A core outcome set for the treatment of pregnant women with pregestational diabetes: an international consensus study.

OBJECTIVE: To develop a core outcome set (COS) for randomised controlled trials (RCTs) evaluating the effectiveness of interventions for the treatment of pregnant women with pregestational diabetes mellitus (PGDM). DESIGN: A consensus developmental study. SETTING: International. POPULATION: Two hundred and five stakeholders completed the first round. METHODS: The study consisted of three components. (1) A systematic review of the literature to produce a list of outcomes reported in RCTs assessing the effectiveness of interventions for the treatment of pregnant women with PGDM. (2) A three-round, online eDelphi survey to prioritise these outcomes by international stakeholders (including healthcare professionals, researchers and women with PGDM). (3) A consensus meeting where stakeholders from each group decided on the final COS. MAIN OUTCOME MEASURES: All outcomes were extracted from the literature. RESULTS: We extracted 131 unique outcomes from 67 records meeting the full inclusion criteria. Of the 205 stakeholders who completed the first round, 174/205 (85%) and 165/174 (95%) completed rounds 2 and 3, respectively. Participants at the subsequent consensus meeting chose 19 outcomes for inclusion into the COS: trimester-specific haemoglobin A1c, maternal weight gain during pregnancy, severe maternal hypoglycaemia, diabetic ketoacidosis, miscarriage, pregnancy-induced hypertension, pre-eclampsia, maternal death, birthweight, large for gestational age, small for gestational age, gestational age at birth, preterm birth, mode of birth, shoulder dystocia, neonatal hypoglycaemia, congenital malformations, stillbirth and neonatal death. CONCLUSIONS: This COS will enable better comparison between RCTs to produce robust evidence synthesis, improve trial reporting and optimise research efficiency in studies assessing treatment of pregnant women with PGDM. TWEETABLE ABSTRACT: 165 key stakeholders have developed #Treatment #CoreOutcomes in pregnant women with #diabetes existing before pregnancy.

Improving Outcomes Among Young Adults with type 1 diabetes: The D1 Now Randomised Pilot Study Protocol.

BACKGROUND: Young adults (18-25 years old) living with type 1 diabetes mellitus often have sub-optimal glycaemic levels which can increase their risk of long term diabetes complications. Informed by health psychology theory and using a (public and patient involvement) young adult-centred approach, we have developed a complex intervention, entitled D1 Now, to improve outcomes in this target group. The D1 Now intervention includes three components; 1) a support-worker, 2) an interactive messaging system and 3) an agenda setting tool for use during clinic consultations. AIMS: The aim of the D1 Now pilot study is to gather and analyse acceptability and feasibility data to allow us to (1) refine the D1 Now intervention, and (2) determine the feasibility of a definitive Randomised Control Trial (RCT) of the intervention. METHODS: Diabetes clinics on the island of Ireland will be recruited and randomised to a D1 Now intervention arm or a usual care control arm. For a participant to be eligible they should be 18-25 years old and living with type 1 diabetes for at least 12 months. Participant outcomes (influenced by a Core Outcome Set) include change in HbA1c, clinic attendance, number of episodes of severe hypoglycaemia and of diabetic ketoacidosis, diabetes distress, self-management, quality of life and perceived level of control over diabetes; these will be will be measured at baseline and after 12 months follow-up for descriptive statistics only. An assessment of treatment fidelity, a health economic analysis and a qualitative sub-study will also be incorporated into the pilot study. ISRCTN (ref: ISRCTN74114336).

The preferences of young adults with Type 1 diabetes at clinics using a discrete choice experiment approach: the D1 Now Study.

AIM: Attending routine outpatient clinic appointments is a central self-management behaviour of individuals living with Type 1 diabetes. A large number of young adults with Type 1 diabetes disengage from diabetes services, which may contribute to poor psychosocial and diabetes outcomes. The aim of this study is to elicit preferences from young adults with Type 1 diabetes regarding clinic-related services to inform service delivery. METHODS: A discrete choice experiment was developed to understand the preferences of young adults with Type 1 diabetes for clinic-related services. RESULTS: Young adults recruited from young adult Type 1 diabetes clinics in 2016 completed the experiment (n = 105). Young adults with Type 1 diabetes showed a preference for shorter waiting times, seeing a nurse and a consultant, relative to a nurse alone, and a flexible booking system compared with fixed appointment times. Results suggest no preference for a nurse and a doctor, relative to a nurse alone, or other optional services (e.g. seeing dietitians or psychologists), type of HbA1c test and digital blood glucose diaries over paper-based diaries. CONCLUSION: This study highlights aspects of routine clinic appointments that are valued by young adults living with Type 1 diabetes, namely shorter waiting times at clinic, the option to see both a nurse and consultant at each visit and a flexible clinic appointment booking system. These findings suggest young adults with Type 1 diabetes value convenience and should help services to restructure their clinics to be more responsive to the needs of young adults.

Factors influencing health-related quality of life in patients with Type 1 diabetes.

AIMS: Generic, preference-based measures of health-related quality of life (HRQoL) are a common input to the economic evaluation of new health technologies. As such, it is important to explore what characteristics of patients with Type 1 diabetes might impact scores on such measures. METHODS: This study utilizes baseline data from a cluster-randomized trial that recruited patients with Type 1 diabetes at six centers across Ireland. Health-related quality of life was assessed using the three-level EuroQol EQ-5D (EQ-5D) measure. Patients' responses to individual dimensions of the EQ-5D were explored. To see which patient factors influenced EQ-5D scores, multivariate regression analysis was conducted with EQ-5D scores as the outcome variable. RESULTS: Data was available for 437 Type 1 diabetes patients. The median age of these patients was 40 (IQR: 31-49) years and 53.8% were female. Overall, patients reported a high HRQoL based on EQ-5D scores (0.87 (SD: 0.19). Fifty-four percent of patients reported a perfect HRQoL. For those that reported problems, the most common dimension was the anxiety/depression dimension of the EQ-5D (29.6%). In the multivariate regression analysis, self-reported mental illness (- 0.22 (95% CI: -0.34, - 0.10)) and being unemployed (- 0.07 (95% CI: -0.13, - 0.02)) were negatively associated with EQ-5D scores (p < 0.05). The influence of self-reported mental illness was persistent in sensitivity analyses. CONCLUSIONS: The study results indicate that patients with Type 1 diabetes report a high HRQoL based on responses to the EQ-5D. However, there are a substantial number of Type 1 diabetes patients that report problems in the anxiety/depression dimension, which may provide avenues to improve patients' HRQoL. TRIAL REGISTRATION: Current Controlled Trials ISRCTN79759174 .

Short- and long-term effects of gestational diabetes mellitus on healthcare cost: a cross-sectional comparative study in the ATLANTIC DIP cohort.

AIMS: This paper examines the association between gestational diabetes mellitus and costs of care during pregnancy and 2-5 years post pregnancy. METHODS: Healthcare utilization during pregnancy was measured for a sample of 658 women drawn from the Atlantic Diabetes in Pregnancy (ATLANTIC DIP) network. Healthcare utilization 2-5 years post pregnancy was assessed for a subsample of 348 women via a postal questionnaire. A vector of unit costs was applied to healthcare activity to calculate the costs of care at both time points. Differences in cost for women with gestational diabetes mellitus compared with those with normal glucose tolerance during the pregnancy were examined using univariate and multivariate regression analyses. RESULTS: Gestational diabetes mellitus was independently associated with an additional €817.60 during pregnancy (€1192.1 in the gestational diabetes mellitus group, €511.6 in the normal glucose tolerance group), in the form of additional delivery and neonatal care costs, and an additional €680.50 in annual healthcare costs 2-5 years after the index pregnancy (€6252.4 in the gestational diabetes mellitus group, €5434.8 in the normal glucose tolerance group). CONCLUSIONS: These results suggest that gestational diabetes mellitus is associated with increased costs of care during and post pregnancy. They provide indication of the associated cost that can be avoided or reduced by the screening, prevention and management of gestational diabetes mellitus in pregnancy. These estimates are useful for further studies that examine the cost and cost-effectiveness of such programmes.

Distance as a risk factor for amputation in patients with diabetes: a case-control study.

We studied the association between amputation and distance of patients' residences to a diabetes care centre. We performed a case-control study matching each case (amputation) with 5 controls (no amputation) by age and sex. We compared the distance of residence to the diabetes centre, duration and type of diabetes, haemoglobin-A1c levels and foot examination findings for cases and controls. We analysed the association between distance and the strongest predictors of amputation. Sixty-six cases of amputation and 313 controls were identified. Distance of residence was 12.1km greater for cases (p = 0.028). In multivariate analysis, only diabetes duration (OR/year 1.07, 1.03 to 1.11) and neuropathy (OR 10.73, 4.55 to 25.74) were significantly associated with amputation. Patients with neuropathy resided 97 km further than those without neuropathy (p = 0.01). Patients requiring amputation reside at greater distances from the diabetes centre, possibly due to higher rates of neuropathy.

The effects of dependence and function on costs of care for Alzheimer's disease and mild cognitive impairment in Ireland.

OBJECTIVE: To explore the incremental effects of patient dependence and function on costs of care for patients with Alzheimer's disease (AD) and amnestic mild cognitive impairment (MCI) in Ireland. METHODS: Cost analysis based on reported resource use for a cross-section of 100 community-based people with AD and MCI. Formal care included general practice visits, hospitalizations, outpatient clinic consultations, accident and emergency visits, respite care, meals on wheels services and other health and social care professional consultations. Informal care included time input provided by caregivers. Resource unit costs were applied to value formal care and the opportunity cost method was used to value informal care. Patient dependence on others was measured using the Dependence Scale and patient functional capacity using the Disability Assessment for Dementia scale. Multivariate regression analysis was used to model the cost of care. RESULTS: Both dependence and function were independently and significantly associated with total formal and informal care cost: a one point increase in dependence was associated with a €796 increase in total cost and a one point improvement in function with a €417 reduction in total cost over 6 months. Patient function was significantly associated with formal care costs, whereas patient function and dependence were both significantly associated with informal care costs. CONCLUSION: The costs of care for patients with AD and MCI in Ireland are substantial. Interventions that reduce patient dependence on others and functional decline may be associated with important economic benefits.

Expression, immunogenicity, histopathology, and potency of a mosquito-based malaria transmission-blocking recombinant vaccine.

Vaccines have been at the forefront of global research efforts to combat malaria, yet despite several vaccine candidates, this goal has yet to be realized. A potentially effective approach to disrupting the spread of malaria is the use of transmission-blocking vaccines (TBV), which prevent the development of malarial parasites within their mosquito vector, thereby abrogating the cascade of secondary infections in humans. Since malaria is transmitted to human hosts by the bite of an obligate insect vector, mosquito species in the genus Anopheles, targeting mosquito midgut antigens that serve as ligands for Plasmodium parasites represents a promising approach to breaking the transmission cycle. The midgut-specific anopheline alanyl aminopeptidase N (AnAPN1) is highly conserved across Anopheles vectors and is a putative ligand for Plasmodium ookinete invasion. We have developed a scalable, high-yield Escherichia coli expression and purification platform for the recombinant AnAPN1 TBV antigen and report on its marked vaccine potency and immunogenicity, its capacity for eliciting transmission-blocking antibodies, and its apparent lack of immunization-associated histopathologies in a small-animal model.

Fusion of Na-ASP-2 with human immunoglobulin Fcγ abrogates histamine release from basophils sensitized with anti-Na-ASP-2 IgE.

Na-ASP-2 is a major protein secreted by infective third-stage larvae (L3) of the human hookworm Necator americanus upon host entry. It was chosen as a lead vaccine candidate for its ability to elicit protective immune responses. However, clinical development of this antigen as a recombinant vaccine was halted because it caused allergic reactions among some of human volunteers previously infected with N. americanus. To prevent IgE-mediated allergic reactions induced by Na-ASP-2 but keep its immunogenicity as a vaccine antigen, we designed and tested a genetically engineered fusion protein, Fcγ/Na-ASP-2, composed of full-length Na-ASP-2 and truncated human IgG Fcγ1 that targets the negative signalling receptor FcγRIIb expressed on pro-allergic cells. The chimeric recombinant Fcγ/Na-ASP-2 protein was expressed in Pichia pastoris and shared the similar antigenicity as native Na-ASP-2. Compared to Na-ASP-2, the chimeric fusion protein efficiently reduced the release of histamine in human basophils sensitized with anti-Na-ASP-2 IgE obtained from individuals living in a hookworm-endemic area. In dogs infected with canine hookworm, Fcγ/Na-ASP-2 resulted in significantly reduced immediate-type skin reactivity when injected intradermally compared with Na-ASP-2. Hamsters vaccinated with Fcγ/Na-ASP-2 formulated with Alhydrogel(®) produced specific IgG that recognized Na-ASP-2 and elicited similar protection level against N. americanus L3 challenge as native Na-ASP-2.

Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing.

All organisms need to ensure that no DNA segments are rereplicated in a single cell cycle. Eukaryotes achieve this through a process called origin licensing, which involves tight spatiotemporal control of the assembly of prereplicative complexes (pre-RCs) onto chromatin. Cdt1 is a key component and crucial regulator of pre-RC assembly. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent DNA rereplication. Here, we address the mechanism of DNA licensing inhibition by Geminin, by combining X-ray crystallography, small-angle X-ray scattering, and functional studies in Xenopus and mammalian cells. Our findings show that the Cdt1:Geminin complex can exist in two distinct forms, a "permissive" heterotrimer and an "inhibitory" heterohexamer. Specific Cdt1 residues, buried in the heterohexamer, are important for licensing. We postulate that the transition between the heterotrimer and the heterohexamer represents a molecular switch between licensing-competent and licensing-defective states.

The impact of travel distance on the decision to attend for screening for gestational diabetes mellitus.

This paper estimates the impact of travel distance on the decision to attend for screening for gestational diabetes mellitus (GDM), controlling for a range of personal, clinical and lifestyle characteristics. The results suggest that women who live further away from a screening site are less likely to attend for screening. In particular, the probability of attending for screening is reduced by 1.8% [95% CI: 1.2% to 2.4%] for every additional 10 kms of travel. This is consistent wth previous research that shows geographic inequalities in access to GDM screening in Ireland. We also find that older women, those with a family history of diabetes, and those who are obese are more likely to accept the screening offer, suggesting that certain higher-risk groups may be either self-selecting into the screening programme or are being targeted by health care professionals through specific initiatives.

New estimates of the costs of universal screening for gestational diabetes mellitus in Ireland.

The new International Association of Diabetes and Pregnancy Study Groups (IADPSG) diagnostic criteria have been predicted to increase the prevalence of gestational diabetes mellitus 2-to-3 fold and will have important resource implications for healthcare systems. A bottom-up, prevalence-based analysis was undertaken to estimate the costs of universal screening for gestational diabetes mellitus in Ireland using the new criteria. Healthcare activity was identified from the Atlantic Diabetes in Pregnancy database and grouped into five categories: (i) screening and testing, (ii) GDM treatment, (iii) prenatal care, (iv) delivery care, and (v) neonatal care. When individual resource components were valued using unit cost data and aggregated, the total healthcare cost was estimated at Euro 46,311,301 (95% CI: Euro 36,381,038, Euro 68,007,432). The average cost per case detected was Euro 351 (95% CI: (Euro 126, Euro 558) and the average total cost per case detected and treated was Euro 9,325 (95% CI: Euro 5,982, Euro 13,996). Further research is required to determine the cost effectiveness of screening in the region with a view to improving resource allocation in this area in the future.

Is there a socioeconomic gradient in the prevalence of gestational diabetes mellitus?

Previous studies have shown an association between Type 2 diabetes and lower socioeconomic status. This link is less clear in those with gestational diabetes mellitus (GDM). We test for a socioeconomic gradient in the prevalence of GDM by analysing data on 9,842 pregnant women who were offered testing for GDM in the Atlantic Diabetes in Pregnancy universal screening programme. A bivariate probit model relating GDM prevalence to socioeconomic status was estimated, controlling for variation in screening uptake rates across socioeconomic groups. The estimated increased prevalence of GDM is 8.6% [95% CI 2.7%-12.0%] for women in the lowest socioeconomic group when compared to the highest, suggesting a strong socioeconomic gradient in the prevalence of GDM. This gradient is found to be driven by differences in personal, clinical and lifestyle factors across socioeconomic groups.

A randomised placebo-controlled trial of the effectiveness of early metformin in addition to usual care in the reduction of gestational diabetes mellitus effects (EMERGE): study protocol.

BACKGROUND: Pregnancies affected by gestational diabetes mellitus (GDM) are associated with an increased risk of adverse maternal and foetal outcomes. Current treatments for GDM involve initial medical nutritional therapy (MNT) and exercise and pharmacotherapy in those with persistent hyperglycaemia. Insulin is considered first-line pharmacotherapy but is associated with hypoglycaemia, excessive gestational weight gain (GWG) and an increased caesarean delivery rate. Metformin is safe in selected groups of women with GDM but is not first-line therapy in many guidelines due to a lack of long-term data on efficacy. The EMERGE trial will evaluate the effectiveness of early initiation of metformin in GDM. METHODS: EMERGE is a phase III, superiority, parallel, 1:1 randomised, double-blind, placebo-controlled trial comparing the effectiveness of metformin versus placebo initiated by 28 weeks (+6 days) plus usual care. Women aged 18-50 years will be recruited. Women with established diabetes, multiple pregnancies, known major congenital malformation or small for gestational age (<10th centile), intolerance or contraindication to the use of metformin, shock or sepsis, current gestational hypertension or pre-eclampsia, significant gastrointestinal problems, congestive heart failure, severe mental illness or galactose intolerance are excluded. INTERVENTION: Immediate introduction of metformin or placebo in addition to MNT and usual care. Metformin is initiated at 500mg/day and titrated to a maximum dose of 2500mg over 10 days. Women are followed up at 4 and 12 weeks post-partum to assess maternal and neonatal outcomes. The composite primary outcome measure is initiation of insulin or fasting blood glucose ≥ 5.1 mmol/L at gestational weeks 32 or 38. The secondary outcomes are the time to insulin initiation and insulin dose required; maternal morbidity at delivery; mode and time of delivery; postpartum glucose status; insulin resistance; postpartum body mass index (BMI); gestational weight gain; infant birth weight; neonatal height and head circumference at delivery; neonatal morbidities (neonatal care unit admission, respiratory distress, jaundice, congenital anomalies, Apgar score); neonatal hypoglycaemia; cost-effectiveness; treatment acceptability and quality of life determined by the EQ5D-5L scale. DISCUSSION: The EMERGE trial will determine the effectiveness and safety of early and routine use of metformin in GDM. TRIAL REGISTRATION: EudraCT Number 2016-001644-19l; NCT NCT02980276 . Registered on 6 June 2017.

The cost of universal screening for gestational diabetes mellitus in Ireland.

AIMS: To estimate the costs associated with universal screening for gestational diabetes mellitus in Ireland. METHODS: Bottom-up, prevalence-based cost analysis. Healthcare activity identified using the Atlantic Diabetes in Pregnancy (ATLANTIC DIP) database was grouped into five categories: screening and testing, gestational diabetes treatment, prenatal care, delivery care and neonatal care. A vector of unit cost data (euros in 2008 prices) was applied to specified resource use and the total healthcare cost calculated. A series of one-way and probabilistic sensitivity analyses were undertaken to explore the uncertainty in the analysis. RESULTS: When individual resource components were valued and aggregated, the total healthcare cost of gestational diabetes in Ireland was estimated at €12 433 320 (95% CI €9 298 228-16 778 193). The average cost per case detected was €1621 (95% CI €524-2603) and the average total cost per case detected and treated was €11 903 (95% CI €7645-16 121). CONCLUSIONS: This research provides the first estimates of the healthcare costs associated with gestational diabetes mellitus in Ireland. Further research is required to determine the cost-effectiveness of gestational diabetes screening in the region with a view to improving resource allocation in this area in the future.

Where do young adults want opportunistic chlamydia screening services to be located?

BACKGROUND: This study measured the acceptability of urine-based chlamydia screening to young adults, where young adults wanted opportunistic chlamydia screening services to be located, and by whom they wanted to be offered screening. METHODS: A cross-sectional survey of 5685 university students and 400 young adult healthcares setting attendees (age: 18-29 years). RESULTS: Ninety-six percent of males and 93% of females said that they would find it acceptable to be offered chlamydia screening. Seventy-six percent of males and 77% of females wanted to be offered screening by a doctor or nurse. Young women would prefer female staff. Most respondents preferred that screening be located in traditional healthcare settings such as General Practices, and offered by either doctors or nurses. More than 90% of respondents did not want screening services to be located in pharmacies and almost all rejected public non-health care screening settings. CONCLUSIONS: Opportunistic chlamydia screening services should be located in traditional healthcare/medical settings, and screening should be offered by doctors and nurses.

Development of amplified fragment length polymorphism-derived functional strain-specific markers to assess the persistence of 10 bacterial strains in soil microcosms.

To augment the information on commercial microbial products, we investigated the persistence patterns of high-priority bacterial strains from the Canadian Domestic Substance List (DSL). Specific DNA markers for each of the 10 DSL bacterial strains were developed using the amplified fragment length polymorphism (AFLP) technique, and the fates of DSL strains introduced in soil were assessed by real-time quantitative PCR (qPCR). The results indicated that all DNA markers had high specificity at the functional strain level and that detection of the target microorganisms was sensitive at a detection limitation range from 1.3 × 10² to 3.25 × 105 CFU/g of dry soil. The results indicated that all introduced strains showed a trend toward a declining persistence in soil and could be categorized into three pattern types. The first type was long-term persistence exemplified by Pseudomonas stutzeri (ATCC 17587) and Pseudomonas denitrificans (ATCC 13867) strains. In the second pattern, represented by Bacillus subtilis (ATCC 6051) and Escherichia hermannii (ATCC 700368), the inoculated strain populations dropped dramatically below the detection threshold after 10 to 21 days, while in the third pattern there was a gradual decrease, with the population falling below the detectable level within the 180-day incubation period. These patterns indicate a selection effect of a microbial community related to the ecological function of microbial strains introduced in soil. As a key finding, the DSL strains can be quantitatively tracked in soil with high sensitivity and specificity at the functional strain level. This provides the basic evidence for further risk assessment of the priority DSL strains.

Si complexes in calcium phosphate biomaterials.

Silicon complexes in silicon doped calcium phosphate bioceramics have been studied using (29)Si magic angle spinning nuclear magnetic resonance spectroscopy with the objective of identifying the charge compensation mechanisms of silicon dopants. Three different materials have been studied: a multiphase material composed pre-dominantly of a silicon stabilized alpha-tricalcium phosphate(alpha-TCP) phase plus a hydroxyapatite (HA) phase, a single phase Si-HA material and a single phase silicon stabilized alpha-TCP material. NMR results showed that in all three materials the silicon dopants formed Q(1) structures in which two silicate tetrahedra share an oxygen, creating an oxygen vacancy which compensated the substitution of two silicon for phosphorus. This finding may explain the phase evolution previously found where silicon stabilized alpha-TCP is found at low temperature after sintering.

High-purity isolation of bullfrog hair bundles and subcellular and topological localization of constituent proteins.

The small number of hair cells in auditory and vestibular organs severely impedes the biochemical characterization of the proteins involved in mechano-electrical transduction. By developing an efficient and clean "twist-off" method of hair bundle isolation, and by devising a sensitive, nonradioactive method to detect minute quantities of protein, we have partially overcome this limitation and have extensively classified the proteins of the bundles. To isolate hair bundles, we glue the saccular macula of the bullfrog to a glass coverslip, expose the tissue to a molten agarose solution, and allow the agarose to solidify to a firm gel. By rotating the gel disk with respect to the fixed macula, we isolate the hair bundles by shearing them at their mechanically weak bases. The plasma membranes of at least 80% of the stereocilia reseal. To visualize the proteins of the hair bundle, we covalently label them with biotin, separate them by SDS-PAGE, and transfer them to a charged nylon membrane. We can detect less than 500 fg of protein by probing the membrane with streptavidin-alkaline phosphatase and detecting the chemiluminescent product from the hydrolysis of the substrate 3-(4-methoxyspiro-(1,2-dioxetane-3,2'-tricyclo-[3.3.1. 1(3.7)]decan)-4-yl) phenyl phosphate (AMPPD). These techniques reveal a distinct constellation of proteins in and associated with hair bundles. Several proteins, such as calmodulin, calbindin, actin, tubulin, and fimbrin, have previously been described. A second class of proteins in the preparation appears to be derived from extracellular sources. Finally, several heretofore undescribed bundle proteins are identified and characterized by their membrane topology, subcellular localization, and glycosidase and protease sensitivities.

Replication origins in Xenopus egg extract Are 5-15 kilobases apart and are activated in clusters that fire at different times.

When Xenopus eggs and egg extracts replicate DNA, replication origins are positioned randomly with respect to DNA sequence. However, a completely random distribution of origins would generate some unacceptably large interorigin distances. We have investigated the distribution of replication origins in Xenopus sperm nuclei replicating in Xenopus egg extract. Replicating DNA was labeled with [(3)H]thymidine or bromodeoxyuridine and the geometry of labeled sites on spread DNA was examined. Most origins were spaced 5-15 kb apart. This regular distribution provides an explanation for how complete chromosome replication can be ensured although origins are positioned randomly with respect to DNA sequence. Origins were grouped into small clusters (typically containing 5-10 replicons) that fired at approximately the same time, with different clusters being activated at different times in S phase. This suggests that a temporal program of origin firing similar to that seen in somatic cells also exists in the Xenopus embryo. When the quantity of origin recognition complexes (ORCs) on the chromatin was restricted, the average interorigin distance increased, and the number of origins in each cluster decreased. This suggests that the binding of ORCs to chromatin determines the regular spacing of origins in this system.