Influence of improved antenatal detection on the outcomes of complete atrioventricular block diagnosed in fetal-neonatal life and childhood periods - a single-centre experience in South Wales for 55 years.

OBJECTIVE: This study aimed to analyse the influence of improved antenatal detection on the course, contemporary outcomes, and mortality risk factors of the complete atrioventricular block during fetal-neonatal and childhood periods in South Wales. METHODS: The clinical characteristics and outcomes of complete atrioventricular block in patients without structural heart disease at the University Hospital of Wales from January 1966 to April 2021 were studied. Patients were divided into two groups according to their age at diagnosis: I-fetal-neonatal and II-childhood. Contemporary outcomes during the post-2001 era were compared with historical data preceding fetal service development and hence earlier detection. RESULTS: There were 64 patients: 26 were identified in the fetal-neonatal period and the remaining 38 in the childhood period. Maternal antibodies/systemic lupus erythematosus disease (anti-Ro/Sjögren's-syndrome-related Antigen A and/or anti-La/Sjögren's-syndrome-related Antigen B) were present in 15 (57.7%) of the fetal-neonatal. Fetal/neonatal and early diagnosis increased after 2001 with an incidence of 1:25000 pregnancies. Pacemaker implantation was required in 34 patients, of whom 13 were diagnosed in the fetal-neonatal group. Survival rates in cases identified before 2001 were at 96.3% (26/27), whereas it was 83.8% (31/37) in patients diagnosed after 2001 (P > 0.05). Other mortality risk factors comprised a lower gestational week at birth, maternal antibodies, and an average ventricular heart rate of < 55 bpm. CONCLUSIONS: Fetal diagnosis of complete atrioventricular block is still portends high fetal and neonatal mortality and morbidity despite significantly improved antenatal detection after 2001. Pacemaker intervention is needed earlier in the fetal-neonatal group. Whether routine antenatal medical treatment might alter this outcome calls for further prospective multicentre studies.

Predictors of Respiratory Support Use in Emergency Department Patients With COVID-19-Related Respiratory Failure.

BACKGROUND: Given the known downstream implications of choice of respiratory support on patient outcomes, all factors influencing these decisions, even those not limited to the patient, warrant close consideration. We examined the effect of emergency department (ED)-specific system factors, such as work load and census, on the use of noninvasive versus invasive respiratory support. METHODS: We conducted a multi-center retrospective cohort study of all adult subjects with severe COVID-19 requiring an ICU admission from 5 EDs within a single urban health care system. Subject demographics, severity of illness, and the type of respiratory support used were obtained. Using continuous measures of ED census, boarding, and active management, we estimated ED work load for each subjects' ED stay. The subjects were categorized by type(s) of respiratory support used: low-flow oxygen, noninvasive respiratory support (eg, noninvasive ventilation [NIV] and/or high-flow nasal cannula [HFNC]), invasive mechanical ventilation, or invasive mechanical ventilation after trial of NIV/HFNC. We used multivariable logistic regression to examine system factors associated with the type of respiratory support used in the ED. RESULTS: A total of 634 subjects were included. Of these, 431 (70.0%) were managed on low-flow oxygen alone, 108 (17.0%) on NIV/HFNC, 54 (8.5%) on invasive mechanical ventilation directly, and 41 (6.5%) on NIV/HFNC prior to invasive mechanical ventilation in the ED. Higher severity of illness and underlying lung disease increased the odds of requiring invasive mechanical ventilation compared to low-flow oxygen (odds ratio 1.05 [95% CI 1.03-1.07] and odds ratio 3.47 [95% CI 1.37-8.78], respectively). Older age decreased odds of being on invasive mechanical ventilation compared to low-flow oxygen (odds ratio 0.96 [95% CI 0.94-0.99]). As ED work load increased, the odds for subjects to be managed initially with NIV/HFNC prior to invasive mechanical ventilation increased 6-8-fold. CONCLUSIONS: High ED work load was associated with higher odds on HFNC/NIV prior to invasive mechanical ventilation.

Training: improving antenatal detection and outcomes of congenital heart disease.

OBJECTIVES: This study describes the design, delivery and efficacy of a regional fetal cardiac ultrasound training programme. This programme aimed to improve the antenatal detection of congenital heart disease (CHD) and its effect on fetal and postnatal outcomes. DESIGN SETTING AND PARTICIPANTS: This was a prospective study that compared antenatal CHD detection rates by professionals from 13 hospitals in Wales before and after engaging in our 'skills development programme'. Existing fetal cardiac practice and perinatal outcomes were continuously audited and progressive targets were set. The work was undertaken by the Welsh Fetal Cardiovascular Network, Antenatal Screening Wales (ASW), a superintendent sonographer and a fetal cardiologist. INTERVENTIONS: A core professional network was established, engaging all stakeholders (including patients, health boards, specialist commissioners, ASW, ultrasonographers, radiologists, obstetricians, midwives and paediatricians). A cardiac educational lead (midwife, superintendent sonographer, radiologist, obstetrician, or a fetal medicine specialist) was established in each hospital. A new cardiac anomaly screening protocol ('outflow tract view') was created and training on the new protocol was systematically delivered at each centre. Data were prospectively collected and outcomes were continuously audited: locally by the lead fetal cardiologist; regionally by the Congenital Anomaly Register and Information Service in Wales; and nationally by the National Institute for Cardiac Outcomes and Research (NICOR) in the UK. MAIN OUTCOME MEASURES: Patient satisfaction; improvements in individual sonographer skills, confidence and competency; true positive referral rate; local hospital detection rate; national detection rate of CHD; clinical outcomes of selected cardiac abnormalities; reduction of geographical health inequality; cost efficacy. RESULTS: High levels of patient satisfaction were demonstrated and the professional skill mix in each centre was improved. The confidence and competency of sonographers was enhanced. Each centre demonstrated a reduction in the false-positive referral rate and a significant increase in cardiac anomaly detection rate. According to the latest NICOR data, since implementing the new training programme Wales has sustained its status as UK lead for CHD detection. Health outcomes of children with CHD have improved, especially in cases of transposition of the great arteries (for which no perinatal mortality has been reported since 2008). Standardised care led to reduction of geographical health inequalities with substantial cost saving to the National Health Service due to reduced false-positive referral rates. Our successful model has been adopted by other fetal anomaly screening programmes in the UK. CONCLUSIONS: Antenatal cardiac ultrasound mass training programmes can be delivered effectively with minimal impact on finite healthcare resources. Sustainably high CHD detection rates can only be achieved by empowering the regional screening workforce through continuous investment in lifelong learning activities. These should be underpinned by high quality service standards, effective care pathways, and robust clinical governance and audit practices.

Diagnostic ultrasound features and outcome of restrictive foramen ovale in fetuses with structurally normal hearts.

Intrauterine foramen ovale (FO) restriction in association with congenital heart disease (CHD) carries a poor prognosis. However, in the absence of CHD, the clinical importance of restrictive FO in the fetus is not well understood. We evaluated the antenatal prevalence, clinical presentation, diagnostic ultrasound features, and outcome of restrictive FO in fetuses without CHD. We reviewed the echocardiographic and clinical records of 23 fetuses diagnosed with a restrictive FO and structurally normal heart between 2001 and 2012. The atrial septum, dimensions of cardiac structures, left and right cardiac output and Doppler interrogation of cardiac flows were examined. The clinical outcomes of all fetuses with restrictive FO were analysed. Restrictive FO was identified in 23 of 1,682 (1.4%) fetuses with no CHD. Enlarged right heart structures (100%), hypermobile or redundant primum atrial septum (91%), increased right-to-left ventricular cardiac output ratio (91%), and posteriorly angulated ductus arteriosus (68%) were the most common echocardiographic findings associated with this rare phenomenon. Additional noncardiac systemic abnormalities were identified in 13 (56%) babies. Seven (30%) neonates developed persistent pulmonary hypertension, and 7 infants died. Antenatal restrictive FO is an underrecognised entity despite being a common cause of right heart dilatation in the fetus. In the absence of CHD, restrictive FO is well tolerated antenatally, but its frequent association with noncardiac abnormalities and pulmonary hypertension in the neonate are noteworthy.

The SH2B1 adaptor protein associates with a proximal region of the erythropoietin receptor.

Gene targeting experiments have shown that the cytokine erythropoietin (EPO), its cognate erythropoietin receptor (EPO-R), and associated Janus tyrosine kinase, JAK2, are all essential for erythropoiesis. Structural-functional and murine knock-in experiments have suggested that EPO-R Tyr-343 is important in EPO-mediated mitogenesis. Although Stat5 binds to EPO-R phosphotyrosine 343, the initial Stat5-deficient mice did not have profound erythroid abnormalities suggesting that additional Src homology 2 (SH2) domain-containing effectors may bind to EPO-R Tyr-343 and couple to downstream signaling pathways. We have utilized cloning of ligand target (COLT) screening to demonstrate that EPO-R Tyr(P)-343 and Tyr(P)-401 bind to the SH2 domain-containing adaptor protein SH2B1ß. Immunoprecipitation and in vitro mixing experiments reveal that EPO-R binds to SH2B1 in an SH2 domain-dependent manner and that the sequence that confers SH2B1 binding to the EPO-R is pYXXL. Previous studies have shown that SH2B1 binds directly to JAK2, but we show that in hematopoietic cells, SH2B1ß preferentially associates with the EPO-R. SH2B1 is capable of constitutive association with EPO-R, which is necessary for its optimal SH2-dependent recruitment to EPO-R-Tyr(P)-343/Tyr(P)-401. We also demonstrate that SH2B1 is responsive to EPO stimulation and becomes phosphorylated, most likely on serines/threonines, in an EPO dose- and time-dependent manner. In the absence of SH2B1, we observe enhanced activation of signaling pathways downstream of the EPO-R, indicating that SH2B1 is a negative regulator of EPO signaling.

Rapid control of foetal supraventricular tachycardia with digoxin and flecainide combination treatment.

OBJECTIVES: To evaluate the efficacy of flecainide and digoxin combination in foetal supraventricular tachycardia. SETTING: This study was carried out in a tertiary referral centre. METHODS: We conducted a retrospective review of 29 patients diagnosed with supraventricular foetal tachycardia between 2001 and 2009. Mode of presentation, foetal cardiac function, maternal anti-arrhythmic serum levels, drug tolerance, and maternal electrocardiogram recordings were assessed. The postnatal outcome of each infant was also evaluated for tachycardia recurrence. RESULTS: In all, 27 foetuses were treated with digoxin and flecainide combination, and two foetuses were delivered without any treatment. Of the 27 foetuses treated, six [corrected] had atrial flutter and the remaining 21 [corrected] had atrioventricular re-entry tachycardia. There were eight foetuses with hydrops (27%), of whom three had atrial flutter and five had atrioventricular re-entry tachycardia; 26 foetuses (96%) responded to flecainide and digoxin combination, with restoration of sinus rhythm in 22 (81.4%) and rate control in the other four. In one severely hydropic foetus, there was no response to treatment. In all, 26 treated infants were delivered alive, but one pregnancy was terminated for non-cardiac causes when the foetus was in sinus rhythm. There was no intrauterine death due to tachycardia. Although there were minor side effects to anti-arrhythmic medications, none of the pregnant women developed proarrhythmia. CONCLUSION: Flecainide and digoxin combination treatment offers a safe and effective treatment for foetal supraventricular tachycardia with fast restoration of sinus rhythm.

Neural tube defects in Wales: changing demographics from 1998 to 2009.

OBJECTIVE: To establish if there is evidence for the perceived increase in the number of live births with a Neural tube defect (NTD) in South Wales from 1998 to 2009. METHODS: Data was obtained from the Congenital Abnormalities Register Information Services (CARIS), which recorded 305 cases of pregnancies involving neural tube defects. Descriptive analysis was carried out for each year in this period to obtain the number of live births, the proportion of live births compared to NTD pregnancies, plus the number and the percentage terminated each year. RESULTS: From the 305 cases, 66 resulted in live births, 230 in terminations. There was an increase in live births from 13.8% in 1998 to 33.3% in 2009, and a decrease in terminations from 82.76% in 1998 to 62.50% in 2009. The data also showed that this increase occurred mainly in the South of Wales. Discussion. Over this time period, there has been a decrease in the number of pregnancies affected by a neural tube defect and a decline in the proportion of pregnancies with neural tube defects resulting in terminations. Consequently, there has been an increase in the number of live births. The reasons for this change in trend is unclear, it may be that prospective parents are more willing to proceed with the pregnancy due to better support and services and the improved prognosis for children with neural tube defects. CONCLUSION: This data clearly shows that the number of children born with a NTD is increasing, and if this trend persists, services will have to expand and adapt to the change in the demographic of this population.

Safety concerns for the use of calcium channel blockers in pregnancy for the treatment of spontaneous preterm labour and hypertension: a systematic review and meta-regression analysis.

BACKGROUND: Calcium channel blockers (CCBs) are not licensed for use in pregnancy but are used without robust surveillance to treat hypertension in pregnancy and preterm labour. The objective of this study was to evaluate the fetomaternal safety of CCB in pregnancy by a quantitative systematic review. METHODS: Medline (1996-2005), EMBASE (1996-2003), BIOSIS (1993-2003), Current contents (1995-2003), DERWENT DRUGFILE (1983-2003) and Cochrane Library (2005: issue 3). The number of women reporting an adverse event was used to compute a percentage of the total number of women in whom the occurrence of that event or confirmation of its absence was reported. Meta-regression with generalised estimation equations modelling explored reasons for heterogeneity, seeking factors that increased the rates of the most commonly reported adverse events. FINDINGS: Of 269 relevant reports, including 5607 women, adverse fetomaternal events varied according to the total dose of nifedipine and study design. Adverse events were highest amongst women given more than 60 mg total dose of nifedipine [odds ratio (OR) 3.78, 95% confidence interval (CI) 1.27-11.2, p = 0.017] and in reports from case series compared to controlled studies (OR 2.45, 95% CI 1.17-5.15, p = 0.018). INTERPRETATION: Adverse event rates generated from this study provide an evidence base for clinical guidelines and informed patient consent for CCB use in pregnancy.

Rational protein engineering in action: the first crystal structure of a phenylalanine tRNA synthetase from Staphylococcus haemolyticus.

In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality--they only diffracted X-rays to 3-5A resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2A or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.

Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.

Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.

Expression profiling of herpesvirus and vaccinia virus proteins using a high-throughput baculovirus screening system.

We have developed a high-throughput system for generating baculoviruses and testing the expression, solubility, and affinity column purification of encoded proteins. We have used this system to generate baculoviruses for and analyze the expression of 337 proteins from three different herpesviruses (HSV-1, EBV, and CMV) and vaccinia virus. Subsets of these proteins were also tested for expression and solubility in E. coli. Comparisons of the results in the two systems are presented for each virus.

The quality of nifedipine studies used to assess tocolytic efficacy: a systematic review.

OBJECTIVE: To assess the quality of studies of nifedipine used to treat spontaneous preterm labor. DESIGN: A systematic review of study quality using a novel validity assessment tool, examining method-specific and topic-specific items in the domains of selection, performance and measurement biases. DATA SOURCES: Medline (1996-2003), EMBASE (1996-2003), BIOSIS (1993-2003), Current Contents (1995-2003), DERWENT DRUGFILE (1983-2003), Cochrane Database of Systematic Reviews. Bibliographies of existing meta-analyses and systematic reviews of nifedipine as a tocolytic. METHODS OF STUDY SELECTION: Forty-five studies evaluating the effectiveness of nifedipine were identified. DATA EXTRACTION: Each study was assessed for 40 method-specific and topic-specific items of quality in duplicate using piloted data extraction forms. Disagreements between assessors were settled by consensus/arbitration. DATA SYNTHESIS: Very few of the studies complied with adequacy criteria of quality for either method-specific or topic-specific items. There was no improvement in quality over time. The quality of method-specific items was significantly poorer when compared with topic-specific items of quality overall (P<0.0001) and in the domains of selection bias (P<0.0001) and performance bias (P<0.0001). CONCLUSION: Studies of the effectiveness of nifedipine as a tocolytic are of poorer quality with respect to method-specific items than topic-specific items. These deficiencies should be highlighted in meta-analyses or systematic reviews which measure efficacy and should influence the generation of guideline statements or recommendations for the use of nifedipine as a tocolytic. A large randomized trial fulfilling the quality items is necessary to assess the real efficacy of nifedipine in preterm labor.

Erythropoietin receptor Y479 couples to ERK1/2 activation via recruitment of phospholipase Cgamma.

Red blood cell development is primarily controlled by erythropoietin (EPO). Several studies have revealed the importance of EPO-R Y343 and Y479 for erythroid cell growth, differentiation, and survival. In order to isolate critical signaling proteins that bind to EPO-R, we initiated a Cloning of Ligand Target (COLT) screen using a murine embryonic day 16 phage library and a biotinylated EPO-R Y343 phosphopeptide. One of the clones isolated encodes Phospholipase C (PLC)gamma1. PLCgamma1 is rapidly tyrosine phosphorylated upon EPO stimulation and associates with EPO-R in an SH2-domain-dependent manner. Although PLCgamma1 bound EPO-R Y343, Y401, Y429, Y431, and Y479 in the COLT screen, PLCgamma1 required Y479 for association with EPO-R in Ba/F3-EPO-R cells. Studies have identified EPO-R Y479 as important for ERK activation. Since PI3-kinase binds EPO-R Y479, one group has suggested that ERK activation downstream of PI3-kinase accounts for the importance of this residue in EPO signaling. However, we show that inhibition of PI3-kinase does not abolish ERK activation. Furthermore, we demonstrate interaction of PLCgamma1 with Grb2 and SOS2. Hence, we have identified a novel adapter function for PLCgamma1 in EPO signaling in which recruitment of PLCgamma1 to EPO-R may lead to activation of the ERK pathway.

Interaction network containing conserved and essential protein complexes in Escherichia coli.

Proteins often function as components of multi-subunit complexes. Despite its long history as a model organism, no large-scale analysis of protein complexes in Escherichia coli has yet been reported. To this end, we have targeted DNA cassettes into the E. coli chromosome to create carboxy-terminal, affinity-tagged alleles of 1,000 open reading frames (approximately 23% of the genome). A total of 857 proteins, including 198 of the most highly conserved, soluble non-ribosomal proteins essential in at least one bacterial species, were tagged successfully, whereas 648 could be purified to homogeneity and their interacting protein partners identified by mass spectrometry. An interaction network of protein complexes involved in diverse biological processes was uncovered and validated by sequential rounds of tagging and purification. This network includes many new interactions as well as interactions predicted based solely on genomic inference or limited phenotypic data. This study provides insight into the function of previously uncharacterized bacterial proteins and the overall topology of a microbial interaction network, the core components of which are broadly conserved across Prokaryota.

RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits.

We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.

Sequential Peptide Affinity (SPA) system for the identification of mammalian and bacterial protein complexes.

A vector system is described that combines reliable, very low level, regulated protein expression in human cells with two affinity purification tags (Sequential Peptide Affinity, or SPA, system). By avoiding overproduction of the target protein, this system allows for the efficient purification of natural protein complexes and their identification by mass spectrometry. We also present an adaptation of the SPA system for the efficient purification and identification of protein complexes in E. coli and, potentially, other bacteria.

High-definition macromolecular composition of yeast RNA-processing complexes.

A remarkably large collection of evolutionarily conserved proteins has been implicated in processing of noncoding RNAs and biogenesis of ribonucleoproteins. To better define the physical and functional relationships among these proteins and their cognate RNAs, we performed 165 highly stringent affinity purifications of known or predicted RNA-related proteins from Saccharomyces cerevisiae. We systematically identified and estimated the relative abundance of stably associated polypeptides and RNA species using a combination of gel densitometry, protein mass spectrometry, and oligonucleotide microarray hybridization. Ninety-two discrete proteins or protein complexes were identified comprising 489 different polypeptides, many associated with one or more specific RNA molecules. Some of the pre-rRNA-processing complexes that were obtained are discrete sub-complexes of those previously described. Among these, we identified the IPI complex required for proper processing of the ITS2 region of the ribosomal RNA primary transcript. This study provides a high-resolution overview of the modular topology of noncoding RNA-processing machinery.

A panoramic view of yeast noncoding RNA processing.

Predictive analysis using publicly available yeast functional genomics and proteomics data suggests that many more proteins may be involved in biogenesis of ribonucleoproteins than are currently known. Using a microarray that monitors abundance and processing of noncoding RNAs, we analyzed 468 yeast strains carrying mutations in protein-coding genes, most of which have not previously been associated with RNA or RNP synthesis. Many strains mutated in uncharacterized genes displayed aberrant noncoding RNA profiles. Ten factors involved in noncoding RNA biogenesis were verified by further experimentation, including a protein required for 20S pre-rRNA processing (Tsr2p), a protein associated with the nuclear exosome (Lrp1p), and a factor required for box C/D snoRNA accumulation (Bcd1p). These data present a global view of yeast noncoding RNA processing and confirm that many currently uncharacterized yeast proteins are involved in biogenesis of noncoding RNA.