AutoRNC: An automated modeling program for building atomic models of ribosome-nascent chain complexes.

The interpretation of experimental studies of co-translational protein folding often benefits from the use of computational methods that seek to model or simulate the nascent chain and its interactions with the ribosome. Building realistic 3D models of ribosome-nascent chain (RNC) constructs often requires expert knowledge, so to circumvent this issue, we describe here AutoRNC, an automated modeling program capable of constructing large numbers of plausible atomic models of RNCs within minutes. AutoRNC takes input from the user specifying any regions of the nascent chain that contain secondary or tertiary structure and attempts to build conformations compatible with those specifications-and with the constraints imposed by the ribosome-by sampling and progressively piecing together dipeptide conformations extracted from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB). Despite using only modest computational resources, we show here that AutoRNC can build plausible conformations for a wide range of RNC constructs for which experimental data have already been reported.

AutoRNC: an automated modeling program for building atomic models of ribosome-nascent chain complexes.

The interpretation of experimental studies of co-translational protein folding often benefits from the use of computational methods that seek to model the nascent chain and its interactions with the ribosome. Ribosome-nascent chain (RNC) constructs studied experimentally can vary significantly in size and the extent to which they contain secondary and tertiary structure, and building realistic 3D models of them therefore often requires expert knowledge. To circumvent this issue, we describe here AutoRNC, an automated modeling program capable of constructing large numbers of plausible atomic models of RNCs within minutes. AutoRNC takes input from the user specifying any regions of the nascent chain that contain secondary or tertiary structure and attempts to build conformations compatible with those specifications - and with the constraints imposed by the ribosome - by sampling and progressively piecing together dipeptide conformations extracted from the RCSB. We first show that conformations of completely unfolded proteins built by AutoRNC in the absence of the ribosome have radii of gyration that match well with the corresponding experimental data. We then show that AutoRNC can build plausible conformations for a wide range of RNC constructs for which experimental data have already been reported. Since AutoRNC requires only modest computational resources, we anticipate that it will prove to be a useful hypothesis generator for experimental studies, for example, in providing indications of whether designed constructs are likely to be capable of folding, as well as providing useful starting points for downstream atomic or coarse-grained simulations of the conformational dynamics of RNCs.

Modeling the 3D structure and conformational dynamics of very large RNAs using coarse-grained molecular simulations.

We describe a computational approach to building and simulating realistic 3D models of very large RNA molecules (>1000 nucleotides) at a resolution of one "bead" per nucleotide. The method starts with a predicted secondary structure and uses several stages of energy minimization and Brownian dynamics (BD) simulation to build 3D models. A key step in the protocol is the temporary addition of a 4 th spatial dimension that allows all predicted helical elements to become disentangled from each other in an effectively automated way. We then use the resulting 3D models as input to Brownian dynamics simulations that include hydrodynamic interactions (HIs) that allow the diffusive properties of the RNA to be modelled as well as enabling its conformational dynamics to be simulated. To validate the dynamics part of the method, we first show that when applied to small RNAs with known 3D structures the BD-HI simulation models accurately reproduce their experimental hydrodynamic radii (Rh). We then apply the modelling and simulation protocol to a variety of RNAs for which experimental Rh values have been reported ranging in size from 85 to 3569 nucleotides. We show that the 3D models, when used in BD-HI simulations, produce hydrodynamic radii that are usually in good agreement with experimental estimates for RNAs that do not contain tertiary contacts that persist even under very low salt conditions. Finally, we show that sampling of the conformational dynamics of large RNAs on timescales of 100 µs is computationally feasible with BD-HI simulations.

Atomic Models of All Major Trans-Envelope Complexes Involved in Lipid Trafficking in Escherichia Coli Constructed Using a Combination of AlphaFold2, AF2Complex, and Membrane Morphing Simulations.

In Gram-negative bacteria, several trans-envelope complexes (TECs) have been identified that span the periplasmic space in order to facilitate lipid transport between the inner- and outer- membranes. While partial or near-complete structures of some of these TECs have been solved by conventional experimental techniques, most remain incomplete. Here we describe how a combination of computational approaches, constrained by experimental data, can be used to build complete atomic models for four TECs implicated in lipid transport in Escherichia coli . We use DeepMind's protein structure prediction algorithm, AlphaFold2, and a variant of it designed to predict protein complexes, AF2Complex, to predict the oligomeric states of key components of TECs and their likely interfaces with other components. After obtaining initial models of the complete TECs by superimposing predicted structures of subcomplexes, we use the membrane orientation prediction algorithm OPM to predict the likely orientations of the inner- and outer- membrane components in each TEC. Since, in all cases, the predicted membrane orientations in these initial models are tilted relative to each other, we devise a novel molecular mechanics-based strategy that we call "membrane morphing" that adjusts each TEC model until the two membranes are properly aligned with each other and separated by a distance consistent with estimates of the periplasmic width in E. coli . The study highlights the potential power of combining computational methods, operating within limits set by both experimental data and by cell physiology, for producing useable atomic structures of very large protein complexes.

Identification of Iron-Sulfur (Fe-S) Cluster and Zinc (Zn) Binding Sites Within Proteomes Predicted by DeepMind's AlphaFold2 Program Dramatically Expands the Metalloproteome.

DeepMind's AlphaFold2 software has ushered in a revolution in high quality, 3D protein structure prediction. In very recent work by the DeepMind team, structure predictions have been made for entire proteomes of twenty-one organisms, with >360,000 structures made available for download. Here we show that thousands of novel binding sites for iron-sulfur (Fe-S) clusters and zinc (Zn) ions can be identified within these predicted structures by exhaustive enumeration of all potential ligand-binding orientations. We demonstrate that AlphaFold2 routinely makes highly specific predictions of ligand binding sites: for example, binding sites that are comprised exclusively of four cysteine sidechains fall into three clusters, representing binding sites for 4Fe-4S clusters, 2Fe-2S clusters, or individual Zn ions. We show further: (a) that the majority of known Fe-S cluster and Zn binding sites documented in UniProt are recovered by the AlphaFold2 structures, (b) that there are occasional disputes between AlphaFold2 and UniProt with AlphaFold2 predicting highly plausible alternative binding sites, (c) that the Fe-S cluster binding sites that we identify in E. coli agree well with previous bioinformatics predictions, (d) that cysteines predicted here to be part of ligand binding sites show little overlap with those shown via chemoproteomics techniques to be highly reactive, and (e) that AlphaFold2 occasionally appears to build erroneous disulfide bonds between cysteines that should instead coordinate a ligand. These results suggest that AlphaFold2 could be an important tool for the functional annotation of proteomes, and the methodology presented here is likely to be useful for predicting other ligand-binding sites.

Trends in congenital anomalies in Europe from 1980 to 2012.

BACKGROUND: Surveillance of congenital anomalies is important to identify potential teratogens. METHODS: This study analysed the prevalence of 61 congenital anomaly subgroups (excluding chromosomal) in 25 population-based EUROCAT registries (1980-2012). Live births, fetal deaths and terminations of pregnancy for fetal anomaly were analysed with multilevel random-effects Poisson regression models. RESULTS: Seventeen anomaly subgroups had statistically significant trends from 2003-2012; 12 increasing and 5 decreasing. CONCLUSIONS: The annual increasing prevalence of severe congenital heart defects, single ventricle, atrioventricular septal defects and tetralogy of Fallot of 1.4% (95% CI: 0.7% to 2.0%), 4.6% (1.0% to 8.2%), 3.4% (1.3% to 5.5%) and 4.1% (2.4% to 5.7%) respectively may reflect increases in maternal obesity and diabetes (known risk factors). The increased prevalence of cystic adenomatous malformation of the lung [6.5% (3.5% to 9.4%)] and decreased prevalence of limb reduction defects [-2.8% (-4.2% to -1.5%)] are unexplained. For renal dysplasia and maternal infections, increasing trends may be explained by increased screening, and deceases in patent ductus arteriosus at term and increases in craniosynostosis, by improved follow up period after birth and improved diagnosis. For oesophageal atresia, duodenal atresia/stenosis and ano-rectal atresia/stenosis recent changes in prevalence appeared incidental when compared with larger long term fluctuations. For microcephaly and congenital hydronephrosis trends could not be interpreted due to discrepancies in diagnostic criteria. The trends for club foot and syndactyly disappeared once registries with disparate results were excluded. No decrease in neural tube defects was detected, despite efforts at prevention through folic acid supplementation.

Optimization of folic acid supplementation in the prevention of neural tube defects.

Background: We examined the relationship between timing and duration of folic acid (FA) supplementation in achieving red blood cell (RBC) folate levels in early pregnancy which are optimal (>906 nmol/l) for the prevention of neural tube defects (NTDs). Methods: Clinical, FA supplementation and dietary folate details were computerized at the first antenatal visit. Maternal blood samples were analysed for RBC and serum folate. Results: Of the 502 women, 98.2% (n = 493) reported taking FA. There was a positive correlation between duration of supplementation and both RBC folate (r = 0.43, P < 0.001) and serum folate (rho = 0.29, P < 0.001). The optimal RBC folate level was achieved in 80.4% (n = 46) of women who started FA 400 µg 4-8 weeks before their LMP compared with only 53.6% (n = 153) in women who started 4-8 weeks after their LMP (P < 0.001). Conclusions: This study provides, for the first time, information on both the timing and duration of FA that will achieve the optimum RBC folate levels associated with the prevention of NTDs. Women who are taking FA (400 µg) need to start before they conceive.

Prevalence of microcephaly in Europe: population based study.

OBJECTIVES:  To provide contemporary estimates of the prevalence of microcephaly in Europe, determine if the diagnosis of microcephaly is consistent across Europe, and evaluate whether changes in prevalence would be detected using the current European surveillance performed by EUROCAT (the European Surveillance of Congenital Anomalies). DESIGN:  Questionnaire and population based observational study. SETTING:  24 EUROCAT registries covering 570 000 births annually in 15 countries. PARTICIPANTS:  Cases of microcephaly not associated with a genetic condition among live births, fetal deaths from 20 weeks' gestation, and terminations of pregnancy for fetal anomaly at any gestation. MAIN OUTCOME MEASURES:  Prevalence of microcephaly (1 Jan 2003-31 Dec 2012) analysed with random effects Poisson regression models to account for heterogeneity across registries. RESULTS:  16 registries responded to the questionnaire, of which 44% (7/16) used the EUROCAT definition of microcephaly (a reduction in the size of the brain with a skull circumference more than 3 SD below the mean for sex, age, and ethnic origin), 19% (3/16) used a 2 SD cut off, 31% (5/16) were reliant on the criteria used by individual clinicians, and one changed criteria between 2003 and 2012. Prevalence of microcephaly in Europe was 1.53 (95% confidence interval 1.16 to 1.96) per 10 000 births, with registries varying from 0.4 (0.2 to 0.7) to 4.3 (3.6 to 5.0) per 10 000 (χ(2)=338, df=23, I(2)=93%). Registries with a 3 SD cut off reported a prevalence of 1.74 per 10 000 (0.86 to 2.93) compared with those with the less stringent 2 SD cut off of 1.21 per 10 000 (0.21 to 2.93). The prevalence of microcephaly would need to increase in one year by over 35% in Europe or by over 300% in a single registry to reach statistical significance (P<0.01). CONCLUSIONS:  EUROCAT could detect increases in the prevalence of microcephaly from the Zika virus of a similar magnitude to those observed in Brazil. Because of the rarity of microcephaly and discrepant diagnostic criteria, however, the smaller increases expected in Europe would probably not be detected. Clear diagnostic criteria for microcephaly must be adopted across Europe.

Congenital anomalies associated with trisomy 18 or trisomy 13: A registry-based study in 16 European countries, 2000-2011.

The aim of this study was to examine the prevalence of trisomies 18 and 13 in Europe and the prevalence of associated anomalies. Twenty-five population-based registries in 16 European countries provided data from 2000-2011. Cases included live births, fetal deaths (20+ weeks' gestation), and terminations of pregnancy for fetal anomaly (TOPFAs). The prevalence of associated anomalies was reported in live births. The prevalence of trisomy 18 and trisomy 13 were 4.8 (95%CI: 4.7-5.0) and 1.9 (95%CI: 1.8-2.0) per 10,000 total births. Seventy three percent of cases with trisomy 18 or trisomy 13 resulted in a TOPFA. Amongst 468 live born babies with trisomy 18, 80% (76-83%) had a cardiac anomaly, 21% (17-25%) had a nervous system anomaly, 8% (6-11%) had esophageal atresia and 10% (8-13%) had an orofacial cleft. Amongst 240 Live born babies with trisomy 13, 57% (51-64%) had a cardiac anomaly, 39% (33-46%) had a nervous system anomaly, 30% (24-36%) had an eye anomaly, 44% (37-50%) had polydactyly and 45% (39-52%) had an orofacial cleft. For babies with trisomy 18 boys were less likely to have a cardiac anomaly compared with girls (OR = 0.48 (0.30-0.77) and with trisomy 13 were less likely to have a nervous system anomaly [OR = 0.46 (0.27-0.77)]. Babies with trisomy 18 or trisomy 13 do have a high proportion of associated anomalies with the distribution of anomalies being different in boys and girls.

Epidemiology of orofacial clefts in the East of ireland in the 25-year period 1984-2008.

Objective : To describe the epidemiology of orofacial clefts in the east of Ireland. Design and Setting : A descriptive epidemiologic study on 851 cases of orofacial cleft identified over a 25-year period from 1984 to 2008 from more than 500,000 births. Results : There were 438 (51.5%) cases of cleft lip with or without cleft palate and 413 (48.5%) cases of cleft palate. The total birth prevalence was 16.0 per 10,000 births for all orofacial clefts, 8.2 for cleft lip with or without cleft palate, and 7.8 for cleft palate. Of all cases, 63.7% (542/851) occurred as isolated anomalies, 21.5% (183/851) were associated with multiple anomalies, and 14.8% (126/851) were associated with a syndrome or chromosomal anomaly. A significantly increasing trend over the 25-year period was observed for cleft lip with or without cleft palate associated with syndromes or chromosomal anomalies among mothers younger than 35 years but not in those older than 35 years. Conclusion : A slightly higher rate of orofacial clefts was observed in the east of Ireland than was observed in European and multinational studies during the study period, and there were higher rates of cleft palate. The rising trend in the proportion of mothers aged 35 years or older in Ireland is not contributing significantly to orofacial clefts associated with chromosomal syndromes.

Prevalence of esophageal atresia among 18 international birth defects surveillance programs.

BACKGROUND: The prevalence of esophageal atresia (EA) has been shown to vary across different geographical settings. Investigation of geographical differences may provide an insight into the underlying etiology of EA. METHODS: The study population comprised infants diagnosed with EA during 1998 to 2007 from 18 of the 46 birth defects surveillance programs, members of the International Clearinghouse for Birth Defects Surveillance and Research. Total prevalence per 10,000 births for EA was defined as the total number of cases in live births, stillbirths, and elective termination of pregnancy for fetal anomaly (ETOPFA) divided by the total number of all births in the population. RESULTS: Among the participating programs, a total of 2943 cases of EA were diagnosed with an average prevalence of 2.44 (95% confidence interval [CI], 2.35-2.53) per 10,000 births, ranging between 1.77 and 3.68 per 10,000 births. Of all infants diagnosed with EA, 2761 (93.8%) were live births, 82 (2.8%) stillbirths, 89 (3.0%) ETOPFA, and 11 (0.4%) had unknown outcomes. The majority of cases (2020, 68.6%), had a reported EA with fistula, 749 (25.5%) were without fistula, and 174 (5.9%) were registered with an unspecified code. CONCLUSIONS: On average, EA affected 1 in 4099 births (95% CI, 1 in 3954-4251 births) with prevalence varying across different geographical settings, but relatively consistent over time and comparable between surveillance programs. Findings suggest that differences in the prevalence observed among programs are likely to be attributable to variability in population ethnic compositions or issues in reporting or registration procedures of EA, rather than a real risk occurrence difference. Birth Defects Research (Part A), 2012.

How valid are the rates of Down syndrome internationally? Findings from the International Clearinghouse for Birth Defects Surveillance and Research.

Rates of Down syndrome (DS) show considerable international variation, but a systematic assessment of this variation is lacking. The goal of this study was to develop and test a method to assess the validity of DS rates in surveillance programs, as an indicator of quality of ascertainment. The proposed method compares the observed number of cases with DS (livebirths plus elective pregnancy terminations, adjusted for spontaneous fetal losses that would have occurred if the pregnancy had been allowed to continue) in each single year of maternal age, with the expected number of cases based on the best-published data on rates by year of maternal age. To test this method we used data from birth years 2000 to 2005 from 32 surveillance programs of the International Clearinghouse for Birth Defects Surveillance and Research. We computed the adjusted observed versus expected ratio (aOE) of DS birth prevalence among women 25-44 years old. The aOE ratio was close to unity in 13 programs (the 95% confidence interval included 1), above 1 in 2 programs and below 1 in 18 programs (P < 0.05). These findings suggest that DS rates internationally can be evaluated simply and systematically, and underscores how adjusting for spontaneous fetal loss is crucial and feasible. The aOE ratio can help better interpret and compare the reported rates, measure the degree of under- or over-registration, and promote quality improvement in surveillance programs that will ultimately provide better data for research, service planning, and public health programs.

Trends of selected malformations in relation to folic acid recommendations and fortification: an international assessment.

BACKGROUND: Two crucial issues relative to the benefits and impact of folic acid in the prevention of birth defects are whether supplementation recommendations alone, without fortification, are effective in reducing the population-wide rates of neural tube defects (NTDs), and whether such policies can reduce the occurrence of other birth defects. Using data from 15 registries, we assessed rates and trends of 14 major defects, including NTDs, in areas with official recommendations or fortification to assess the effectiveness of recommendations and fortification on a wide range of major birth defects. METHODS: We evaluated surveillance data through 2003 on major birth defects from population-based registries from Europe, North America, and Australia. All included ascertainment of pregnancy terminations (where legal). Trends before and after policies or fortification were assessed via Poisson regression and were compared via rate ratios. RESULTS: Significant changes in trends were seen for NTDs in areas with fortification but not in areas with supplementation recommendations alone. For other major birth defects, there was an overall lack of major trend changes after recommendations or fortification. However, some significant declines were observed for select birth defects in individual areas. CONCLUSIONS: Recommendations alone remain an ineffective approach in translating the known protective effect of folic acid in population-wide decline in NTD rates. Fortification appears to be effective in reducing NTDs. The effect on other birth defects remains unclear.

Preventing neural tube defects in Europe: a missed opportunity.

Each year, more than 4500 pregnancies in the European Union are affected by neural tube defects (NTD). Unambiguous evidence of the effectiveness of periconceptional folic acid in preventing the majority of neural tube defects has been available since 1991. We report on trends in the total prevalence of neural tube defects up to 2002, in the context of a survey in 18 European countries of periconceptional folic acid supplementation (PFAS) policies and their implementation. EUROCAT is a network of population-based registries in Europe collaborating in the epidemiological surveillance of congenital anomalies. Representatives from 18 participating countries provided information about policy, health education campaigns and surveys of PFAS uptake. The yearly total prevalence of neural tube defects including livebirths, stillbirths and terminations of pregnancy was calculated from 1980 to 2002 for 34 registries, with UK and Ireland estimated separately from the rest of Europe. A meta-analysis of changes in NTD total prevalence between 1989-1991 and 2000-2002 according to PFAS policy was undertaken for 24 registries. By 2005, 13 countries had a government recommendation that women planning a pregnancy should take 0.4mg folic acid supplement daily, accompanied in 7 countries by government-led health education initiatives. In the UK and Ireland, countries with PFAS policy, there was a 30% decline in NTD total prevalence (95% CI 16-42%) but it was difficult to distinguish this from the pre-existing strong decline. In other European countries with PFAS policy, there was virtually no decline in NTD total prevalence whether a policy was in place by 1999 (2%, 95% CI 28% reduction to 32% increase) or not (8%, 95% CI 26% reduction to 16% increase). The potential for preventing NTDs by periconceptional folic acid supplementation is still far from being fulfilled in Europe. Only a public health policy including folic acid fortification of staple foods is likely to result in large-scale prevention of NTDs.