Impact of Cognitive Intervention on Neurocognitive Development of Schoolchildren Exposed to Lead in a Semi-Urban Community in Mexico.

Lead exposure is a severe public health issue that can adversely affect children's neurocognitive development. A semi-urban community in Mexico has been exposed to lead from food cooked in glazed clay pots. A cognitive intervention was conducted from 2015 to 2016 to minimize this negative impact. This intervention aimed to improve the neurocognitive development of the affected children. METHODS: A quasi-experimental study with a control group was conducted in children aged 7 to 12 years from 2 communities in Morelos, Mexico. Blood lead levels were determined, and the neurocognitive function was assessed pre- and postintervention with the Wechsler Intelligence Scale for Children and Children's Auditory Verbal Learning Test-2. A cognitive intervention was conducted at the school. The difference-in-differences method adjusted for variables known as priori and evaluated the impact of cognitive intervention. RESULTS: The differences-in-differences models indicated a significant average increase in scores on the Verbal Comprehension Index (9.58 points), Processing Speed Index (5.33 points), intelligence quotient (5.63 points) level of learning (7.66 points), interference trial (10.12 points), immediate memory span (7.98 points), and recognition accuracy (1.18 points) subtests after the cognitive intervention. CONCLUSION: The results suggest that cognitive intervention improves neurocognitive development in schoolchildren exposed to Pb.

Repurposing of rabeprazole as an anti-Trypanosoma cruzi drug that targets cellular triosephosphate isomerase.

Trypanosoma cruzi is the causative agent of American trypanosomiasis, which mainly affects populations in Latin America. Benznidazole is used to control the disease, with severe effects in patients receiving this chemotherapy. Previous studies have demonstrated the inhibition of triosephosphate isomerase from T. cruzi, but cellular enzyme inhibition has yet to be established. This study demonstrates that rabeprazole inhibits both cell viability and triosephosphate isomerase activity in T. cruzi epimastigotes. Our results show that rabeprazole has an IC50 of 0.4 µM, which is 14.5 times more effective than benznidazole. Additionally, we observed increased levels of methyl-glyoxal and advanced glycation end products after the inhibition of cellular triosephosphate isomerase by rabeprazole. Finally, we demonstrate that the inactivation mechanisms of rabeprazole on triosephosphate isomerase of T. cruzi can be achieved through the derivatization of three of its four cysteine residues. These results indicate that rabeprazole is a promising candidate against American trypanosomiasis.

Foreign multinationals affiliates and countries' carbon upstreamness. How could these firms support the fulfilment of emissions reduction targets?

Climate emergency requires urgent actions to reduce carbon emissions. In this paper we calculate the countries' carbon upstreamness and evaluate its linkage to the presence of foreign MNE affiliates, by using a multiregional input-output model with firm heterogeneity. We find a mismatch between carbon upstreamness, emissions reduction targets and income per capita between countries. OECD countries, which are located in the final stages of carbon production, have lower carbon intensity than the world average and have committed strongly to reducing their total emissions. On the contrary, non-OECD countries, which are located mainly in the initial stages of carbon production, maintain higher carbon intensity than the world average and they are less ambitiously committed, as they have lower per capita income. In that context, multinational enterprises (MNEs) could play a key role in supporting the fulfilment of emission reduction targets in host countries, so we propose a simulation to evaluate this role. Specifically, if the MNE affiliates adopt the Intended Nationally Determined Contributions (INDC) set by the controlling country regardless of where they are located, the emissions of MNEs would be reduced by 15.6% (395,864 KtCO2), 4% more than they would be reduced under current emission reduction targets in 2016. However, if MNEs apply the more ambitious INDC, regardless of origin or destination, the emissions would be reduced by 18% (455,910 KtCO2), 7% more than scenario 1 and 1.7% of global emissions in 2016.

Improved yield, stability, and cleavage reaction of a novel tobacco etch virus protease mutant.

The protease catalytic subunit of the nuclear inclusion protein A from tobacco etch virus (TEVp) is widely used to remove tags and fusion proteins from recombinant proteins. Some intrinsic drawbacks to its recombinant production have been studied for many years, such as low solubility, auto-proteolysis, and instability. Some point mutations have been incorporated in the amino acid protease sequence to improve its production. Here, a comprehensive review of each mutation reported so far has been made to incorporate them into a mutant called TEVp7M with a total of seven changes. This mutant with a His7tag at N-terminus was produced with remarkable purification yields (55 mg/L of culture) from the soluble fraction in a single step affinity purification. The stability of His7-TEVp7M was analyzed and compared with the single mutant TEVp S219V, making evident that His7-TEVp7M shows very constant thermal stability against pH variation, whereas TEVp S219V is highly sensitive to this change. The cleavage reaction was optimized by determining the amount of protease that could cleave a 100-fold excess substrate in the shortest possible time at 30 °C. Under these conditions, His7-TEVp7M was able to cleave His-tag in the buffers commonly used for affinity purification. Finally, a structural analysis of the mutations showed that four of them increased the polarity of the residues involved and, consequently, showed increased solubility of TEVp and fewer hydrophobic regions exposed to the solvent. Taken together, the seven changes studied in this work improved stability, solubility, and activity of TEVp producing enough protease to digest large amounts of tags or fusion proteins. KEY POINTS: • Production of excellent yields of a TEVp (TEVp7M) by incorporation of seven changes. • His-tag removal in an excess substrate in the common buffers used for purification. • Incorporated mutations improve polarity, stability, and activity of TEVp7M.

Epsilon-aminocaproic acid prevents high glucose and insulin induced-invasiveness in MDA-MB-231 breast cancer cells, modulating the plasminogen activator system.

Obesity and type II diabetes mellitus have contributed to the increase of breast cancer incidence worldwide. High glucose concentration promotes the proliferation of metastatic cells, favoring the activation of the plasminogen/plasmin system, thus contributing to tumor progression. The efficient formation of plasmin is dependent on the binding of plasminogen to the cell surface. We studied the effect of ε-aminocaproic acid (EACA), an inhibitor of the binding of plasminogen to cell surface, on proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and plasminogen activation system, in metastatic MDA-MB-231 breast cancer cells grown in a high glucose microenvironment and treated with insulin. MDA-MB-231 cells were treated with EACA 12.5 mmol/L under high glucose 30 mmol/L (HG) and high glucose and insulin 80 nmol/L (HG-I) conditions, evaluating: cell population growth, % of viability, migratory, and invasive abilities, as well as the expression of uPA, its receptor (uPAR), and its inhibitor (PAI-1), by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, MMP-2 and MMP-9 mRNAs were evaluated by RT-PCR. Markers of EMT were evaluated by Western blot. Additionally, the presence of active uPA was studied by gel zymography, using casein-plasminogen as substrates. EACA prevented the increase in cell population, migration and invasion induced by HG and insulin, which was associated with the inhibition of EMT and the attenuation of HG- and insulin-dependent expression of uPA, uPAR, PAI-1, MMP-2, MMP-9, α-enolase (ENO A), and HCAM. The interaction of plasminogen to the cell surface and plasmin formation are mediators of the prometastasic action of hyperglycemia and insulin, potentially, EACA can be employed in the prevention and as adjuvant treatment of breast tumorigenesis promoted by hyperglycemia and insulin.

Dissection of a major QTL qhir1 conferring maternal haploid induction ability in maize.

KEY MESSAGE: Among the qhir11 and qhir12 sub-regions of a major QTL qhir1, only qhir11 has significant effect on maternal haploid induction, segregation distortion and kernel abortion. In vivo haploid induction in maize can be triggered in high frequencies by pollination with special genetic stocks called haploid inducers. Several genetic studies with segregating populations from non-inducer x inducer crosses identified a major QTL, qhir1, on chromosome 1.04 contributing to in vivo haploid induction. A recent Genome Wide Association Study using 51 inducers and 1482 non-inducers also identified two sub-regions within the qhir1 QTL region, named qhir11 and qhir12; qhir12 was proposed to be mandatory for haploid induction because the haplotype of qhir11 was also present in some non-inducers and putative candidate genes coding for DNA and amino acid binding proteins were identified in the qhir12 region. To characterize the effects of each sub-region of qhir1 on haploid induction rate, F2 recombinants segregating for one of the sub-regions and fixed for the other were identified in a cross between CML269 (non-inducer) and a tropicalized haploid inducer TAIL8. To quantify the haploid induction effects of qhir11 and qhir12, selfed progenies of recombinants between these sub-regions were genotyped. F3 plants homozygous for qhir11 and/or qhir12 were identified, and crossed to a ligueless tester to determine their haploid induction rates. The study revealed that only the qhir11 sub-region has a significant effect on haploid induction ability, besides causing significant segregation distortion and kernel abortion, traits that are strongly associated with maternal haploid induction. The results presented in this study can guide fine mapping efforts of qhir1 and in developing new inducers efficiently using marker assisted selection.

Glazed clay pottery and lead exposure in Mexico: Current experimental evidence.

OBJECTIVES: Lead exposure remains a significant environmental problem; lead is neurotoxic, especially in developing humans. In Mexico, lead in human blood is still a concern. Historically, much of the lead exposure is attributed to the use of handcrafted clay pottery for cooking, storing and serving food. However, experimental cause-and-effect demonstration is lacking. The present study explores this issue with a prospective experimental approach. METHODS: We used handcrafted clay containers to prepare and store lemonade, which was supplied as drinking water to pregnant rats throughout the gestational period. RESULTS AND DISCUSSION: We found that clay pots, jars, and mugs leached on average 200 µg/l lead, and exposure to the lemonade resulted in 2.5 µg/dl of lead in the pregnant rats' blood. Neonates also showed increased lead content in the hippocampus and cerebellum. Caspase-3 activity was found to be statistically increased in the hippocampus in prenatally exposed neonates, suggesting increased apoptosis in that brain region. Glazed ceramics are still an important source of lead exposure in Mexico, and our results confirm that pregnancy is a vulnerable period for brain development.

Financial crisis, virtual carbon in global value chains, and the importance of linkage effects. The Spain-china case.

Trade has a disproportionate environmental impact, while the international fragmentation of production promotes different patterns of intermediate inputs and final goods. Therefore, we split up the balance of domestic embodied emissions in trade (BDEET) to assess it. We find that Spain has a significant emissions deficit with China between 2005 and 2011. The Global Financial Crisis of 2008 reduced Spanish imports of pollution-intensive inputs from China and slightly improved the BDEET. China primarily exports indirect virtual carbon, representing 86% of the total, especially from Production of electricity, gas, and water sector. These linkages effects in China indicate that post-Kyoto agreements must focus not only on traded goods but also on the environmental efficiency of all domestic production chains. The methodology proposed allows us to identify the agents responsible for this trade in both Spain and China, namely the sectors importing intermediate inputs (Construction and Transport equipment) and industries and consumers importing final goods (Textiles, Other manufactures, Computers, and Machinery). The relevant sectors uncertainties found when we compare the results for BDEET and emissions embodied in bilateral trade (BEET) lead us to recommend the former methodology to evaluate the implications of environmental and energy policy for different industries and agents.

Pancreatic ß­cell apoptosis in type 2 diabetes is related to post­translational modifications of p53 (Review).

Pancreatic ß­cells are the only cells that synthesize insulin to regulate blood glucose levels. Various conditions can affect the mass of pancreatic ß­cells and decrease insulin levels. Diabetes mellitus is a disease characterized by insulin resistance and chronic hyperglycemia, mainly due to the loss of pancreatic ß­cells caused by an increase in the rate of apoptosis. Additionally, hyperglycemia has a toxic effect on ß­cells. Although the precise mechanism of glucotoxicity is not fully understood, several mechanisms have been proposed. The most prominent changes are increases in reactive oxygen species, the loss of mitochondrial membrane potential and the activation of the intrinsic pathway of apoptosis due to p53. The present review analyzed the location of p53 in the cytoplasm, mitochondria and nucleus in terms of post­translational modifications, including phosphorylation, O­GlcNAcylation and poly­ADP­ribosylation, under hyperglycemic conditions. These modifications protect p53 from degradation by the proteasome and, in turn, enable it to regulate the intrinsic pathway of apoptosis through the regulation of anti­apoptotic and pro­apoptotic elements. Degradation of p53 occurs in the proteasome and depends on its ubiquitination by Mdm2. Understanding the mechanisms that activate the death of pancreatic ß­cells will allow the proposal of treatment alternatives to prevent the decrease in pancreatic ß­cells.

Preliminary exploration of the expression of acetylcholinesterase in normal human T lymphocytes and leukemic Jurkat T cells.

The classic enzymatic function of acetylcholinesterase (AChE) is the hydrolysis of acetylcholine (ACh) in the neuronal synapse. However, AChE is also present in nonneuronal cells such as lymphocytes. Various studies have proposed the participation of AChE in the development of cancer. The ACHE gene produces three mRNAs (T, H and R). AChE-T encodes amphiphilic monomers, dimers, tetramers (G1 A, G2 A and G4 A) and hydrophilic tetramers (G4 H). AChE-H encodes amphiphilic monomers and dimers (G1 A and G2 A). AChE-R encodes a hydrophilic monomer (G1 H). The present study considered the differences in the mRNA expression (T, H and R) and protein levels of AChE, as well as the molecular forms of AChE, the glycosylation pattern and the enzymatic activity of AChE present in normal T lymphocytes and leukemic Jurkat E6-1 cells. The results revealed that AChE enzymatic activity was higher in normal T lymphocytes than in Jurkat cells. Normal T cells expressed AChE-H transcripts, whereas Jurkat cells expressed AChE-H and AChE-T. The molecular forms identified in normal T cells were G2 A (5.2 S) and G1 A (3.5 S), whereas those in Jurkat cells were G2 A (5.2 S), G1 A (3.5 S) and G4 H (10.6S). AChE in Jurkat cells showed altered posttranslational maturation since a decrease in the incorporation of galactose and sialic acid into its structure was observed. In conclusion, the content and composition of AChE were altered in Jurkat cells compared with those in normal T lymphocytes. The present study opened new avenues for exploring the development of novel therapeutic strategies against T-cell leukemia and for identifying potential molecular targets for the early detection of this type of cancer.

Effectiveness of R1-nj Anthocyanin Marker in the Identification of In Vivo Induced Maize Haploid Embryos.

Doubled haploid (DH) technology has become integral to maize breeding programs to expedite inbred line development and increase the efficiency of breeding operations. Unlike many other plant species that use in vitro methods, DH production in maize uses a relatively simple and efficient in vivo haploid induction method. However, it takes two complete crop cycles for DH line generation, one for haploid induction and the other one for chromosome doubling and seed production. Rescuing in vivo induced haploid embryos has the potential to reduce the time for DH line development and improve the efficiency of DH line production. However, the identification of a few haploid embryos (~10%) resulting from an induction cross from the rest of the diploid embryos is a challenge. In this study, we demonstrated that an anthocyanin marker, namely R1-nj, which is integrated into most haploid inducers, can aid in distinguishing haploid and diploid embryos. Further, we tested conditions that enhance R1-nj anthocyanin marker expression in embryos and found that light and sucrose enhance anthocyanin expression, while phosphorous deprivation in the media had no affect. Validating the use of the R1-nj marker for haploid and diploid embryo identification using a gold standard classification based on visual differences among haploids and diploids for characteristics such as seedling vigor, erectness of leaves, tassel fertility, etc., indicated that the R1-nj marker could lead to significantly high false positives, necessitating the use of additional markers for increased accuracy and reliability of haploid embryo identification.

Energy-socio-economic-environmental modelling for the EU energy and post-COVID-19 transitions.

Relevant energy questions have arisen because of the COVID-19 pandemic. The pandemic shock leads to emissions' reductions consistent with the rates of decrease required to achieve the Paris Agreement goals. Those unforeseen drastic reductions in emissions are temporary as long as they do not involve structural changes. However, the COVID-19 consequences and the subsequent policy response will affect the economy for decades. Focusing on the EU, this discussion article argues how recovery plans are an opportunity to deepen the way towards a low-carbon economy, improving at the same time employment, health, and equity and the role of modelling tools. Long-term alignment with the low-carbon path and the development of a resilient transition towards renewable sources should guide instruments and policies, conditioning aid to energy-intensive sectors such as transport, tourism, and the automotive industry. However, the potential dangers of short-termism and carbon leakage persist. The current energy-socio-economic-environmental modelling tools are precious to widen the scope and deal with these complex problems. The scientific community has to assess disparate, non-equilibrium, and non-ordinary scenarios, such as sectors and countries lockdowns, drastic changes in consumption patterns, significant investments in renewable energies, and disruptive technologies and incorporate uncertainty analysis. All these instruments will evaluate the cost-effectiveness of decarbonization options and potential consequences on employment, income distribution, and vulnerability.

Deamidated Human Triosephosphate Isomerase is a Promising Druggable Target.

Therapeutic strategies for the treatment of any severe disease are based on the discovery and validation of druggable targets. The human genome encodes only 600-1500 targets for small-molecule drugs, but posttranslational modifications lead to a considerably larger druggable proteome. The spontaneous conversion of asparagine (Asn) residues to aspartic acid or isoaspartic acid is a frequent modification in proteins as part of the process called deamidation. Triosephosphate isomerase (TIM) is a glycolytic enzyme whose deamidation has been thoroughly studied, but the prospects of exploiting this phenomenon for drug design remain poorly understood. The purpose of this study is to demonstrate the properties of deamidated human TIM (HsTIM) as a selective molecular target. Using in silico prediction, in vitro analyses, and a bacterial model lacking the tim gene, this study analyzed the structural and functional differences between deamidated and nondeamidated HsTIM, which account for the efficacy of this protein as a druggable target. The highly increased permeability and loss of noncovalent interactions of deamidated TIM were found to play a central role in the process of selective enzyme inactivation and methylglyoxal production. This study elucidates the properties of deamidated HsTIM regarding its selective inhibition by thiol-reactive drugs and how these drugs can contribute to the development of cell-specific therapeutic strategies for a variety of diseases, such as COVID-19 and cancer.

[Capillary blood gas test usefulness to evaluate gas exchange with 21% and 100% of oxygen inspired fractions in subjects with stable cardiopulmonary disease at 2,240 meters above sea level]. / Utilidad de la gasometría capilar para evaluar el intercambio gaseoso con FiO2 al 21% y al 100% en el sujeto con enfermedad cardiopulmonar estable a 2,240 metros sobre el nivel del mar.

OBJECTIVE: Capillary blood gas test has had ample use in the infantile population. In the adult population, the information is limited and controversial. The agreement between capillary-arterial gases seems to parallel the pH and the carbon dioxide pressure in different studied populations. In order to know the degree of agreement between these gases, we evaluate them at breathing room air and at 100% of oxygen fractions at 2,240 meters above sea level. METHODS: We obtained capillary-arterial blood gases simultaneously from subjects with stable cardiopulmonary disease in both conditions of inspired oxygen. Demographic, hemodynamic, diagnostic, and laboratory variables were gathered. STATISTICAL ANALYSIS: agreement was analyzed with the intraclass correlation coefficient and the Bland-Altman procedure. RESULTS: We studied 101 subjects, 48 men and 53 women, whose respective ages were 55 +/- 16 and 56 +/- 16. Mean systemic arterial pressure was 94.96 +/- 10.57 mmHg. Hemoglobin was 15.94 +/- 2.48 g/dl. The agreement between the variables with the inspired oxygen fractions, 21%, 100%, and the mean difference in parenthesis was respectively: potential hydrogen, 0.94 (0.0091), 0.94 (0.0039); oxygen pressure, 0.90 (2.94), 0.84 (74.99); carbon dioxide pressure, 0.97 (0.079), 0.97 (0.179); bicarbonate, 0.93 (-0.067), 0,96 (0.262); total dissolved carbon dioxide, 0.94 (-0.142), 0.93 (0.161); base excess: 0.94, (-0.125), 0.92 (0.235); oxygen saturation, 0.98 (0.764), 0.97(0.202). CONCLUSIONS: Capillary blood gas test could be a useful alternative to the arterial one, nevertheless, it is limited by its low agreement with the oxygen pressure in both oxygen inspired fractions.

The carbon footprint of the U.S. multinationals' foreign affiliates.

Multinational enterprises (MNE) need to be a part of the solution in the fight against climate change, as claimed by investors and consumers, reducing emissions within their operations and supply chains. This paper measures the carbon footprint of U.S. MNE foreign affiliates (US-MNE) operating beyond the U.S. borders. Using a multiregional input-output model and information about US-MNE activities, the US-MNE carbon footprint ranks US-MNE as the 12th top emitter of the world. In relative terms, one dollar of value added generated by US-MNE affiliates operating abroad requires higher emissions than the domestic average and the ratio increases when only developing host countries are considered. Only 8% of total carbon footprint returns to the U.S. as virtual carbon embodied in the U.S. final consumption. Potential technology transfers between the U.S. parent company and affiliates to reduce US-MNE carbon footprint have been performed to evaluate potential rippled effects of mitigation actions.

On the molecular and cellular effects of omeprazole to further support its effectiveness as an antigiardial drug.

Research on Giardia lamblia has accumulated large information about its molecular cell biology and infection biology. However, giardiasis is still one of the commonest parasitic diarrheal diseases affecting humans. Additionally, an alarming increase in cases refractory to conventional treatment has been reported in low prevalence settings. Consequently, efforts directed toward supporting the efficient use of alternative drugs, and the study of their molecular targets appears promising. Repurposing of proton pump inhibitors is effective in vitro against the parasite and the toxic activity is associated with the inhibition of the G. lamblia triosephosphate isomerase (GlTIM) via the formation of covalent adducts with cysteine residue at position 222. Herein, we evaluate the effectiveness of omeprazole in vitro and in situ on GlTIM mutants lacking the most superficial cysteines. We studied the influence on the glycolysis of Giardia trophozoites treated with omeprazole and characterized, for the first time, the morphological effect caused by this drug on the parasite. Our results support the effectiveness of omeprazole against GlTIM despite of the possibility to mutate the druggable amino acid targets as an adaptive response. Also, we further characterized the effect of omeprazole on trophozoites and discuss the possible mechanism involved in its antigiardial effect.

Marker-Assisted Breeding of Improved Maternal Haploid Inducers in Maize for the Tropical/Subtropical Regions.

For efficient production of doubled haploid (DH) lines in maize, maternal haploid inducer lines with high haploid induction rate (HIR) and good adaptation to the target environments is an important requirement. In this study, we present second-generation Tropically Adapted Inducer Lines (2GTAILs), developed using marker assisted selection (MAS) for qhir1, a QTL with a significant positive effect on HIR from the crosses between elite tropical maize inbreds and first generation Tropically Adapted Inducers Lines (TAILs). Evaluation of 2GTAILs for HIR and agronomic performance in the tropical and subtropical environments indicated superior performance of 2GTAILs over the TAILs for both HIR and agronomic performance, including plant vigor, delayed flowering, grain yield, and resistance to ear rots. One of the new inducers 2GTAIL006 showed an average HIR of 13.1% which is 48.9% higher than the average HIR of the TAILs. Several other 2GTAILs also showed higher HIR compared to the TAILs. While employing MAS for qhir1 QTL, we observed significant influence of the non-inducer parent on the positive effect of qhir1 QTL on HIR. The non-inducer parents that resulted in highest mean HIR in the early generation qhir1+ families also gave rise to highest numbers of candidate inducers, some of which showed transgressive segregation for HIR. The mean HIR of early generation qhir1+ families involving different non-inducer parents can potentially indicate recipient non-inducer parents that can result in progenies with high HIR. Our study also indicated that the HIR associated traits (endosperm abortion rate, embryo abortion rate, and proportion of haploid plants among the inducer plants) can be used to differentiate inducers vs. non-inducers but are not suitable for differentiating inducers with varying levels of haploid induction rates. We propose here an efficient methodology for developing haploid inducer lines combining MAS for qhir1 with HIR associated traits.

A Hypothesis of the Interaction of the Nitrergic and Serotonergic Systems in Aggressive Behavior Induced by Exposure to Lead.

The effects caused by exposure to lead (Pb) are still considered as a relevant health risk despite public policies aimed to restricting the use of this element. The toxicity limit in the blood (10 µg/dL, established by the Center for Disease Control and Prevention) has been insufficient to prevent adverse effects and even lower values have been related to neurobehavioral dysfunctions in children. Currently, there is not a safe limit of exposure to Pb. A large body of evidence points to environmental pollutant exposure as the cause of predisposition to violent behavior, among others. Considering the evidence by our group and others, we propose that Pb exposure induces alterations in the brain vasculature, specifically in nitric oxide synthases (NOS), affecting in turn the serotonergic system and leading to heightened aggressive behavior in the exposed individuals. This review article describes the consequences of Pb exposure on the nitrergic and serotonergic systems as well as its relationship with aggressive behavior. In addition, it summarizes the available therapy to prevent damage in gestation and among infants.