single center study of single ventricle patients undergoing PAB between Jan 2000 and Dec 2020. The association with reintervention and successful S2P was modeled using exploratory cause-specific hazard regression. A multivariable model was developed adjusting for clinical and statistically relevant predictors. The cumulative proportion of patients undergoing reintervention were summarized using a competing risk model. 77 patients underwent PAB at median (IQR) 47 (24-66) days and 3.73 (3.2-4.5) kg. Within18 months of PAB, 60 (78%) reached S2P, 9 (12%) died, 1 (1%) transplanted and 7 (9%) were alive without S2P. Within 18 months of PAB 10 (13%) patients underwent reintervention related to pulmonary blood flow modification: PAB adjustment (n = 6) and conversion to Damus-Kaye-Stansel/Blalock-Taussig-Thomas shunt (n = 4). 6/10 (60%) reached S2P following reintervention. A trend toward higher intervention in patients with a genetic syndrome (p-0.06) and weight < 3 kg (p-0.057) at time of PAB was noted. Only genetic syndrome was a risk factor associated with poor outcome (p-0.025). PAB has a reasonable outcome in SV patients with unobstructed systemic and pulmonary blood flow, but with a high reintervention rate. Only a quarter of patients with genetic syndromes reach S2P and further study is required to explore the benefits from an alternative palliative strategy.

Structure and Dynamics of the Flexible Cardiac Troponin T Linker Domain in a Fully Reconstituted Thin Filament.

The structural analysis of large protein complexes has been greatly enhanced through the application of electron microscopy techniques. One such multiprotein complex, the cardiac thin filament (cTF), has cyclic interactions with thick filament proteins to drive contraction of the heart that has recently been the subject of such studies. As important as these studies are, they provide limited or no information on highly flexible regions that in isolation would be characterized as inherently disordered. One such region is the extended cardiac troponin T (cTnT) linker between the regions of cTnT which have been labeled TNT1 and TNT2. It comprises a hinge region (residues 158-166) and a highly flexible region (residues 167-203). Critically, this region modulates the troponin/tropomyosin complex's position across the actin filament. Thus, the cTnT linker structure and dynamics are central to the regulation of the function of cardiac muscles, but up to now, it was ill-understood. To establish the cTnT linker structure, we coupled an atomistic computational cTF model with time-resolved fluorescence resonance energy transfer measurements in both ±Ca2+ conditions utilizing fully reconstituted cTFs. We mapped the cTnT linker's positioning across the actin filament, and by coupling the experimental results to computation, we found mean structures and ranges of motion of this part of the complex. With this new insight, we can now address cTnT linker structural dynamics in both myofilament activation and disease.

The pediatric heart network's study on long-term outcomes of children with HLHS and the impact of Norwood Shunt type in the single ventricle reconstruction trial cohort (SVRIII): Design and adaptations.

BACKGROUND: The Single Ventricle Reconstruction (SVR) Trial was the first randomized clinical trial of a surgical approach for treatment of congenital heart disease. Infants with hypoplastic left heart syndrome (HLHS) and other single right ventricle (RV) anomalies were randomized to a modified Blalock Taussig Thomas shunt (mBTTS) or a right-ventricular-to-pulmonary-artery shunt (RVPAS) at the time of the Norwood procedure. The aim of the Long-term Outcomes of Children with HLHS and the Impact of Norwood Shunt Type (SVR III) study is to compare early adolescent outcomes including measures of cardiac function, transplant-free survival, and neurodevelopment, between those who received a mBTTS and those who received an RVPAS. METHODS: Transplant-free survivors of the SVR cohort were enrolled at 10 to 15 years of age for multifaceted in-person evaluation of cardiac function (cardiac magnetic resonance [CMR], echocardiogram and exercise test) and neurodevelopmental evaluation. Right ventricular ejection fraction measured by CMR served as the primary outcome. Development of arrhythmias, protein losing enteropathy, and other comorbidities were assessed through annual medical history interview. Through the course of SVR III, protocol modifications to engage SVR trial participants were designed to enhance recruitment and retention. CONCLUSIONS: Evaluation of long-term outcomes will provide important data to inform decisions about the shunt type placed at the Norwood operation and will improve the understanding of cardiovascular and neurodevelopmental outcomes for early adolescents with HLHS.

Epiretinal proliferation after rhegmatogenous retinal detachment.

PURPOSE: To determine the characteristics and appearance rate of epiretinal proliferation (ERP) on SD-OCT after surgery for rhegmatogenous retinal detachment (RRD) repair. METHODS: One hundred eight eyes of 108 patients who underwent one or more surgeries for RRD were enrolled. The eyes with other maculopathies that were directly related to RRD were excluded. Image acquisition was performed with SD-OCT (Heidelberg Engineering, Germany). Clinical charts were reviewed to assess clinical and surgical findings. Statistical analyses were performed using XLSTAT (Assinsoft, Paris, France). RESULTS: ERP was found in 9.3% eyes (n = 10). The mean initial visual acuity (logMAR) was 1.34 ± 0.82 in the ERP group compared to 0.49 ± 0.70 in the non-ERP group. PVR was present in 70.0% and chronic macular edema was found in 80.0% of eyes which developed ERP. The mean number of vitreoretinal surgeries in eyes with ERP was 3.3 ± 1.19 and only 1.44 ± 1.02 in eyes without. Silicone oil was used in 60.0% of eyes which developed ERP compared to 13.9% in the non-ERP group. CONCLUSION: ERP is a late-onset postoperative finding in eyes with RRD and can occur in absence of macular holes. Overall, ERP is more frequent in eyes with complicated courses of RRD including multiple operations, PVR, usage of silicone oil, and chronic macular edema.

RETINAL DETACHMENT IN EYES WITH BOSTON TYPE 1 KERATOPROSTHESIS: Surgical Techniques and Mid-Term Outcomes.

PURPOSE: To evaluate the mid-term outcomes of pars plana vitrectomy performed for retinal detachment (RD) repair after Boston Type 1 keratoprosthesis (KPro) implantation. METHODS: Retrospective review of medical records of KPro implanted at the Stein Eye Institute presenting with RD and treated by pars plana vitrectomy. Functional success was defined as a postoperative visual acuity maintained within 2 Snellen lines of the corrected distance visual acuity measured before the development of the RD (baseline) and anatomical success as an attached retina after the pars plana vitrectomy. Kaplan-Meyer survival analyses were performed. RESULTS: Among the 224 KPro performed, 28 (15.2%) RD were identified; of which, 21 (9.4%) were included. The mean follow-up was 42.5 ± 27.3 months. Vitreoretinal proliferation was present in 18 of 21 eyes (85.7%). Surgical techniques were adapted to the complex anterior segment anatomy of KPro eyes. Anatomical success was achieved in 18 of 21 eyes (85.7%). Functional success occurred in 17 of 21 eyes (81.0%), and 5 of 21 eyes (23.8%) reached 20/400 or better visual acuity at the final follow-up. The KPro was retained in 11 in 21 eyes (52.4%). The retention rate decreased from 94.7% at 1 year to 53.5% at 5 years. The most frequent complications were retroprosthetic membrane (47.6%) and corneal melt (23.8%). CONCLUSION: Modified pars plana vitrectomy techniques resulted in relatively good mid-term anatomical, functional, and retention rate outcomes, given the severity of RD at presentation and the numerous preoperative comorbidities of KPro eyes.

Development of the Residual Lesion Score for congenital heart surgery: the RAND Delphi methodology.

BACKGROUND AND OBJECTIVE: The Residual Lesion Score is a novel tool for assessing the achievement of surgical objectives in congenital heart surgery based on widely available clinical and echocardiographic characteristics. This article describes the methodology used to develop the Residual Lesion Score from the previously developed Technical Performance Score for five common congenital cardiac procedures using the RAND Delphi methodology. METHODS: A panel of 11 experts from the field of paediatric and congenital cardiology and cardiac surgery, 2 co-chairs, and a consultant were assembled to review and comment on validity and feasibility of measuring the sub-components of intraoperative and discharge Residual Lesion Score for five congenital cardiac procedures. In the first email round, the panel reviewed and commented on the Residual Lesion Score and provided validity and feasibility scores for sub-components of each of the five procedures. In the second in-person round, email comments and scores were reviewed and the Residual Lesion Score revised. The modified Residual Lesion Score was scored independently by each panellist for validity and feasibility and used to develop the "final" Residual Lesion Score. RESULTS: The Residual Lesion Score sub-components with a median validity score of ≥7 and median feasibility score of ≥4 that were scored without disagreement and with low absolute deviation from the median were included in the "final" Residual Lesion Score. CONCLUSION: Using the RAND Delphi methodology, we were able to develop Residual Lesion Score modules for five important congenital cardiac procedures for the Pediatric Heart Network's Residual Lesion Score study.

Carbon Dots for Efficient Small Interfering RNA Delivery and Gene Silencing in Plants.

The initiation of RNA interference (RNAi) by topically applied small interfering RNA has potential applications for plant functional genomics, crop improvement and crop protection, but the primary obstacle for the development of this technology is the efficient delivery of RNAi effectors into the cell. The plant cell wall is a particularly challenging barrier for the delivery of macromolecules because many of the transfection agents that are commonly used with animal cells produce nanocomplexes that are significantly larger than the size exclusion limit of the cell wall. Here, we illustrate the use of a class of very small nanoparticles, called carbon dots, for delivering small interfering RNA into the model plants Nicotiana benthamiana and tomato (Solanum lycopersicum). Low-pressure spray application of these formulations with a spreading surfactant resulted in strong silencing of GFP transgenes in both species. The delivery efficacy of carbon dot formulations was also demonstrated by the silencing of endogenous genes that encode two subunits of magnesium chelatase, an enzyme necessary for chlorophyll synthesis. The strong visible phenotypes observed with the carbon dot-facilitated delivery were validated by measuring significant reductions in the target gene transcript and/or protein levels. Methods for the delivery of RNAi effectors into plants, such as the carbon dot formulations described here, could become valuable tools for gene silencing in plants with practical applications in plant functional genomics and agriculture.

Clinical characteristics and visual outcomes of non-resolving subretinal fluid in neovascular AMD despite continuous monthly anti-VEGF injections: a long-term follow-up.

PURPOSE: To describe the clinical characteristics and visual outcomes of neovascular age-related macular degeneration (NV-AMD) patients with irregular pigment epithelium detachment (PED) and non-resolving subretinal fluid (SRF) despite continuous monthly injections of anti-vascular endothelial growth factor (VEGF). METHODS: This is a retrospective case series, including NV-AMD patients treated in a tertiary academic practice. Inclusion criteria were NV-AMD diagnosis, with irregular PED, and non-resolving SRF treated with continuous monthly anti-VEGF intravitreal injections. Data collection included best corrected visual acuity (BCVA), central macular thickness (CMT), sub-foveal choroidal thickness (SFCT), and type and location of PED as seen on optical coherence tomography (OCT). RESULTS: A total of 738 patients with NV-AMD underwent anti-VEGF injections during the follow-up period and 20 eyes of 19 patients (14 females and 5 males) met the inclusion criteria. Average age was 81.7 ± 6.6 years, mean follow-up time was 32.1 ± 23.5 months, and mean number of injections was 31.3 ± 24.2. Mean VA was 0.26 ± 0.21 logMAR (Snellen 20/36) at baseline versus 0.20 ± 0.23 logMAR (Snellen 20/32) at the end of the follow-up (P = 0.28). All eyes presented with sub-foveal, type 1 macular neovascularization (MNV). Average sub-foveal choroidal thickness changed from 189.70 ± 68.46 µm at baseline to 169.00 ± 63.06 µm (P < 0.001) at last follow-up. CONCLUSION: Patients with type 1 NV-AMD, irregular PED, and non-resolving SRF and under continuous treatment of monthly anti-VEGF injections may maintain good visual acuity after long period of time.

PIGMENT EPITHELIAL DETACHMENT IN AGE-RELATED MACULAR DEGENERATION: Long-Term Visual Acuity May Improve With Higher Injection Index.

PURPOSE: To define injection index (II) and assess its impact on visual acuity (VA) in pigment epithelial detachment from age-related macular degeneration over 5 years. METHODS: Injection index is defined as the mean anti-vascular endothelial growth factor injections per year from presentation. A retrospective study of 256 eyes in 213 patients was performed. Patients were stratified by II (high: ≥9, low: <9). RESULTS: Baseline characteristics showed no differences across II groups. Mean (range) follow-up, in years, was 5.02 (1.04-12.74) for all patients. Mean logMAR VA (Snellen VA) were 0.60 (20/80) and 0.56 (20/73) at baseline, 0.52 (20/66) and 0.59 (20/78) at Year 1, 0.45 (20/56) and 0.67 (20/94) at Year 2, 0.38 (20/48) and 0.66 (20/91) at Year 3, 0.41 (20/51) and 0.89 (20/155) at Year 4, and 0.35 (20/45) and 0.79 (20/123) at Year 5 for the high and low II groups, respectively. Linear regression analysis showed a gain of 0.5 approxETDRS letters with each additional injection per year. CONCLUSION: Increased II was associated with better mean VA, suggesting that long-term continuous vascular endothelial growth factor suppression may improve VA in eyes thought to carry poor prognoses.

Inverse enzyme isotope effects in human purine nucleoside phosphorylase with heavy asparagine labels.

Transition path-sampling calculations with several enzymes have indicated that local catalytic site femtosecond motions are linked to transition state barrier crossing. Experimentally, femtosecond motions can be perturbed by labeling the protein with amino acids containing 13C, 15N, and nonexchangeable 2H. A slowed chemical step at the catalytic site with variable effects on steady-state kinetics is usually observed for heavy enzymes. Heavy human purine nucleoside phosphorylase (PNP) is slowed significantly (kchemlight/kchemheavy = 1.36). An asparagine (Asn243) at the catalytic site is involved in purine leaving-group activation in the PNP catalytic mechanism. In a PNP produced with isotopically heavy asparagines, the chemical step is faster (kchemlight/kchemheavy = 0.78). When all amino acids in PNP are heavy except for the asparagines, the chemical step is also faster (kchemlight/kchemheavy = 0.71). Substrate-trapping experiments provided independent confirmation of improved catalysis in these constructs. Transition path-sampling analysis of these partially labeled PNPs indicate altered femtosecond catalytic site motions with improved Asn243 interactions to the purine leaving group. Altered transition state barrier recrossing has been proposed as an explanation for heavy-PNP isotope effects but is incompatible with these isotope effects. Rate-limiting product release governs steady-state kinetics in this enzyme, and kinetic constants were unaffected in the labeled PNPs. The study suggests that mass-constrained femtosecond motions at the catalytic site of PNP can improve transition state barrier crossing by more frequent sampling of essential catalytic site contacts.

Standardisation of management after Norwood operation has not improved 1-year outcomes.

INTRODUCTION: Treatment of hypoplastic left heart syndrome varies across institutions. This study examined the impact of introducing a standardised programme. METHODS: This retrospective cohort study evaluated the effects of a comprehensive strategy on 1-year transplant-free survival with preserved ventricular and atrioventricular valve (AVV) function following a Norwood operation. This strategy included standardised operative and perioperative management and dedicated interstage monitoring. The post-implementation cohort (C2) was compared to historic controls (C1). Outcomes were assessed using logistic regression and Kaplan-Meier analysis. RESULTS: The study included 105 patients, 76 in C1 and 29 in C2. Groups had similar baseline characteristics, including percentage with preserved ventricular (96% C1 versus 100% C2, p = 0.28) and AVV function (97% C1 versus 93% C2, p = 0.31). Perioperatively, C2 had higher indexed oxygen delivery (348 ± 67 ml/minute/m2 C1 versus 402 ± 102ml/minute/m2 C2, p = 0.015) and lower renal injury (47% C1 versus 3% C2, p = 0.004). The primary outcome was similar in both groups (49% C1 and 52% C2, p = 0.78), with comparable rates of death and transplantation (36% C1 versus 38% C2, p = 0.89) and ventricular (2% C1 versus 0% C2, p = 0.53) and AVV dysfunction (11% C1 versus 11% C2, p = 0.96) at 1-year. When accounting for cohort and 100-day freedom from hospitalisation, female gender (OR 3.7, p = 0.01) increased and ventricular dysfunction (OR 0.21, p = 0.02) and CPR (OR 0.11, p = 0.002) or ECMO use (OR 0.15, p = 001) decreased the likelihood of 1-year transplant-free survival. CONCLUSIONS: Standardised perioperative management was not associated with improved 1-year transplant-free survival. Post-operative ventricular or AVV dysfunction was the strongest predictor of 1-year mortality.

FRET-based analysis of the cardiac troponin T linker region reveals the structural basis of the hypertrophic cardiomyopathy-causing Δ160E mutation.

Mutations in the cardiac thin filament (TF) have highly variable effects on the regulatory function of the cardiac sarcomere. Understanding the molecular-level dysfunction elicited by TF mutations is crucial to elucidate cardiac disease mechanisms. The hypertrophic cardiomyopathy-causing cardiac troponin T (cTnT) mutation Δ160Glu (Δ160E) is located in a putative "hinge" adjacent to an unstructured linker connecting domains TNT1 and TNT2. Currently, no high-resolution structure exists for this region, limiting significantly our ability to understand its role in myofilament activation and the molecular mechanism of mutation-induced dysfunction. Previous regulated in vitro motility data have indicated mutation-induced impairment of weak actomyosin interactions. We hypothesized that cTnT-Δ160E repositions the flexible linker, altering weak actomyosin electrostatic binding and acting as a biophysical trigger for impaired contractility and the observed remodeling. Using time-resolved FRET and an all-atom TF model, here we first defined the WT structure of the cTnT-linker region and then identified Δ160E mutation-induced positional changes. Our results suggest that the WT linker runs alongside the C terminus of tropomyosin. The Δ160E-induced structural changes moved the linker closer to the tropomyosin C terminus, an effect that was more pronounced in the presence of myosin subfragment (S1) heads, supporting previous findings. Our in silico model fully supported this result, indicating a mutation-induced decrease in linker flexibility. Our findings provide a framework for understanding basic pathogenic mechanisms that drive severe clinical hypertrophic cardiomyopathy phenotypes and for identifying structural targets for intervention that can be tested in silico and in vitro.

Rapid Advancement in Enteral Nutrition Does Not Affect Systemic Inflammation and Insulin Homeostasis Following Pediatric Cardiopulmonary Bypass Surgery.

OBJECTIVES: To determine impact of enteral nutrition delivery on the relationship among inflammation, insulin resistance, and outcomes following pediatric cardiopulmonary bypass surgery. DESIGN: Pilot, randomized study analyzed according to intention-to-treat analysis. SETTING: Pediatric cardiac ICU. PATIENTS: Infants (≤ 6 mo) undergoing cardiopulmonary bypass. INTERVENTIONS: Patients randomly assigned to receive rapid escalation to enteral nutrition reaching goal feeds by 27 hours or standard feeding practice reaching goal feeds by 63 hours. Feeds were initiated on the first postoperative day. MEASUREMENTS AND MAIN RESULTS: Fifty patients were randomized equally to study arms. Patients were a median (interquartile range) of 16 days old (7-110 d old), undergoing biventricular surgery (88%) with a median cardiopulmonary bypass time of 125 minutes (105-159 min). Serial blood samples were drawn before and after cardiopulmonary bypass, cardiac ICU admission, and every 12 hours (up to 96 hr) for glucose, insulin, and cytokines (interleukin-1α, interleukin-6, interleukin-8, interleukin-10, and tumor necrosis factor-α) levels. Glucose-insulin ratio was calculated to quantify insulin resistance. Patient characteristics, time to enteral nutrition initiation, enteral nutrition interruptions, and insulin administration were similar across intervention arms. FF reached goal feeds at similar intervals as standard feeding (39 hr [30-60 hr] vs 60 hr [21-78 hr]; p = 0.75). No difference in cytokine, insulin, or glucose-insulin ratio was noted between groups. Higher inflammation was associated with increased glucose-insulin ratio and higher risk of adverse events. In multivariable models of interleukin-8, FF was associated with increased glucose-insulin ratio (estimate of effect [95% CI], 0.152 [0.033-0.272]; p = 0.013). Although higher interleukin-8 was associated with an elevated risk of adverse event, this relationship was possibly mitigated by FF (odds ratio [95% CI], 0.086 [0.002-1.638]; p = 0.13). CONCLUSIONS: A FF strategy was not associated with changes to early enteral nutrition delivery. Inflammation, insulin resistance, and morbidity were similar, but FF may modify the relationship between inflammation and adverse event. Multicenter nutrition studies are possible and necessary in this vulnerable population.

Catecholaminergic Polymorphic Ventricular Tachycardia: An Unusual Case of Fright-Induced Prehospital Cardiac Arrest in a Healthy 6-Year-Old Child.

Catecholaminergic Polymorphic Ventricular Tachycardia is a rare but often lethal genetic disorder that affects approximately 1 in 10,000 people. It often first manifests as stress or exercise-related syncope or sudden unexplained cardiac death, primarily in the pediatric and young adult population. We present a case of a 6-year-old male who had a sudden unexplained prehospital cardiac arrest after being scared by a domestic animal and who presented in ventricular fibrillation. The patient was subsequently defibrillated with a return of spontaneous circulation. During the course of care, medications with beta-1 and -2 agonist properties were administered, followed by multiple further episodes of polymorphic ventricular tachycardia (PVT)/ventricular fibrillation (VF). Once these medications were discontinued and beta blockers were administered, the patient had no further episodes of PVT/VF and was subsequently discharged from hospital 7 days later, completely neurologically intact. This case suggests the need for caution when considering administering beta agonists in a pediatric cardiac arrest patient with no known history of heart disease who presents in VF or PVT after an incident of extreme stress or strenuous physical activity.

DETECTION OF NEUROSENSORY RETINAL DETACHMENT COMPLICATING DEGENERATIVE RETINOSCHISIS BY ULTRA-WIDEFIELD FUNDUS AUTOFLUORESCENCE IMAGING.

PURPOSE: To determine whether neurosensory retinal detachment complicating degenerative retinoschisis (RS) can be reliably detected with ultra-widefield fundus autofluorescence evaluation. METHODS: Consecutive patients diagnosed with RS who had ultra-widefield fundus autofluorescence imaging were included in this retrospective case series. According to the fundus autofluorescence patterns, we divided the eyes into two groups: 1) eyes with RS and a hyperautofluorescent leading edge and 2) eyes with RS and without hyperautofluorescence. Peripheral spectral domain optical coherence tomography images at the level of RS were obtained. RESULTS: Thirty-eight eyes that met eligibility criteria were identified. Review of ultra-widefield fundus autofluorescence demonstrated 21/39 (55%) eyes with distinctive hyperautofluorescence over the area of RS (Group A) and 17/38 (45%) eyes without any form of hyperautofluorescence (Group B). Spectral domain optical coherence tomography images confirmed the presence of full-thickness neurosensory retina separation from the underlying retinal pigment epithelium in the areas of hyperautofluorescence in 10/10 eyes (100%) from Group A. None (0/11; 0%) of the eyes from Group B showed full-thickness neurosensory retina separation on the spectral domain optical coherence tomography imaging of the retina-RS interface. CONCLUSION: Hyperautofluorescent findings suggest the presence of a neurosensory retinal detachment. Retinal detachment associated with RS can be reliably detected on ultra-widefield fundus autofluorescence and may be a useful diagnostic imaging modality.

Catalytic-site design for inverse heavy-enzyme isotope effects in human purine nucleoside phosphorylase.

Heavy-enzyme isotope effects (15N-, 13C-, and 2H-labeled protein) explore mass-dependent vibrational modes linked to catalysis. Transition path-sampling (TPS) calculations have predicted femtosecond dynamic coupling at the catalytic site of human purine nucleoside phosphorylase (PNP). Coupling is observed in heavy PNPs, where slowed barrier crossing caused a normal heavy-enzyme isotope effect (kchemlight/kchemheavy > 1.0). We used TPS to design mutant F159Y PNP, predicted to improve barrier crossing for heavy F159Y PNP, an attempt to generate a rare inverse heavy-enzyme isotope effect (kchemlight/kchemheavy < 1.0). Steady-state kinetic comparison of light and heavy native PNPs to light and heavy F159Y PNPs revealed similar kinetic properties. Pre-steady-state chemistry was slowed 32-fold in F159Y PNP. Pre-steady-state chemistry compared heavy and light native and F159Y PNPs and found a normal heavy-enzyme isotope effect of 1.31 for native PNP and an inverse effect of 0.75 for F159Y PNP. Increased isotopic mass in F159Y PNP causes more efficient transition state formation. Independent validation of the inverse isotope effect for heavy F159Y PNP came from commitment to catalysis experiments. Most heavy enzymes demonstrate normal heavy-enzyme isotope effects, and F159Y PNP is a rare example of an inverse effect. Crystal structures and TPS dynamics of native and F159Y PNPs explore the catalytic-site geometry associated with these catalytic changes. Experimental validation of TPS predictions for barrier crossing establishes the connection of rapid protein dynamics and vibrational coupling to enzymatic transition state passage.