Dynamic Factors Affecting Gaseous Ligand Binding in an Artificial Oxygen Transport Protein

We report the functional analysis of an artificial hexacoordinate oxygen transport protein, HP7, which operates via a mechanism similar to that of human neuroglobin and cytoglobin: the destabilization of one of two heme-ligating histidine residues. In the case of HP7, this is the result of the coupling of histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Here we compare gaseous ligand binding, including rates, affinities, and oxyferrous state lifetimes, of both heme binding sites in HP7. We find that despite the identical sequence of helices in both binding sites, there are differences in oxygen affinity and oxyferrous state lifetime that may be the result of differences in the freedom of motion imposed by the candelabra fold on the two sites of the protein. We further examine the effect of mutational removal of the buried glutamates on function. Heme iron in the ferrous state of this mutant is rapidly oxidized when exposed to oxygen. Compared to that of HP7, the distal histidine affinity is increased by a 22-fold decrease in the histidine ligand off rate. Electron paramagnetic resonance comparison of these ferric hemoproteins demonstrates that the mutation increases the level of disorder at the heme binding site. Nuclear magnetic resonance-detected deuterium exchange demonstrates that the mutation greatly increases the degree of penetration of water into the protein core. The inability of the mutant protein to bind oxygen may be due to an increased level of water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors. Together, these data underline the importance of the control of protein dynamics in the design of functional artificial proteins

Excretion of Urinary Metabolites of the Phthalate Esters DEP and DEHP in 16 Volunteers after Inhalation and Dermal Exposure

Phthalate esters are suspected endocrine disruptors that are found in a wide range of applications. The aim of this study was to determine the excretion of urinary metabolites in 16 individuals after inhalation and/or dermal exposure to 100⁻300 µg/m³ of deuterium-labelled diethyl phthalate (D₄-DEP) and bis(2-ethylhexyl) phthalate (D₄-DEHP). Dermal exposure in this study represents a case with clean clothing acting as a barrier. After inhalation, D₄-DEP and D₄-DEHP metabolites were excreted rapidly, though inter-individual variation was high. D₄-DEP excretion peaked 3.3 h (T½ of 2.1 h) after combined inhalation and dermal exposure, with total excreted metabolite levels ranging from 0.055 to 2.351 nmol/nmol/m³ (nmol of urinary metabolites per phthalates air concentration in (nmol/m³)). After dermal exposure to D₄-DEP, metabolite excretion peaked 4.6 h (T½ of 2.7 h) after exposure, with excreted metabolite levels in between 0.017 and 0.223 nmol/nmol/m³. After combined inhalation and dermal exposure to D₄-DEHP, the excretion of all five analysed metabolites peaked after 4.7 h on average (T½ of 4.8 h), and metabolite levels ranged from 0.072 to 1.105 nmol/nmol/m³ between participants. No dermal uptake of particle phase D₄-DEHP was observed. In conclusion, the average excreted levels of metabolites after combined inhalation and dermal exposure to D₄-DEP was three times higher than after combined exposure to D₄-DEHP; and nine times higher than after dermal exposure of D₄-DEP. This study was made possible due to the use of novel approaches, i.e., the use of labelled phthalate esters to avoid the background concentration, and innovative technique of phthalate generation, both in the particle and the gas phase

The angiogenic switch leads to a metabolic shift in human glioblastoma

Invasion and angiogenesis are major hallmarks of glioblastoma (GBM) growth. While invasive tumor cells grow adjacent to blood vessels in normal brain tissue, tumor cells within neovascularized regions exhibit hypoxic stress and promote angiogenesis. The distinct microenvironments likely differentially affect metabolic processes within the tumor cells. In the present study, we analyzed gene expression and metabolic changes in a human GBM xenograft model that displayed invasive and angiogenic phenotypes. In addition, we used glioma patient biopsies to confirm the results from the xenograft model. We demonstrate that the angiogenic switch in our xenograft model is linked to a proneural-to-mesenchymal transition that is associated with upregulation of the transcription factors BHLHE40, CEBPB, and STAT3. Metabolic analyses revealed that angiogenic xenografts employed higher rates of glycolysis compared with invasive xenografts. Likewise, patient biopsies exhibited higher expression of the glycolytic enzyme lactate dehydrogenase A and glucose transporter 1 in hypoxic areas compared with the invasive edge and lower-grade tumors. Analysis of the mitochondrial respiratory chain showed reduction of complex I in angiogenic xenografts and hypoxic regions of GBM samples compared with invasive xenografts, nonhypoxic GBM regions, and lower-grade tumors. In vitro hypoxia experiments additionally revealed metabolic adaptation of invasive tumor cells, which increased lactate production under long-term hypoxia. The use of glycolysis versus mitochondrial respiration for energy production within human GBM tumors is highly dependent on the specific microenvironment. The metabolic adaptability of GBM cells highlights the difficulty of targeting one specific metabolic pathway for effective therapeutic interventio

Preterm birth, infant weight gain, and childhood asthma risk: A meta-analysis of 147,000 European children

Background: Preterm birth, low birth weight, and infant catch-up growth seem associated with an increased risk of respiratory diseases in later life, but individual studies showed conflicting results. Objectives: We performed an individual participant data meta-analysis for 147,252 children of 31 birth cohort studies to determine the associations of birth and infant growth characteristics with the risks of preschool wheezing (1-4 years) and school-age asthma (5-10 years). Methods: First, we performed an adjusted 1-stage random-effect meta-analysis to assess the combined associations of gestational age, birth weight, and infant weight gain with childhood asthma. Second, we performed an adjusted 2-stage random-effect meta-analysis to assess the associations of preterm birth (gestational age < 37 weeks) and low birth weight (< 2500 g) with childhood asthma outcomes. Results: Younger gestational age at birth and higher infant weight gain were independently associated with higher risks of preschool wheezing and school-age asthma (P <. 05). The inverse associations of birth weight with childhood asthma were explained by gestational age at birth. Compared with term-born children with normal infant weight gain, we observed the highest risks of school-age asthma in children born preterm with high infant weight gain (odds ratio [OR], 4.47; 95% CI, 2.58-7.76). Preterm birth was positively associated with an increased risk of preschool wheezing (pooled odds ratio [pOR], 1.34; 95% CI, 1.25-1.43) and school-age asthma (pOR, 1.40; 95% CI, 1.18-1.67) independent of birth weight. Weaker effect estimates were observed for the associations of low birth weight adjusted for gestational age at birth with preschool wheezing (pOR, 1.10; 95% CI, 1.00-1.21) and school-age asthma (pOR, 1.13; 95% CI, 1.01-1.27). Conclusion: Younger gestational age at birth and higher infant weight gain were associated with childhood asthma outcomes. The associations of lower birth weight with childhood asthma were largely explained by gestational age at birth

Preterm birth, infant weight gain, and childhood asthma risk: a meta-analysis of 147,000 European children

Background: Preterm birth, low birth weight, and infant catch-up growth seem associated with an increased risk of respiratory diseases in later life, but individual studies showed conflicting results. Objectives: We performed an individual participant data meta-analysis for 147,252 children of 31 birth cohort studies to determine the associations of birth and infant growth characteristics with the risks of preschool wheezing (1-4 years) and school-age asthma (5-10 years). Methods: First, we performed an adjusted 1-stage random-effect meta-analysis to assess the combined associations of gestational age, birth weight, and infant weight gain with childhood asthma. Second, we performed an adjusted 2-stage random-effect meta-analysis to assess the associations of preterm birth (gestational age <37 weeks) and low birth weight (<2500 g) with childhood asthma outcomes. Results: Younger gestational age at birth and higher infant weight gain were independently associated with higher risks of preschool wheezing and school-age asthma (P < .05). The inverse associations of birth weight with childhood asthma were explained by gestational age at birth. Compared with term-born children with normal infant weight gain, we observed the highest risks of school-age asthma in children born preterm with high infant weight gain (odds ratio [OR], 4.47; 95% CI, 2.58-7.76). Preterm birth was positively associated with an increased risk of preschool wheezing (pooled odds ratio [pOR], 1.34; 95% CI, 1.25-1.43) and school-age asthma (pOR, 1.40; 95% CI, 1.18-1.67) independent of birth weight. Weaker effect estimates were observed for the associations of low birth weight adjusted for gestational age at birth with preschool wheezing (pOR, 1.10; 95% CI, 1.00-1.21) and school-age asthma (pOR, 1.13; 95% CI, 1.01-1.27). Conclusion: Younger gestational age at birth and higher infant weight gain were associated with childhood asthma outcomes. The associations of lower birth weight with childhood asthma were largely explained by gestational age at birth.Per cohort. ABIS: Data used for this research was provided by the Cohort Study, which is supported in part by JDRF-Wallenberg foundations (K 98-99D-12813-01A), the Swedish Medical Research Council (MFR; Vetenskapsrådet; K99-72X-11242-05A), the Swedish Child Diabetes Foundation (Barndiabetesfonden), and the Swedish Diabetes Association, Medical Research Council of South East Sweden (FORSS), Novo Nordisk Foundation, Prevention of Diabetes, and its Complications Strategic Area-LiU. ALSPAC: We are extremely grateful to all the families who took part in the study, the midwives for their help in recruiting them, and the whole ALSPAC team, which includes interviewers, computer and laboratory technicians, clerical workers, research scientists, volunteers, managers, receptionist, and nurses. The UK Medical Research Council and the Wellcome Trust (grant reference 092731) and the University of Bristol provide core support for ALSPAC. BILD: Data used for this research were provided by the Cohort Study, which is supported in part by funds of the Swiss National Science Foundation; the European Respiratory Society (ERS); the Austrian, German and Swiss Paediatric respiratory Society; and the Swiss Governmental Anti-Tobacco Fund. CONER: Data used for this research were provided by the Cohort Study, which is supported in part by funds of the Italian ministry of health. COPSAC: COPSAC is funded by private and public research funds listed on www.copsac.com. The Lundbeck Foundation, the Danish Strategic Research Council, the Pharmacy Foundation of 1991, the Augustinus Foundation, the Danish Medical Research Council, and the Danish Pediatric Asthma Centre provided the core support for the COPSAC research center. No pharmaceutical company was involved in the study. The funding agencies did not have any role in design and conduct of the study; collection, management, and interpretation of the data; or preparation, review, or approval of the manuscript. CZECH: Data used for this research was provided by the Cohort Study, which is supported in part by funds of the Ministry of Environment of the Czech Republic (SP/1b3/8/08). DNBC: The Danish National Research Foundation has established the Danish Epidemiology Science Centre that initiated and created the Danish National Birth Cohort. The cohort is furthermore a result of a major grant from this foundation. Additional support for the Danish National Birth Cohort is obtained from the Pharmacy Foundation, the Egmont Foundation, the March of Dimes Birth Defects Foundation, and the Augustinus Foundation. EDEN: We acknowledge all the funding sources for the EDEN study: Fondation pour la Recherche Médicale (FRM), the French Ministry of Research: IFR program, the INSERM Nutrition Research program, the French Ministry of Health Perinatality Program, the French Agency for Environment security (AFFSET), the French National Institute for Population Health Surveillance (INVS), Paris-Sud University, the French National Institute for Health Education (INPES), Nestlé, Mutuelle Générale de l'Education Nationale {MGEN), the French-speaking Association for the Study of Diabetes and Metabolism (Alfediam), and the National Agency for Research (ANR). GASPII: Data used for this research was provided by the Cohort Study, which is supported in part by funds of the Italian Ministry of Health, 2001. GECKO Drenthe: The GECKO Drenthe cohort is supported and funded by an unrestricted grant from Hutchison Whampoa, the University of Groningen, and Well Baby Clinic Foundation Icare. GENERATION R: The Generation R Study is made possible by financial support from the Erasmus Medical Center, Rotterdam; the Erasmus University Rotterdam; and the Netherlands Organization for Health Research and Development. The researchers are independent from the funders. The study sponsors had no role in study design, data analysis, interpretation of data, or writing of this report. Additional support was available from the Netherlands Organization for Health Research and Development (VIDI) and the Dutch Asthma Foundation. GENERATION XXI: Data used for this research were provided by the Cohort Study, which is supported in part by funds of the Programa Operacional de Saúde–Saúde XXI, Quadro Comunitário de Apoio III (FEDER), the Northern Regional Administration of Health, the Portuguese Foundation for Science and Technology (PTDC/SAUESA/105033/2008), and the Calouste Gulbenkian Foundation. HUMIS: The research leading to these results has received funding from the Norwegian Research Council under grant agreement 213148 (MILPAAHEL) and the European Union's Seventh Framework Programme (FP7/2007-2013), project Early Nutrition under grant agreement number 289346, and project OBELIX under grant agreement number 22739. INMA: Gipuzkoa/Sabadell/Valencia/Menorca Data used for this research were provided by the INMA–Environment and Childhood Project (www.proyectoinma.org), which is supported in part by funds. This study was funded by grants from Instituto de Salud Carlos III (Red INMA G03/176 and CB06/02/0041), the Spanish Ministry of Health (FIS- PI041436, PI042018, PI06/0867 PI07/0252, PI081151, and PI09/02311,and FIS-FEDER 03/1615, 04/1509, 04/1112, 04/1931, 05/1079, 05/1052, 06/1213, 07/0314, and 09/02647), Generalitat de Catalunya-CIRIT 1999SGR 00241, the Conselleria de Sanitat Generalitat Valenciana, the Department of Health of the Basque Government (2005111093 and 2009111069), the Provincial Government of Gipuzkoa (DFG06/004 and DFG08/001), Obra Social Cajastur, Universidad de Oviedo, the EU Commission (QLK4-1999-01422, QLK4-2002-00603 and CONTAMED FP7-ENV-212502), Consejería de Salud de la Junta de Andalucía (grant number 183/07), and Fundació Roger Torné. ISLE OF WIGHT: Data used for this research were provided by the Cohort Study, which is supported in part by funds of the National Institute of Health, the British Medical Association, and David Hide Asthma and Allergy Research Centre Trustees. KOALA: Data used for this research were provided by the Cohort Study, which is supported in part by funds from the Netherlands Asthma Foundation (grant nos. 3.2.03.48 and 3.2.07.022). LEICESTER 1990/1998: Data used for this research were provided by the Leicester Cohort Studies, which are supported by funds from Asthma UK (grant no. 07/048), the Swiss National Science Foundation (grant no. 32003B-144068), the Wellcome Trust, and many others. LIFEWAYS: Data used for this research were provided by the Cohort Study, which is supported in part by funds of the Health Research Board, Republic of Ireland. MAS: Data for this research question were obtained by the study centre of the cohort study. The Multicentre Allergy Study (1990) was supported by grants from the German Federal Ministry for Education and Research (BMBF) under reference numbers 07015633, 07 ALE 27, 01EE9405/5, and 01EE9406.NINFEA: Data used for this research were provided by the Cohort Study, which is supported in part by funds of Compagnia di SanPaolo Foundation, Piedmont Region, and the Italian Ministry of University and Research. PCB: Data used for this research was provided by the Cohort Study, which is supported in part by funds from National Institutes of Health grant R01-CA096525 and EU project OBELIX (no. 227391). PIAMA: The PIAMA study has been funded by the Netherlands Organization for Health Research and Development; the Netherlands Organization for Scientific Research; the Netherlands Asthma Fund; the Netherlands Ministry of Spatial Planning, Housing, and the Environment; and the Netherlands Ministry of Health, Welfare and Sport. REPRO PL: Data used for this research were provided by the Cohort Study, which is supported in part by funds from the National Center for Research and Development, Poland (grant no. PBZ-MEiN-/8/2//2006; contract no. K140/P01/2007/1.3.1.1.) and grant PNRF-218-AI-1/07 from Norway through the Norwegian Financial Mechanism within the Polish-Norwegian Research Fund. RHEA: Data used for this research were provided by the Cohort Study, which is supported in part by funds of European Commission. SEATON: Data used for this research were provided by the university, which is supported in part by funds from Asthma UK and the Medical Research Council. SWS: The Southampton Women's Survey is supported by grants from the Medical Research Council, the British Heart Foundation, the Food Standards Agency, the British Lung Foundation, Arthritis Research UK, NIHR Southampton Biomedical Research Centre, the University of Southampton and University Hospital Southampton NHS Foundation Trust, and the Commission of the European Community, specific RTD Programme “Quality of Life and Management of Living Resources,” within the 7th Framework Programme, research grant no. FP7/2007-13 (Early Nutrition Project). This manuscript does not necessarily reflect the views of the funders and in no way anticipates the future policy in this area. WHISTLER: Data used for this research were provided by the Cohort Study, which is supported in part by funds from the Netherlands Organization for health Research and Development (ZON-MW), the University Medical Center Utrecht, and an unrestricted research grant from GlaxoSmithKline, The Netherland