Crystallization of a nonclassical Kazal-type Carcinoscorpius rotundicauda serine protease inhibitor, CrSPI-1, complexed with subtilisin. Corrigendum.

The name of the first author in the article by Tulisas et al. [(2009), Acta Cryst. F65, 533-535] is corrected.

Catalytically important damage-free structures of a copper nitrite reductase obtained by femtosecond X-ray laser and room-temperature neutron crystallography.

Copper-containing nitrite reductases (CuNiRs) that convert NO2 - to NO via a CuCAT-His-Cys-CuET proton-coupled redox system are of central importance in nitrogen-based energy metabolism. These metalloenzymes, like all redox enzymes, are very susceptible to radiation damage from the intense synchrotron-radiation X-rays that are used to obtain structures at high resolution. Understanding the chemistry that underpins the enzyme mechanisms in these systems requires resolutions of better than 2 Å. Here, for the first time, the damage-free structure of the resting state of one of the most studied CuNiRs was obtained by combining X-ray free-electron laser (XFEL) and neutron crystallography. This represents the first direct comparison of neutron and XFEL structural data for any protein. In addition, damage-free structures of the reduced and nitrite-bound forms have been obtained to high resolution from cryogenically maintained crystals by XFEL crystallography. It is demonstrated that AspCAT and HisCAT are deprotonated in the resting state of CuNiRs at pH values close to the optimum for activity. A bridging neutral water (D2O) is positioned with one deuteron directed towards AspCAT Oδ1 and one towards HisCAT N∊2. The catalytic T2Cu-ligated water (W1) can clearly be modelled as a neutral D2O molecule as opposed to D3O+ or OD-, which have previously been suggested as possible alternatives. The bridging water restricts the movement of the unprotonated AspCAT and is too distant to form a hydrogen bond to the O atom of the bound nitrite that interacts with AspCAT. Upon the binding of NO2 - a proton is transferred from the bridging water to the Oδ2 atom of AspCAT, prompting electron transfer from T1Cu to T2Cu and reducing the catalytic redox centre. This triggers the transfer of a proton from AspCAT to the bound nitrite, enabling the reaction to proceed.

Comparison of chloral hydrate and pentobarbital sedation for pediatric echocardiography.

BACKGROUND: In 2013, outpatient use of chloral hydrate (CH) was limited and other alternatives such as oral pentobarbital (PB) were explored to achieve conscious sedation in young children for transthoracic echocardiography (TTE). We aimed to assess efficacy and safety of the two medications. METHODS: Clinical information, from a computerized database, about children who received sedation with either CH or PB for TTE at our center (2008-2015) was reviewed, and the two groups were compared for sedation effectiveness and complications. RESULTS: Three thousand eight hundred fifty one pediatric patients (median age 8 months) underwent conscious sedation during TTE (mean doses CH 50 mg/kg, PB 4 mg/kg). Demographic characteristics of the two groups were similar. Sedation failure rate (CH 2.4%, PB 2.9%, P = NS), need for supplemental doses (CH 17.9%, PB 16.2%, P = NS), and overall adverse event rate (PB 1.4%, CH 1.9%; P = NS) were similar in the two groups. There were fewer episodes of respiratory depression with PB (0.3% vs 1.6%, P < 0.05). The rate of paradoxical reactions was higher with PB (1% vs 0.03%, P < 0.05). Increasing age predicted the need for supplemental doses and for sedation failure in both groups. Neonates (7.5% vs 0%) and infants (2% vs 0.6%) given CH were more likely to develop adverse reactions. CONCLUSION: Chloral hydrate and PB are equally effective. However, CH is associated with an increased incidence of transient desaturation, while PB is associated with an increased incidence of a paradoxical reaction. Increasing age is predictive of the need for supplemental doses and for failure of sedation in both groups.

Risk factors for development of obesity in an ethnically diverse CHD population.

OBJECTIVES: Previous cross-sectional studies have demonstrated obesity rates in children with CHD and the general paediatric population. We reviewed longitudinal data to identify factors predisposing to the development of obesity in children, hypothesising that age may be an important risk factor for body mass index growth.Study designRetrospective electronic health records were reviewed in all 5-20-year-old CHD patients seen between 2011 and 2015, and in age-, sex-, and race/ethnicity-matched controls. Subjects were stratified into aged cohorts of 5-10, 11-15, and 15-20. Annualised change in body mass index percentile (BMI%) over this period was compared using paired Student's t-test. Linear regression analysis was performed with the CHD population. RESULTS: A total of 223 CHD and 223 matched controls met the inclusion criteria for analysis. Prevalence of combined overweight/obesity did not differ significantly between the CHD cohort (24.6-25.8%) and matched controls (23.3-29.1%). Univariate analysis demonstrated a significant difference of BMI% change in the age cohort of 5-10 (CHD +4.1%/year, control +1.7%/year, p=0.04), in male sex (CHD +1.8%/year, control -0.3%/year, p=0.01), and status-post surgery (CHD 2.03%/year versus control 0.37%, p=0.02). Linear regression analysis within the CHD subgroup demonstrated that age 5-10 years (+4.80%/year, p<0.001) and status-post surgery (+3.11%/year, p=0.013) were associated with increased BMI% growth. CONCLUSIONS: Prevalence rates of overweight/obesity did not differ between children with CHD and general paediatric population over a 5-year period. Longitudinal data suggest that CHD patients in the age cohort 5-10 and status-post surgery may be at increased risk of BMI% growth relative to peers with structurally normal hearts.

Unexpected Roles of a Tether Harboring a Tyrosine Gatekeeper Residue in Modular Nitrite Reductase Catalysis.

It is generally assumed that tethering enhances rates of electron harvesting and delivery to active sites in multidomain enzymes by proximity and sampling mechanisms. Here, we explore this idea in a tethered 3-domain, trimeric copper-containing nitrite reductase. By reverse engineering, we find that tethering does not enhance the rate of electron delivery from its pendant cytochrome c to the catalytic copper-containing core. Using a linker that harbors a gatekeeper tyrosine in a nitrite access channel, the tethered haem domain enables catalysis by other mechanisms. Tethering communicates the redox state of the haem to the distant T2Cu center that helps initiate substrate binding for catalysis. It also tunes copper reduction potentials, suppresses reductive enzyme inactivation, enhances enzyme affinity for substrate, and promotes intercopper electron transfer. Tethering has multiple unanticipated beneficial roles, the combination of which fine-tunes function beyond simplistic mechanisms expected from proximity and restrictive sampling models.

Umpolung Synthesis of Vicinal Diamines: Diastereoselective Addition of 2-Azaallyl Anions to Davis-Ellman's Imines.

A highly regioselective and diastereoselective addition of 2-azaallyl anions to N- tert-butanesulfinylimines is reported. This methodology affords the preparation of enantiomerically and diastereomerically pure vicinal diamines bearing two adjacent stereocenters. Reactions proceed efficiently (yield up to 94%), diastereoselectively ( dr values up to 98:2:0:0), and site-selectively to deliver products with differentiated amino groups.

Diastereoselective addition of anisoles to N-tert-butanesulfinyl imines via four-membered lithium cycles.

A highly regio- and diastereo-selective ortho-lithiation/addition of anisoles to N-tert-butanesulfinyl imines resulting in the selective formation of chiral α-branched amines is described. This method is also efficient for highly regioselective benzylic lithiation of o-methylanisoles, followed by diastereoselective addition to N-tert-butanesulfinyl imines.

Asymmetric Synthesis of α-(Diarylmethyl) Alkyl Amines through Regioselective Lithiation of α-Diarylmethanes and the Diastereoselective Addition to Ellman's Imines.

A highly regio- and diastereoselective lithiation/addition of α-diarylmethanes to N-tert-butanesulfinylimines is reported. This methodology also affords the preparation of enantiomerically pure α-(diarylmethyl) alkyl amines bearing quaternary centers.

Abnormalities in Left Ventricular Rotation Are Inherent in Young Children with Repaired Tetralogy of Fallot and Are Independent of Right Ventricular Dilation.

Left ventricular (LV) dysfunction is a risk factor for adverse outcomes in older children and adults with repaired Tetralogy of Fallot (rToF). Pulmonary regurgitation (PR), right ventricular (RV) dilation, and dysfunction have been shown to result in abnormal LV myocardial mechanics and dysfunction. The aim of our study was to evaluate LV rotational mechanics, especially apical rotation in young children with rToF with and without RV dilation. This is a retrospective, single center study in 28 asymptomatic young children with rToF (16 with RV dilation; 12 without RV dilation); 29 age-matched normal controls. RV and LV systolic and diastolic function was studied using conventional two-dimensional echocardiography (2DE) and speckle tracking echocardiography (STE). Rotational mechanics studied included basal and apical rotation (BR, AR), peak twist (calculated by difference between the apical and basal rotation), twist rate (TR), and untwist rate (UnTR). The mean age of the cohort was 4.7 years (± 2.3). Abnormal AR, BR, TR, and UnTR were noted in patients with rToF. The abnormalities were significant in magnitude as well as the direction of rotation; more pronounced in the absence of RV dilation. LV systolic and diastolic dysfunction as evidenced by abnormal AR and degree of untwist is inherent in rToF and not associated with RV dilation in rToF children. Abnormal BR may reflect a lack of maturation to adult type of rotational mechanics. Further longitudinal studies are required to study the progression of these abnormalities and their correlation with clinical outcomes.

Body Weights in Adults With Congenital Heart Disease and the Obesity Frequency.

Obesity may associate with greater cardiovascular risk in adults with congenital heart disease (ACHD) than in the general population. As ACHD often have exercise limitations, they may be uniquely predisposed to obesity. Nevertheless, obesity prevalence in ACHD, compared with the general population, has not been quantified in a large US cohort. Hence, we sought to determine the prevalence of obesity (30 ≤ body mass index <40) and morbid obesity (body mass index ≥40), in a large cohort of ACHD, compared with matched controls. Retrospective analysis was thus performed on all ACHD seen in an academic system in 2013. CHD severity was classified as simple, complex, or unclassified, using recently published criteria. A control group without CHD was randomly generated matching for age, gender, and race/ethnicity; 1,451 ACHD met inclusion criteria; 59.5% of ACHD were overweight to morbidly obese. Compared with controls, ACHD had similar prevalence of overweight (odds ratio [OR] 1.04, 95% confidence interval [CI] 0.89 to 1.22, p = 0.63) and obesity (OR 0.96, 95% CI 0.81 to 1.15, p = 0.69) but lower prevalence of morbid obesity (OR 0.24, 95% CI 0.16 to 0.34, p <0.001). These relationships were not attenuated by adjustment for CHD severity. In conclusion, ACHD are at equal risk as their matched peers to be overweight and obese. This is the largest study of obesity in US ACHD and the highest reported obesity prevalence in ACHD to date. As obesity is associated with significant cardiovascular risk, our findings indicate a need for improved lifestyle counseling in patients with CHD of all ages.

Impact of changing indications and increased utilization of fetal echocardiography on prenatal detection of congenital heart disease.

BACKGROUND: Antenatal diagnosis of congenital heart defects (CHD) can impact outcomes in neonates with severe CHD. Obstetric screening guidelines and the indications for fetal echocardiography (FE) have evolved in an attempt to improve the early prenatal detection of CHD. Analyzing yield for specific indications will help clinicians better stratify at-risk pregnancies. METHODS: Retrospective cohort study of all FE performed between 2000 and 2010 at a single tertiary care academic medical center in New York City. A total of 9878 FE met inclusion criteria for our study. In cases of multiple gestations (MG), each fetus was counted as a separate study. RESULTS: The number of new diagnosis of fetal CHD by FEs increased 200%. There was a statistically significant increase in those referred for suspected CHD, increased nuchal translucency (NT), MG, and suboptimal imaging (P < .001). The indication of "suboptimal imaging" (SO) not only accounted for 5.23% of all referrals from 2000 to 2002, compared to 22.26% of all referrals from 2008 to 2010 (P < .0001), but also had the lowest yield for diagnoses of CHD (P < .02). CONCLUSIONS: Over the past decade, there has been an increase in utilization of FE with a proportional increase in prenatally diagnosed CHD. For indications such as suspected CHD, NT and MG increases in referrals have led to a proportionate increase in fetal diagnosis of CHD. SO as an indication has the lowest yield of fetal diagnosis of CHD. Antenatal detection of CHD may be improved by a change in obstetric imaging protocols to ensure appropriate referrals.

Utility of Multimodality Imaging in the Morphologic Characterization of Anomalous Aortic Origin of a Coronary Artery.

BACKGROUND: Anomalous aortic origin of a coronary artery from the wrong Sinus of Valsalva (AAOCA) is a rare congenital anomaly and is associated with sudden cardiac death. Morphologic features considered to be "high risk" are significant luminal narrowing, acute coronary angulation at its origin, intramural course, and long interarterial course. A consistent approach for characterization of these features is lacking. METHODS: A retrospective single-center review of all patients diagnosed with AAOCA using echocardiogram and computed tomography (CT)/magnetic resonance imaging (MRI) studies was performed. Twenty-nine patients were identified (25 using CT and 4 using MRI) with subsequent three-dimensional data sets. The MRI data sets lacked adequate resolution and were excluded. Twenty-five patients (median age 15.1, range 10-39.5 years, 72% male) were further analyzed using echocardiogram and CT. Morphologic assessment focused on luminal stenosis, coronary angulation, and interarterial length. Additional morphologic features focusing on cross-sectional area and degree of ellipticity were also assessed. RESULTS: Echocardiography tended to yield smaller measurements compared to CT and had poor interobserver reproducibility for measurements pertaining to the narrowest proximal and distal coronary segments. Computed tomography showed good inter-/intraobserver reproducibility for the same. Agreement between both modalities for coronary angulation at its origin was excellent. There was good agreement for measurements of interarterial length between echocardiography and CT, but echocardiography had superior reproducibility. Assessment of luminal cross-sectional area and elliptical shape by CT had excellent inter-/intraobserver reproducibility. CONCLUSION: The combination of echocardiography and CT characterizes morphologic features of anomalous origin of the coronary artery more reliably than either modality alone.

Use of a Three Dimensional Printed Cardiac Model to Assess Suitability for Biventricular Repair.

Three dimensional (3D) printing is rapidly gaining interest in the medical field for use in presurgical planning. We present the case of a seven-year-old boy with double outlet right ventricle who underwent a bidirectional Glenn anastomosis. We used a 3D cardiac model to assess his suitability for a biventricular repair. He underwent a left ventricle-to-aorta baffle with a right ventricle-to-pulmonary artery conduit placement. He did well postoperatively and was discharged home with no evidence of baffle obstruction and good biventricular function. A 3D printed model can provide invaluable intracardiac spatial information in these complex patients.

The safety of postnatal transport of newborns prenatally diagnosed with duct-dependent congenital heart disease.

OBJECTIVE: Critical congenital heart disease is increasingly recognized prenatally. Following the diagnosis, families are advised to deliver in a facility where neonatal cardiac interventions are available. We studied the safety of transport of neonates who had been prenatally diagnosed with duct-dependent congenital heart lesions. METHODS: We performed a retrospective chart review of all fetuses diagnosed with duct-dependent congenital heart disease in our fetal program between 2007 and 2011. Demographic data, dose of prostaglandin infusion, respiratory status, blood gas data, as well as complications of the transport were collected. RESULTS: Twenty-nine neonates qualified for inclusion in the study. Ten were intubated (7 electively) prior to the transport. One of these required intervention for desaturation during the transport. One of the 19 unintubated patients required emergent intubation during the transport. Thus, the overall rate of incidents was 6.9%. All 4 patients who developed apnea requiring intubation did so within 1 h of starting prostaglandin. CONCLUSIONS: Elective postnatal transport of neonates prenatally diagnosed with duct-dependent congenital heart lesions is safe. Prophylactic intubation of these infants may not be necessary. It would be advantageous to schedule the elective transport an hour after starting prostaglandin or later.

A proteomic study of mitotic phase-specific interactors of EB1 reveals a role for SXIP-mediated protein interactions in anaphase onset.

Microtubules execute diverse mitotic events that are spatially and temporally separated; the underlying regulation is poorly understood. By combining drug treatments, large-scale immunoprecipitation and mass spectrometry, we report the first comprehensive map of mitotic phase-specific protein interactions of the microtubule-end binding protein, EB1. EB1 interacts with some, but not all, of its partners throughout mitosis. We show that the interaction of EB1 with Astrin-SKAP complex, a key regulator of chromosome segregation, is enhanced during prometaphase, compared to anaphase. We find that EB1 and EB3, another EB family member, can interact directly with SKAP, in an SXIP-motif dependent manner. Using an SXIP defective mutant that cannot interact with EB, we uncover two distinct pools of SKAP at spindle microtubules and kinetochores. We demonstrate the importance of SKAP's SXIP-motif in controlling microtubule growth rates and anaphase onset, without grossly disrupting spindle function. Thus, we provide the first comprehensive map of temporal changes in EB1 interactors during mitosis and highlight the importance of EB protein interactions in ensuring normal mitosis.

Tetralogy of fallot with dextrocardia and situs inversus in a 7-year-old boy.

Tetralogy of Fallot is one of the most frequently diagnosed congenital cardiac abnormalities; however, its association with dextrocardia and situs inversus is reported only rarely. We report a case of these findings in a 7-year-old boy, describe the patient's postoperative course, and review the medical literature relevant to these combined conditions.

Monopolin subunit Csm1 associates with MIND complex to establish monopolar attachment of sister kinetochores at meiosis I.

Sexually reproducing organisms halve their cellular ploidy during gametogenesis by undergoing a specialized form of cell division known as meiosis. During meiosis, a single round of DNA replication is followed by two rounds of nuclear divisions (referred to as meiosis I and II). While sister kinetochores bind to microtubules emanating from opposite spindle poles during mitosis, they bind to microtubules originating from the same spindle pole during meiosis I. This phenomenon is referred to as mono-orientation and is essential for setting up the reductional mode of chromosome segregation during meiosis I. In budding yeast, mono-orientation depends on a four component protein complex referred to as monopolin which consists of two nucleolar proteins Csm1 and Lrs4, meiosis-specific protein Mam1 of unknown function and casein kinase Hrr25. Monopolin complex binds to kinetochores during meiosis I and prevents bipolar attachments. Although monopolin associates with kinetochores during meiosis I, its binding site(s) on the kinetochore is not known and its mechanism of action has not been established. By carrying out an imaging-based screen we have found that the MIND complex, a component of the central kinetochore, is required for monopolin association with kinetochores during meiosis. Furthermore, we demonstrate that interaction of monopolin subunit Csm1 with the N-terminal domain of MIND complex subunit Dsn1, is essential for both the association of monopolin with kinetochores and for monopolar attachment of sister kinetochores during meiosis I. As such this provides the first functional evidence for a monopolin-binding site at the kinetochore.

Coarctation of the aorta associated with Dandy-Walker variant.

This article reports a rare case of coarctation of the aorta associated with Dandy-Walker variant in a 17-year-old girl. Differential diagnoses of coarctation of the aorta and Dandy-Walker variant are extensively discussed. In addition, standard surgical treatment of coarctation as well as new approaches such as endovascular stenting are described in detail to provide therapeutic insights into her management. Although surgical or endovascular repair of coarctation results in significant improvement of systemic hypertension and is associated with better survival, cardiovascular complications are still very common. Thus, long-term follow-up after repair is required, and high-quality imaging studies such as echocardiography, CT and MRI are warranted.