The Bacillus subtilis primosomal protein DnaD untwists supercoiled DNA

The essential Bacillus subtilis DnaD and DnaB proteins have been implicated in the initiation of DNA replication. Recently, DNA remodeling activities associated with both proteins were discovered that could provide a link between global or local nucleoid remodeling and initiation of replication. DnaD forms scaffolds and opens up supercoiled plasmids without nicking to form open circular complexes, while DnaB acts as a lateral compaction protein. Here we show that DnaD-mediated opening of supercoiled plasmids is accompanied by significant untwisting of DNA. The net result is the conversion of writhe (Wr) into negative twist (Tw), thus maintaining the linking number (Lk) constant. These changes in supercoiling will reduce the considerable energy required to open up closed circular plectonemic DNA and may be significant in the priming of DNA replication. By comparison, DnaB does not affect significantly the supercoiling of plasmids. Binding of the DnaD C-terminal domain (Cd) to DNA is not sufficient to convert Wr into negative Tw, implying that the formation of scaffolds is essential for duplex untwisting. Overall, our data suggest that the topological effects of the two proteins on supercoiled DNA are different; DnaD opens up, untwists and converts plectonemic DNA to a more paranemic form, whereas DnaB does not affect supercoiling significantly and condenses DNA only via its lateral compaction activity. The significance of these findings in the initiation of DNA replication is discussed.

PREVALENCE AND PREDICTORS OF VITAMIN A DEFICIENCY AMONG INFANTS IN WESTERN KENYA USING A CROSS-SECTIONAL ANALYSIS

Vitamin A (VA) deficiency (VAD) is a major nutritional public health problem among children under-5-years-old in the developing world including Kenya. A community-based cross-sectional survey among 1,630 children (aged 6-23 mos) was undertaken in Western Kenya. A questionnaire was administered to collect demographic, socio-economic and dietary intake information. Prevalence of low retinol-binding protein (RBP) concentrations was assessed using Dried Blood Spot (DBS) methodology. Analysis of RBP was carried out using rapid enzyme immunoassay (EIA) and C-reactive protein (CRP) was carried out using enzyme linked immunosorbent assay (ELISA) to estimate VA and sub-clinical inflammation statuses, respectively. Values were adjusted for influence of inflammation using CRP (CRP >5 mg/L) and population prevalence of VAD (RBP <0.825 μmol/L, biologically equivalent to 0.70 μmol/L retinol) estimated. Anthropometric data gave three indices: stunting, wasting and underweight—all of which took age and sex into consideration. Mean (geometric± SD) concentration of RBP was adequate (1.56±0.79μmol/L) but the inflammation-adjusted mean (±SE) prevalence of VAD was high (20.1±1.1%) in this population. The level of CRP was within normal range (1.06±4.95 mg/L) whilst 18.4±0.9% of the children had subclinical inflammation (CRP>5 mg/L). Intake of VA capsule (VAC) by a child was a predictor of VAD with children who have not taken VA during the past 1 year prior to the survey having a 30% increased risk of VAD (OR (CI): 1.3 (1.1-1.7); p=0.025. Additionally, age of the child was a predictor with older children (18-23 mos) having a 30 % increased risk of VAD (OR (CI): 1.3 (1.1-1.9); p=0.035); the caretaker’s knowledge on VA and nutrition was also a predictor of VAD with children whose caretaker’s had poor knowledge having a 40 % increased risk of VAD (OR (CI): 1.4 (1.0-1.9); p=0.027. A child’s district of residence was also a significant predictor of VAD. Prevalence of VAD in this sample of infants was high. Predictors of VAD included child intake of VAC in the last 1 year before the survey, older children, children whose caretakers had poor VA and nutritional knowledge and a child’s district of residence. There is a need to improve knowledge on nutrition and VA of caretakers; undertake a targeted VAC distribution, particularly in children older than 1 year and above and use a sustainable food-based intervention in the areas with severe VAD.

Biochemical studies of postsynaptic density signaling proteins with a focus on synaptic GTPase activating protein and PDZ domains

Memory storage in the brain involves adjustment of the strength of existing synapses and formation of new neural networks. A key process underlying memory formation is synaptic plasticity, the ability of excitatory synapses to strengthen or weaken their connections in response to patterns of activity between their connected neurons. Synaptic plasticity is governed by the precise pattern of Ca²⁺ influx through postsynaptic N-methyl-D-aspartate-type glutamate receptors (NMDARs), which can lead to the activation of the small GTPases Ras and Rap. Differential activation of Ras and Rap acts to modulate synaptic strength by promoting the insertion or removal of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptors (AMPARs) from the synapse. Synaptic GTPase activating protein (synGAP) regulates AMPAR levels by catalyzing the inactivation of GTP-bound (active) Ras or Rap. synGAP is positioned in close proximity to the cytoplasmic tail regions of the NMDAR through its association with the PDZ domains of PSD-95. SynGAP’s activity is regulated by the prominent postsynaptic protein kinase, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5), a known binding partner of CaMKII. Modulation of synGAP’s activity by phosphorylation may alter the ratio of active Ras to Rap in spines, thus pushing the spine towards the insertion or removal of AMPARs, subsequently strengthening or weakening the synapse. To date, all biochemical studies of the regulation of synGAP activity by protein kinases have utilized impure preparations of membrane bound synGAP. Here we have clarified the effects of phosphorylation of synGAP on its Ras and Rap GAP activities by preparing and utilizing purified, soluble recombinant synGAP, Ras, Rap, CaMKII, CDK5, PLK2, and CaM. Using mass spectrometry, we have confirmed the presence of previously identified CaMKII and CDK5 sites in synGAP, and have identified novel sites of phosphorylation by CaMKII, CDK5, and PLK2. We have shown that the net effect of phosphorylation of synGAP by CaMKII, CDK5, and PLK2 is an increase in its GAP activity toward HRas and Rap1. In contrast, there is no effect on its GAP activity toward Rap2. Additionally, by assaying the GAP activity of phosphomimetic synGAP mutants, we have been able to hypothesize the effects of CDK5 phosphorylation at specific sites in synGAP. In the course of this work, we also found, unexpectedly, that synGAP is itself a Ca²⁺/CaM binding protein. While Ca²⁺/CaM binding does not directly affect synGAP activity, it causes a conformational change in synGAP that increases the rate of its phosphorylation and exposes additional phosphorylation sites that are inaccessible in the absence of Ca²⁺/CaM.

The postsynaptic density (PSD) is an electron-dense region in excitatory postsynaptic neurons that contains a high concentration of glutamate receptors, cytoskeletal proteins, and associated signaling enzymes. Within the PSD, three major classes of scaffolding molecules function to organize signaling enzymes and glutamate receptors. PDZ domains present in the Shank and PSD-95 scaffolds families serve to physically link AMPARs and NMDARs to signaling molecules in the PSD. Because of the specificity and high affinity of PDZ domains for their ligands, I reasoned that these interacting pairs could provide the core components of an affinity chromatography system, including affinity resins, affinity tags, and elution agents. I show that affinity columns containing the PDZ domains of PSD-95 can be used to purify active PDZ domain-binding proteins to very high purity in a single step. Five heterologously expressed neuronal proteins containing endogenous PDZ domain ligands (NMDAR GluN2B subunit Tail, synGAP, neuronal nitric oxide synthase PDZ domain, cysteine rich interactor of PDZ three and cypin) were purified using PDZ domain resin, with synthetic peptides having the sequences of cognate PDZ domain ligands used as elution agents. I also show that conjugation of PDZ domain-related affinity tags to Proteins Of Interest (POIs) that do not contain endogenous PDZ domains or ligands does not alter protein activity and enables purification of the POIs on PDZ domain-related affinity resins.

Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response

Tese de Doutoramento em Ciências Veterinárias na especialidade de Sanidade Animal

Correlates of Vitamin D Status in Healthy Older Adults Living in Miami-Dade and its Association with Non-Skeletal Outcomes: A Cross-Sectional Study

Examining factors that affect vitamin D status in the fast-growing elderly population of Miami-Dade, Florida, is needed. Vitamin D deficiency in older adults has been linked to correlates of disability, including falls and fractures, and cardiovascular disease. The purpose of this study was to determine the proportion of vitamin D insufficient individuals and their relationship with vitamin D insufficiency in older adults (n=97) living in Miami-Dade. We evaluated the association between vitamin D status and 1) dual task physical performance to understand the link between vitamin D and cognition in the context of mobility; and 2) cardiometabolic risk, measured by galvanic skin response, pulse oximetry, and blood pressure to create a composite score based on autonomic nervous system and endothelial function. Participants completed baseline assessments that included serum levels of vitamin D, anthropometrics, body composition, dual task physical performance and cardiometabolic risk. Surveys to evaluate vitamin D intake, sun exposure, physical activity, and depressive symptoms were completed. Spearman’s correlations, independent t-tests, paired t-tests, repeated measures ANOVAs, and multiple logistic and linear regressions were used to examine the relationship of vitamin D insufficiency (25(OH)D /ml) and sufficiency (25(OH)D ≥30 ng/ml) with determinants of vitamin D status, dual task physical performance variables and cardiometabolic risk scores. Although the proportion of vitamin D insufficient individuals was lower when compared to the prevalance of the general United States elderly population, it was still common in healthy community-dwelling older adults living in Miami-Dade County, especially among Hispanics. Factors that affected skin synthesis (ethnicity, and sun exposure), and bioavailability/metabolism (obesity) were significant predictors of vitamin D status. Vitamin D insufficiency was not significantly correlated with worse dual task physical performance; however, cognitive performance was worse in the vitamin D insufficient group. Our results suggest a relationship of vitamin D insufficiency with executive dysfunction, and support an association with cardiometabolic risk using an innovative electro-sensor complex, possibly by modulating autonomic nervous system activity and vascular function, thus affecting cardiac performance.

Postprandial serum endotoxin in healthy humans is modulated by dietary fat in a randomized, controlled, cross-over study

Background: High-fat diets may contribute to metabolic disease via postprandial changes in serum endotoxin and inflammation. It is unclear how dietary fat composition may alter these parameters. We hypothesized that a meal rich in n-3 (ω3) fatty acids would reduce endotoxemia and associated inflammation but a saturated or n-6 (ω6) fatty acid-rich meal would increase postprandial serum endotoxin concentrations and systemic inflammation in healthy adults. Methods: Healthy adults (n = 20; mean age 25 ± 3.2 S.D. years) were enrolled in this single-blind, randomized, cross-over study. Participants were randomized to treatment and reported to the laboratory, after an overnight fast, on four occasions separated by at least one week. Participants were blinded to treatment meal and consumed one of four isoenergetic meals that provided: 1) 20 % fat (control; olive oil) or 35 % fat provided from 2) n-3 (ω3) (DHA = 500 mg; fish oil); 3) n-6 (ω6) (7.4 g; grapeseed oil) or 4) saturated fat (16 g; coconut oil). Baseline and postprandial blood samples were collected. Primary outcome was defined as the effect of treatment meal on postprandial endotoxemia. Serum was analyzed for metabolites, inflammatory markers, and endotoxin. Data from all 20 participants were analyzed using repeated-measures ANCOVA. Results: Participant serum endotoxin concentration was increased during the postprandial period after the consumption of the saturated fat meal but decreased after the n-3 meal (p < 0.05). The n-6 meal did not effect a different outcome in participant postprandial serum endotoxin concentration from that of the control meal (p > 0.05). There was no treatment meal effect on participant postprandial serum biomarkers of inflammation. Postprandial serum triacylglycerols were significantly elevated following the n-6 meal compared to the n-3 meal. Non-esterified fatty acids were significantly increased after consumption of the saturated fat meal compared to other treatment meals. Conclusions: Meal fatty acid composition modulates postprandial serum endotoxin concentration in healthy adults. However, postprandial endotoxin was not associated with systemic inflammation in vivo. Trial registration: This study was retrospectively registered at clinicaltrials.gov as NCT02521779 on July 28, 2015.

EU-North Africa relations in cross-border law enforcement: new legal challenges for the EU in the post-Lisbon and post-Stockholm era

The Stockholm Programme, allied to the Lisbon Treaty, heralds a new era of development of the EU provisions on cross-border law enforcement. The focus is shifting from the ongoing internal EU developments to the external relations of the EU. Many North African countries have had long legal relationships with the EU through the Euro-Mediterranean Partnerships. A number of these partnership agreements make express references, at the political level, to the development of cross-border law enforcement provision, as is the case of Morocco and Algeria with regard to drug trafficking and manufacture, or the lengthy references by Egypt to many of the crimes of interest to the EU’s own law enforcement legal framework. Algeria is currently focusing on modernising their own police forces, with both Algeria and Tunisia, reforming their criminal judicial frameworks. Another key player, Libya, currently has no legal agreements with the EU, and at least until the recent conflict, maintained an observer status in the Euro-Mediterranean process. At a practitioner level, the European Police College (CEPOL) is currently involved in the Euromed Police II programme. Clearly momentum is developing, both within the EU and from a number of Euro-Med North African countries to develop closer law enforcement co-operation. This may well develop further with the recent changes in governments of a number of North African countries. The EU approach in the Police and Judicial Cooperation in Criminal Matters (PJCCM) policy area is to develop a legal framework upon which EU cross-border law enforcement will be based. The current EU cross-border law enforcement framework is the product of many years of multi-level negotiations. Challenges will arise as new countries from different legal and policing traditions will attempt to engage with already highly detailed legal and practice frameworks. The shared European legal traditions will not necessarily be reflected in the North African countries. This chapter critically analyses, from an EU legal perspective the problems and issues that will be encountered as the EU’s North African partner countries attempt to articulate into the existing, and still developing EU cross-border law enforcement framework.

Spatio-temporal patterns of herbage availability and livestock movements: A cross-border analysis in the Chinese-Mongolian Altay

Due to increasing population and the recent implementation of policies to intensify the use of land and water resources, the transhumant pastoral systems in the Chinese-Mongolian Altay-Dzungarian region are rapidly changing, leading to modifications of herd size, herd composition and spatial distribution of livestock grazing. This may have major consequences for the supply and quality of rangeland biomass. Despite similar topographic settings, the socio-political framework for Chinese and Mongolian pastoralists differs significantly, leading to differences in rangeland utilization. To substantiate these claims, the long-distance transhumance routes, frequency of pasture changes, daily grazing itineraries and size of pastures were recorded by means of GPS tracking of cattle and goats on 1,535 (China) and 1,396 (Mongolia) observation days. The status quo of the main seasonal pastures was captured by measuring the herbage offer and its nutritive value in 869 sampling spots. In the Altay-Dzungarian region, small ruminant herds covered up to 412 km (Mongolia) and grazed on up to nine pastures per year (China). In Mongolia, the herds’ average duration of stay at an individual pasture was longer than in China, particularly in spring and autumn. Herbage allowance at the onset of a grazing period (kg dry matter per sheep unit and day) ranged from 34/17 to 91/95 (China/Mongolia). Comparing crude protein and phosphorous concentrations of herbage, in China, the highest concentrations were measured for spring and summer pastures, whereas in Mongolia, the highest concentrations were determined for autumn and winter pastures. Based on our data, we conclude that regulation of animal numbers and access to pastures seemingly maintained pasture productivity in China, especially at high altitudes. However, this policy may prohibit flexible adaptation to sudden environmental constraints. In contrast, high stocking densities and grazing of pastures before flowering of herbaceous plants negatively affected rangeland productivity in Mongolia, especially for spring and summer pastures.

tRNA anticodon loop modifications ensure protein homeostasis and cell morphogenesis in yeast

Using budding yeast, we investigated a negative interaction network among genes for tRNA modifications previously implicated in anticodon-codon interaction: 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U34: ELP3, URM1), pseudouridine (Ψ38/39: DEG1) and cyclic N6-threonyl-carbamoyl-adenosine (ct6A37: TCD1). In line with functional cross talk between these modifications, we find that combined removal of either ct6A37 or Ψ38/39 and mcm5U34 or s2U34 results in morphologically altered cells with synthetic growth defects. Phenotypic suppression by tRNA overexpression suggests that these defects are caused by malfunction of tRNALysUUU or tRNAGlnUUG, respectively. Indeed, mRNA translation and synthesis of the Gln-rich prion Rnq1 are severely impaired in the absence of Ψ38/39 and mcm5U34 or s2U34, and this defect can be rescued by overexpression of tRNAGlnUUG. Surprisingly, we find that combined modification defects in the anticodon loops of different tRNAs induce similar cell polarity- and nuclear segregation defects that are accompanied by increased aggregation of cellular proteins. Since conditional expression of an artificial aggregation-prone protein triggered similar cytological aberrancies, protein aggregation is likely responsible for loss of morphogenesis and cytokinesis control in mutants with inappropriate tRNA anticodon loop modifications.

Data assimilation for advanced cross-scale unstructured-grid ocean modeling.

The coastal ocean is a complex environment with extremely dynamic processes that require a high-resolution and cross-scale modeling approach in which all hydrodynamic fields and scales are considered integral parts of the overall system. In the last decade, unstructured-grid models have been used to advance in seamless modeling between scales. On the other hand, the data assimilation methodologies to improve the unstructured-grid models in the coastal seas have been developed only recently and need significant advancements. Here, we link the unstructured-grid ocean modeling to the variational data assimilation methods. In particular, we show results from the modeling system SANIFS based on SHYFEM fully-baroclinic unstructured-grid model interfaced with OceanVar, a state-of-art variational data assimilation scheme adopted for several systems based on a structured grid. OceanVar implements a 3DVar DA scheme. The combination of three linear operators models the background error covariance matrix. The vertical part is represented using multivariate EOFs for temperature, salinity, and sea level anomaly. The horizontal part is assumed to be Gaussian isotropic and is modeled using a first-order recursive filter algorithm designed for structured and regular grids. Here we introduced a novel recursive filter algorithm for unstructured grids. A local hydrostatic adjustment scheme models the rapidly evolving part of the background error covariance. We designed two data assimilation experiments using SANIFS implementation interfaced with OceanVar over the period 2017-2018, one with only temperature and salinity assimilation by Argo profiles and the second also including sea level anomaly. The results showed a successful implementation of the approach and the added value of the assimilation for the active tracer fields. While looking at the broad basin, no significant improvements are highlighted for the sea level, requiring future investigations. Furthermore, a Machine Learning methodology based on an LSTM network has been used to predict the model SST increments.

Computational design of a new type of sandwich cross-section for a defined solution of a tree-like column produced by wire-and-arc additive manufacturing

When it comes to designing a structure, architects and engineers want to join forces in order to create and build the most beautiful and efficient building. From finding new shapes and forms to optimizing the stability and the resistance, there is a constant link to be made between both professions. In architecture, there has always been a particular interest in creating new shapes and types of a structure inspired by many different fields, one of them being nature itself. In engineering, the selection of optimum has always dictated the way of thinking and designing structures. This mindset led through studies to the current best practices in construction. However, both disciplines were limited by the traditional manufacturing constraints at a certain point. Over the last decades, much progress was made from a technological point of view, allowing to go beyond today's manufacturing constraints. With the emergence of Wire-and-Arc Additive Manufacturing (WAAM) combined with Algorithmic-Aided Design (AAD), architects and engineers are offered new opportunities to merge architectural beauty and structural efficiency. Both technologies allow for exploring and building unusual and complex structural shapes in addition to a reduction of costs and environmental impacts. Through this study, the author wants to make use of previously mentioned technologies and assess their potential, first to design an aesthetically appreciated tree-like column with the idea of secondly proposing a new type of standardized and optimized sandwich cross-section to the construction industry. Parametric algorithms to model the dendriform column and the new sandwich cross-section are developed and presented in detail. A catalog draft of the latter and methods to establish it are then proposed and discussed. Finally, the buckling behavior of this latter is assessed considering standard steel and WAAM material properties.

Investigating perceptual multisensory impairments in Autism through a neural network: a possible neural implementation for the shift from cross-modal competition to facilitation

Resolution of multisensory deficits has been observed in teenagers with Autism Spectrum Disorders (ASD) for complex, social speech stimuli; this resolution extends to more basic multisensory processing, involving low-level stimuli. In particular, a delayed transition of multisensory integration (MSI) from a default state of competition to one of facilitation has been observed in ASD children. In other terms, the complete maturation of MSI is achieved later in ASD. In the present study a neuro-computational model is used to reproduce some patterns of behavior observed experimentally, modeling a bisensory reaction time task, in which auditory and visual stimuli are presented in random sequence alone (A or V) or together (AV). The model explains how the default competitive state can be implemented via mutual inhibition between primary sensory areas, and how the shift toward the classical multisensory facilitation, observed in adults, is the result of inhibitory cross-modal connections becoming excitatory during the development. Model results are consistent with a stronger cross-modal inhibition in ASD children, compared to normotypical (NT) ones, suggesting that the transition toward a cooperative interaction between sensory modalities takes longer to occur. Interestingly, the model also predicts the difference between unisensory switch trials (in which sensory modality switches) and unisensory repeat trials (in which sensory modality repeats). This is due to an inhibitory mechanism, characterized by a slow dynamics, driven by the preceding stimulus and inhibiting the processing of the incoming one, when of the opposite sensory modality. These findings link the cognitive framework delineated by the empirical results to a plausible neural implementation.

Lung Age Is Related To Carotid Structural Alterations In Hypertensive Subjects.

Hypertensive patients exhibit higher cardiovascular risk and reduced lung function compared with the general population. Whether this association stems from the coexistence of two highly prevalent diseases or from direct or indirect links of pathophysiological mechanisms is presently unclear. This study investigated the association between lung function and carotid features in non-smoking hypertensive subjects with supposed normal lung function. Hypertensive patients (n = 67) were cross-sectionally evaluated by clinical, hemodynamic, laboratory, and carotid ultrasound analysis. Forced vital capacity, forced expired volume in 1 second and in 6 seconds, and lung age were estimated by spirometry. Subjects with ventilatory abnormalities according to current guidelines were excluded. Regression analysis adjusted for age and prior smoking history showed that lung age and the percentage of predicted spirometric parameters associated with common carotid intima-media thickness, diameter, and stiffness. Further analyses, adjusted for additional potential confounders, revealed that lung age was the spirometric parameter exhibiting the most significant regression coefficients with carotid features. Conversely, plasma C-reactive protein and matrix-metalloproteinases-2/9 levels did not influence this relationship. The present findings point toward lung age as a potential marker of vascular remodeling and indicate that lung and vascular remodeling might share common pathophysiological mechanisms in hypertensive subjects.

Taurine Supplementation Increases K(atp) Channel Protein Content, Improving Ca2+ Handling And Insulin Secretion In Islets From Malnourished Mice Fed On A High-fat Diet.

Pancreatic β-cells are highly sensitive to suboptimal or excess nutrients, as occurs in protein-malnutrition and obesity. Taurine (Tau) improves insulin secretion in response to nutrients and depolarizing agents. Here, we assessed the expression and function of Cav and KATP channels in islets from malnourished mice fed on a high-fat diet (HFD) and supplemented with Tau. Weaned mice received a normal (C) or a low-protein diet (R) for 6 weeks. Half of each group were fed a HFD for 8 weeks without (CH, RH) or with 5% Tau since weaning (CHT, RHT). Isolated islets from R mice showed lower insulin release with glucose and depolarizing stimuli. In CH islets, insulin secretion was increased and this was associated with enhanced KATP inhibition and Cav activity. RH islets secreted less insulin at high K(+) concentration and showed enhanced KATP activity. Tau supplementation normalized K(+)-induced secretion and enhanced glucose-induced Ca(2+) influx in RHT islets. R islets presented lower Ca(2+) influx in response to tolbutamide, and higher protein content and activity of the Kir6.2 subunit of the KATP. Tau increased the protein content of the α1.2 subunit of the Cav channels and the SNARE proteins SNAP-25 and Synt-1 in CHT islets, whereas in RHT, Kir6.2 and Synt-1 proteins were increased. In conclusion, impaired islet function in R islets is related to higher content and activity of the KATP channels. Tau treatment enhanced RHT islet secretory capacity by improving the protein expression and inhibition of the KATP channels and enhancing Synt-1 islet content.

Delays In Receiving Obstetric Care And Poor Maternal Outcomes: Results From A National Multicentre Cross-sectional Study.

The vast majority of maternal deaths in low-and middle-income countries are preventable. Delay in obtaining access to appropriate health care is a fairly common problem which can be improved. The objective of this study was to explore the association between delay in providing obstetric health care and severe maternal morbidity/death. This was a multicentre cross-sectional study, involving 27 referral obstetric facilities in all Brazilian regions between 2009 and 2010. All women admitted to the hospital with a pregnancy-related cause were screened, searching for potentially life-threatening conditions (PLTC), maternal death (MD) and maternal near-miss (MNM) cases, according to the WHO criteria. Data on delays were collected by medical chart review and interview with the medical staff. The prevalence of the three different types of delays was estimated according to the level of care and outcome of the complication. For factors associated with any delay, the PR and 95%CI controlled for cluster design were estimated. A total of 82,144 live births were screened, with 9,555 PLTC, MNM or MD cases prospectively identified. Overall, any type of delay was observed in 53.8% of cases; delay related to user factors was observed in 10.2%, 34.6% of delays were related to health service accessibility and 25.7% were related to quality of medical care. The occurrence of any delay was associated with increasing severity of maternal outcome: 52% in PLTC, 68.4% in MNM and 84.1% in MD. Although this was not a population-based study and the results could not be generalized, there was a very clear and significant association between frequency of delay and severity of outcome, suggesting that timely and proper management are related to survival.