Recent Submissions

  • Tenkanen, Henrikki; Saarsalmi, Perttu; Järv, Olle; Salonen, Maria; Toivonen, Tuuli (BioMed Central, 2016)
    Abstract Background In this paper, we demonstrate why and how both temporality and multimodality should be integrated in health related studies that include accessibility perspective, in this case healthy food accessibility. We provide evidence regarding the importance of using multimodal spatio-temporal accessibility measures when conducting research in urban contexts and propose a methodological approach for integrating different travel modes and temporality to spatial accessibility analyses. We use the Helsinki metropolitan area (Finland) as our case study region to demonstrate the effects of temporality and modality on the results. Methods Spatial analyses were carried out on 250 m statistical grid squares. We measured travel times between the home location of inhabitants and open grocery stores providing healthy food at 5 p.m., 10 p.m., and 1 a.m. using public transportation and private cars. We applied the so-called door-to-door approach for the travel time measurements to obtain more realistic and comparable results between travel modes. The analyses are based on open access data and publicly available open-source tools, thus similar analyses can be conducted in urban regions worldwide. Results Our results show that both time and mode of transport have a prominent impact on the outcome of the analyses; thus, understanding the realities of accessibility in a city may be very different according to the setting of the analysis used. In terms of travel time, there is clear variation in the results at different times of the day. In terms of travel mode, our results show that when analyzed in a comparable manner, public transport can be an even faster mode than a private car to access healthy food, especially in central areas of the city where the service network is dense and public transportation system is effective. Conclusions This study demonstrates that time and transport modes are essential components when modeling health-related accessibility in urban environments. Neglecting them from spatial analyses may lead to overly simplified or even erroneous images of the realities of accessibility. Hence, there is a risk that health related planning and decisions based on simplistic accessibility measures might cause unwanted outcomes in terms of inequality among different groups of people.
  • Chatzidionysiou, Katerina; Lie, Elisabeth; Nasonov, Evgeny; Lukina, Galina; Hetland, Merete Lund; Tarp, Ulrik; Ancuta, Ioan; Pavelka, Karel; Nordstrom, Dan C.; Gabay, Cem; Canhao, Helene; Tomsic, Matija; van Riel, Piet L. C. M.; Gomez-Reino, Juan; Kvien, Tore K.; van Vollenhoven, Ronald F.; Rheumatic Dis Portuguese Register (BioMed Central, 2016)
    Background: The approved dose of rituximab (RTX) in rheumatoid arthritis is 1000 mg x 2, but some data have suggested similar clinical efficacy with 500 mg x 2. The purpose of this study was to compare the effectiveness of the regular and low doses given as first treatment course. Methods: Twelve European registries participating in the CERERRA collaboration (The European Collaborative Registries for the Evaluation of Rituximab in Rheumatoid Arthritis) submitted anonymized datasets with demographic, efficacy and treatment data for patients who had started RTX. Treatment effectiveness was assessed by DAS28 reductions and EULAR responses after 6 months. Results: Data on RTX dose were available for 2,873 patients, of whom 2,625 (91.4 %) and 248 (8.6 %) received 1000 mg x 2 and 500 mg x 2, respectively. Patients treated with 500 mg x 2 were significantly older, had longer disease duration, higher number of prior DMARDs, but lower number of prior biologics and lower baseline DAS28 than those treated with 1000 mg x 2. Fewer patients in the low-dose group received concomitant DMARDs but more frequently received concomitant corticosteroids. Both doses led to significant clinical improvements at 6 months. DAS28 reductions at 6 months were comparable in the 2 dose regimens [mean DeltaDAS28 +/- SD -2.0 +/- 1.3 (high dose) vs. -1.7 +/- 1.4 (low dose), p = 0.23 adjusted for baseline differences]. Similar percentages of patients achieved EULAR good response in the two dose groups, 18.4 % vs. 17.3 %, respectively (p = 0.36). Conclusions: In this large observational cohort initial treatment with RTX at 500 mg x 2 and 1000 mg x 2 led to comparable clinical outcomes at 6 months.
  • Soder, Birgitta; Meurman, Jukka H.; Soder, Per-Osten (PUBLIC LIBRARY OF SCIENCE, 2016)
    Objectives Dental infections, such as periodontitis, associate with atherosclerosis and its complications. We studied a cohort followed-up since 1985 for incidence of angina pectoris with the hypothesis that calculus accumulation, proxy for poor oral hygiene, links to this symptom. Methods In our Swedish prospective cohort study of 1676 randomly selected subjects followed-up for 26 years. In 1985 all subjects underwent clinical oral examination and answered a questionnaire assessing background variables such as socio-economic status and pack-years of smoking. By using data from the Center of Epidemiology, Swedish National Board of Health and Welfare, Sweden we analyzed the association of oral health parameters with the prevalence of in-hospital verified angina pectoris classified according to the WHO International Classification of Diseases, using descriptive statistics and logistic regression analysis. Results Of the 1676 subjects, 51 (28 women/23 men) had been diagnosed with angina pectoris at a mean age of 59.8 +/- 2.9 years. No difference was observed in age and gender between patients with angina pectoris and subjects without. Neither was there any difference in education level and smoking habits (in pack years), Gingival index and Plaque index between the groups. Angina pectoris patients had significantly more often their first maxillary molar tooth extracted (d. 16) than the other subjects (p = 0.02). Patients also showed significantly higher dental calculus index values than the subjects without angina pectoris (p = 0.01). Multiple regression analysis showed odds ratio 2.21 (95% confidence interval 1.17-4.17) in the association between high calculus index and angina pectoris (p = 0.015). Conclusion Our study hypothesis was confirmed by showing for the first time that high dental calculus score indeed associated with the incidence of angina pectoris in this cohort study.
  • Volpers, Michael; Claassens, Nico J.; Noor, Elad; van der Oost, John; de Vos, Willem M.; Kengen, Serve W. M.; dos Santos, Vitor A. P. Martins (PUBLIC LIBRARY OF SCIENCE, 2016)
    The strong advances in synthetic biology enable the engineering of novel functions and complex biological features in unprecedented ways, such as implementing synthetic autotrophic metabolism into heterotrophic hosts. A key challenge for the sustainable production of fuels and chemicals entails the engineering of synthetic autotrophic organisms that can effectively and efficiently fix carbon dioxide by using sustainable energy sources. This challenge involves the integration of carbon fixation and energy uptake systems. A variety of carbon fixation pathways and several types of photosystems and other energy uptake systems can be chosen and, potentially, modularly combined to design synthetic autotrophic metabolism. Prior to implementation, these designs can be evaluated by the combination of several computational pathway analysis techniques. Here we present a systematic, integrated in silico analysis of photo-electro-autotrophic pathway designs, consisting of natural and synthetic carbon fixation pathways, a proton-pumping rhodopsin photosystem for ATP regeneration and an electron uptake pathway. We integrated Flux Balance Analysis of the heterotrophic chassis Escherichia coli with kinetic pathway analysis and thermodynamic pathway analysis (Max-min Driving Force). The photo-electro-autotrophic designs are predicted to have a limited potential for anaerobic, autotrophic growth of E. coli, given the relatively low ATP regenerating capacity of the proton pumping rhodopsin photosystems and the high ATP maintenance of E. coli. If these factors can be tackled, our analysis indicates the highest growth potential for the natural reductive tricarboxylic acid cycle and the synthetic pyruvate synthase-pyruvate carboxylate - glyoxylate bicycle. Both carbon fixation cycles are very ATP efficient, while maintaining fast kinetics, which also results in relatively low estimated protein costs for these pathways. Furthermore, the synthetic bicycles are highly thermodynamic favorable under conditions analysed. However, the most important challenge identified for improving photo-electro-autotrophic growth is increasing the proton-pumping rate of the rhodopsin photosystems, allowing for higher ATP regeneration. Alternatively, other designs of autotrophy may be considered, therefore the herein presented integrated modeling approach allows synthetic biologists to evaluate and compare complex pathway designs before experimental implementation.
  • Galli, Emilia; Harkonen, Taina; Sainio, Markus; Ustav, Mart; Toots, Urve; Urtti, Arto; Yliperttula, Marjo; Lindahl, Maria; Knip, Mikael; Saarma, Mart; Lindholm, Paivi (NATURE PUBLISHING GROUP, 2016)
    Mesencephalic astrocyte-derived neurotrophic factor (MANF) was recently shown to be essential for the survival and proliferation of pancreatic beta-cells in mice, where deletion of MANF resulted in diabetes. The current study aimed at determining whether the concentration of circulating MANF is associated with the clinical manifestation of human type 1 diabetes (T1D). MANF expression in T1D or MANF levels in serum have not been previously studied. We developed an enzyme-linked immunosorbent assay (ELISA) for MANF and measured serum MANF concentrations from 186 newly diagnosed children and adolescents and 20 adults with longer-term T1D alongside with age-matched controls. In healthy controls the mean serum MANF concentration was 7.0 ng/ml. High MANF concentrations were found in children 1-9 years of age close to the diagnosis of T1D. The increased MANF concentrations were not associated with diabetes-predictive autoantibodies and autoantibodies against MANF were extremely rare. Patients with conspicuously high MANF serum concentrations had lower C-peptide levels compared to patients with moderate MANF concentrations. Our data indicate that increased MANF concentrations in serum are associated with the clinical manifestation of T1D in children, but the exact mechanism behind the increase remains elusive.
  • Stock, Kristin; Estrada, Marta F.; Vidic, Suzana; Gjerde, Kjersti; Rudisch, Albin; Santo, Vitor E.; Barbier, Michael; Blom, Sami; Arundkar, Sharath C.; Selvam, Irwin; Osswald, Annika; Stein, Yan; Gruenewald, Sylvia; Brito, Catarina; van Weerden, Wytske; Rotter, Varda; Boghaert, Erwin; Oren, Moshe; Sommergruber, Wolfgang; Chong, Yolanda; de Hoogt, Ronald; Graeser, Ralph (NATURE PUBLISHING GROUP, 2016)
    Two-dimensional (2D) cell cultures growing on plastic do not recapitulate the three dimensional (3D) architecture and complexity of human tumors. More representative models are required for drug discovery and validation. Here, 2D culture and 3D mono-and stromal co-culture models of increasing complexity have been established and cross-comparisons made using three standard cell carcinoma lines: MCF7, LNCaP, NCI-H1437. Fluorescence-based growth curves, 3D image analysis, immunohistochemistry and treatment responses showed that end points differed according to cell type, stromal co-culture and culture format. The adaptable methodologies described here should guide the choice of appropriate simple and complex in vitro models.
  • Airas, Niina; Hautaniemi, Maria; Syrja, Pernilla; Knuuttila, Anna; Putkuri, Niina; Coulter, Lesley; McInnes, Colin J.; Vapalahti, Olli; Huovilainen, Anita; Kinnunen, Paula M. (National Center for Infectious Diseases, Centers for Disease Control and Prevention (CDC), 2016)
    A horse in Finland exhibited generalized granulomatous inflammation and severe proliferative dermatitis. After euthanization, we detected poxvirus DNA from a skin lesion sample. The virus sequence grouped with parapoxviruses, closely resembling a novel poxvirus detected in humans in the United States after horse contact. Our findings indicate horses may be a reservoir for zoonotic parapoxvirus.
  • van Leeuwen, Elisabeth M.; Sabo, Aniko; Bis, Joshua C.; Huffman, Jennifer E.; Manichaikul, Ani; Smith, Albert V.; Feitosa, Mary F.; Demissie, Serkalem; Joshi, Peter K.; Duan, Qing; Marten, Jonathan; van Klinken, Jan B.; Surakka, Ida; Nolte, Ilja M.; Zhang, Weihua; Mbarek, Hamdi; Li-Gao, Ruifang; Trompet, Stella; Verweij, Niek; Evangelou, Evangelos; Lyytikainen, Leo-Pekka; Tayo, Bamidele O.; Deelen, Joris; van der Most, Peter J.; van der Laan, Sander W.; Arking, Dan E.; Morrison, Alanna; Dehghan, Abbas; Franco, Oscar H.; Hofman, Albert; Rivadeneira, Fernando; Sijbrands, Eric J.; Uitterlinden, Andre G.; Mychaleckyj, Josyf C.; Campbell, Archie; Hocking, Lynne J.; Padmanabhan, Sandosh; Brody, Jennifer A.; Rice, Kenneth M.; White, Charles C.; Harris, Tamara; Isaacs, Aaron; Campbell, Harry; Lange, Leslie A.; Rudan, Igor; Kolcic, Ivana; Navarro, Pau; Zemunik, Tatijana; Salomaa, Veikko; Kooner, Angad S.; Kooner, Jaspal S.; Lehne, Benjamin; Scott, William R.; Tan, Sian-Tsung; de Geus, Eco J.; Milaneschi, Yuri; Penninx, Brenda W. J. H.; Willemsen, Gonneke; de Mutsert, Renee; Ford, Ian; Gansevoort, Ron T.; Segura-Lepe, Marcelo P.; Raitakari, Olli T.; Viikari, Jorma S.; Nikus, Kjell; Forrester, Terrence; McKenzie, Colin A.; de Craen, Anton J. M.; de Ruijter, Hester M.; Pasterkamp, Gerard; Snieder, Harold; Oldehinkel, Albertine J.; Slagboom, P. Eline; Cooper, Richard S.; Kahonen, Mika; Lehtimaki, Terho; Elliott, Paul; van der Harst, Pim; Jukema, J. Wouter; Mook-Kanamori, Dennis O.; Boomsma, Dorret I.; Chambers, John C.; Swertz, Morris; Ripatti, Samuli; van Dijk, Ko Willems; Vitart, Veronique; Polasek, Ozren; Hayward, Caroline; Wilson, James G.; Wilson, James F.; Gudnason, Vilmundur; Rich, Stephen S.; Psaty, Bruce M.; Borecki, Ingrid B.; Boerwinkle, Eric; Rotter, Jerome I.; Cupples, L. Adrienne; van Duijn, Cornelia M.; CHARGE Lipids Working Grp (BMJ PUBLISHING GROUP, 2016)
    Background So far, more than 170 loci have been associated with circulating lipid levels through genome-wide association studies (GWAS). These associations are largely driven by common variants, their function is often not known, and many are likely to be markers for the causal variants. In this study we aimed to identify more new rare and low-frequency functional variants associated with circulating lipid levels. Methods We used the 1000 Genomes Project as a reference panel for the imputations of GWAS data from similar to 60 000 individuals in the discovery stage and similar to 90 000 samples in the replication stage. Results Our study resulted in the identification of five new associations with circulating lipid levels at four loci. All four loci are within genes that can be linked biologically to lipid metabolism. One of the variants, rs116843064, is a damaging missense variant within the ANGPTL4 gene. Conclusions This study illustrates that GWAS with high-scale imputation may still help us unravel the biological mechanism behind circulating lipid levels.
  • Broesby-Olsen, Sigurd; Dybedal, Ingunn; Gulen, Theo; Kristensen, Thomas K.; Moller, Michael B.; Ackermann, Leena; Saaf, Maria; Karlsson, Maria; Agertoft, Lone; Brixen, Kim; Hermann, Pernille; Stylianou, Eva; Mortz, Charlotte G.; Torfing, Trine; Havelund, Troels; Sander, Birgitta; Bergstrom, Anna; Bendix, Marie; Garvey, Lene H.; Weis Bjerrum, Ole; Valent, Peter; Bindslev-Jensen, Carsten; Nilsson, Gunnar; Vestergaard, Hanne; Hagglund, Hans (Acta Dermato-Venereologica, 2016)
    Mastocytosis is a heterogeneous group of diseases defined by an increased number and accumulation of mast cells, and often also by signs and symptoms of mast cell activation. Disease subtypes range from indolent to rare aggressive forms. Mastocytosis affects people of all ages and has been considered rare; however, it is probably underdiagnosed with potential severe implications. Diagnosis can be challenging and symptoms may be complex and involve multiple organ-systems. In general it is advised that patients should be referred to centres with experience in the disease offering an individualized, multidisciplinary approach. We present here consensus recommendations from a Nordic expert group for the diagnosis and general management of patients with mastocytosis.
  • Pirneskoski, Jussi; Peräjoki, Katja; Nuutila, Mika; Kuisma, Markku (BioMed Central, 2016)
    Abstract Background The aim of this study was to examine Helsinki Emergency Medical Services (EMS) and hospital records to determine the incidence and possible complications of out-of-hospital deliveries managed by EMS in Helsinki. Methods We retrospectively analysed all urgent ambulance dispatches relating to childbirth in Helsinki from January 1, 2010 to December 31, 2014 with further analysis of hospital records for the out-of-hospital deliveries. Patients were divided in to two groups: those who delivered before reaching hospital and those who did not deliver before reaching hospital and differences between groups were analysed. Deliveries with gestational age of at least 22 + 0 weeks were considered as births in statistical analysis as this is the current national practice. Results There were 799 urgent dispatches during the study period. In 102 (12.8 %) of these delivery took place before reaching the hospital. The incidence of EMS managed out-of-hospital delivery was found to be 3.0/1000 births. The annual number of out-of-hospital deliveries attended by EMS increased from 15 in 2010 to 28 in 2014. No stillbirths were reported. Neither maternal or perinatal deaths nor major maternal complications were noted in the study population. Discussion Out-of-hospital deliveries represent a small minority of EMS calls and remain a challenge to maintaining professional capabilities. Small sample size might have limited the ability of the study to pick up rare complications. Conclusions The amount of out-of-hospital deliveries in Helsinki increased during the five-year study period. There were no maternal or perinatal mortality or major complications resulting in long-term sequelae associated with the EMS-managed out-of-hospital births.
    Legehar, Ashenafi; Xhaard, Henri; Ghemtio, Leo (BIOMED CENTRAL LTD, 2016)
    Background: The disposition of a pharmaceutical compound within an organism, i.e. its Absorption, Distribution, Metabolism, Excretion, Toxicity (ADMET) properties and adverse effects, critically affects late stage failure of drug candidates and has led to the withdrawal of approved drugs. Computational methods are effective approaches to reduce the number of safety issues by analyzing possible links between chemical structures and ADMET or adverse effects, but this is limited by the size, quality, and heterogeneity of the data available from individual sources. Thus, large, clean and integrated databases of approved drug data, associated with fast and efficient predictive tools are desirable early in the drug discovery process. Description: We have built a relational database (IDAAPM) to integrate available approved drug data such as drug approval information, ADMET and adverse effects, chemical structures and molecular descriptors, targets, bioactivity and related references. The database has been coupled with a searchable web interface and modern data analytics platform (KNIME) to allow data access, data transformation, initial analysis and further predictive modeling. Data were extracted from FDA resources and supplemented from other publicly available databases. Currently, the database contains information regarding about 19,226 FDA approval applications for 31,815 products (small molecules and bio-logics) with their approval history, 2505 active ingredients, together with as many ADMET properties, 1629 molecular structures, 2.5 million adverse effects and 36,963 experimental drug-target bioactivity data. Conclusion: IDAAPM is a unique resource that, in a single relational database, provides detailed information on FDA approved drugs including their ADMET properties and adverse effects, the corresponding targets with bioactivity data, coupled with a data analytics platform. It can be used to perform basic to complex drug-target ADMET or adverse effects analysis and predictive modeling. IDAAPM is freely accessible at and can be exploited through a KNIME workflow connected to the database.
  • Hanski, Leena; Ausbacher, Dominik; Tiirola, Terttu M.; Strom, Morten B.; Vuorela, Pia M. (PUBLIC LIBRARY OF SCIENCE, 2016)
    We demonstrate in the current work that small cationic antimicrobial beta(2,2)-amino acid derivatives (Mw <500 Da) are highly potent against Chlamydia pneumoniae at clinical relevant concentrations (<5 mu M, i.e. <3.4 mu g/mL). C. pneumoniae is an atypical respiratory pathogen associated with frequent treatment failures and persistent infections. This gram-negative bacterium has a biphasic life cycle as infectious elementary bodies and proliferating reticulate bodies, and efficient treatment is challenging because of its long and obligate intracellular replication cycle within specialized inclusion vacuoles. Chlamydicidal effect of the beta(2,2)-amino acid derivatives in infected human epithelial cells was confirmed by transmission electron microscopy. Images of infected host cells treated with our lead derivative A2 revealed affected chlamydial inclusion vacuoles 24 hours post infection. Only remnants of elementary and reticulate bodies were detected at later time points. Neither the EM studies nor resazurin-based cell viability assays showed toxic effects on uninfected host cells or cell organelles after A2 treatment. Besides the effects on early intracellular inclusion vacuoles, the ability of these beta(2,2)-amino acid derivatives to suppress Chlamydia pneumoniae infectivity upon treatment of elementary bodies suggested also a direct interaction with bacterial membranes. Synthetic beta(2,2)-amino acid derivatives that target C. pneumoniae represent promising lead molecules for development of antimicrobial agents against this hard-totreat intracellular pathogen.
  • Neittaanmäki-Perttu, Noora; Gronroos, Mari; Tani, Taneli; Snellman, Erna (Acta Dermato-Venereologica, 2016)
  • Lehikoinen, Anni; Orden, Maija-Riitta; Heinonen, Seppo Tapani; Voutilainen, Raimo (Wiley-Blackwell, 2016)
    AimMaternal alcohol abuse is poorly recognised and causes developmental problems. This study explored the foetal central nervous systems (CNS), head circumference and psychomotor development of children exposed to drugs or alcohol during pregnancy up to 2.5years of age. MethodsWe recruited 23 pregnant women referred to Kuopio University Hospital, Finland, by their family doctor because of drug or alcohol abuse, and 22 control mothers. Foetal CNS parameters were measured by three-dimensional ultrasonography at the mean gestational age of 20weeks and the Griffiths Mental Developmental Scales (GMDS), and anthropometric measurements were carried out at the mean ages of one and 2.5years. ResultsThe exposed foetuses had decreased biparietal and occipito-frontal distances and head circumferences, but unchanged cerebellar volume at 20weeks, and decreased head circumferences and length and height at birth, one and 2.5years of age. They scored lower than the controls on the GMDS general quotient and the hearing, language and locomotor subscales at 2.5years of age. ConclusionMaternal alcohol or drug exposure was associated with decreased head size from mid-pregnancy to childhood and reduced development at 2.5years. Foetal head circumference at mid-pregnancy was a useful indicator of substance abuse affecting theCNS.
  • Saarnio, Elisa; Pekkinen, Minna; Itkonen, Suvi T; Kemi, Virpi; Karp, Heini; Kärkkäinen, Merja; Mäkitie, Outi; Lamberg-Allardt, Christel (BioMed Central, 2016)
    Abstract Background Vitamin D binding protein (DBP) binds vitamin D and its plasma metabolites, including 25-hydroxyvitamin D (25(OH)D), in the circulation. Only a small fraction circulates free (free 25(OH)D). Genetic variation of the GC gene, encoding DBP, has been associated with 25(OH)D concentrations. The roles of DBP and free 25(OH)D concentrations in the biological actions of vitamin D remain unclear. Methods We assessed the relationship between GC gene variants rs4588, rs7041, and rs705124, and serum total 25(OH)D, free and bioavailable 25(OH)D, and serum DBP and parathyroid hormone (PTH) concentrations in 622 Caucasian females (421) and males (201) aged 37–47 years. Concentrations of 25(OH)D, DBP, and PTH were measured from fasting blood samples. Dietary intakes of vitamin D and Ca were evaluated using 1-month food use frequency data, which were collected by a validated Food Frequency Questionnaire on vitamin D and calcium intakes. The subjects filled in the questionnaire covering overall health, medications, use of vitamin D and calcium supplements, and holidays in sunny locations. Three SNPs in the GC gene were genotyped: rs4588, rs7041, and rs705124. The SNPs rs4588 and rs7041 combine to form six common diplotypes. Free and bioavailable 25(OH)D were calculated by using specific binding coefficients. Differences among the diplo- and haplotypes of the GC gene in measures of 25(OH)D, DBP, and PTH were tested by analysis of covariance (ANCOVA) using appropriate covariates. Results We found significant variation among the SNPs rs4588 and rs7041 variants in DBP, total, free, and bioavailable 25(OH)D, and PTH. DBP concentration was lowest in genotype GC2/2 in both diplotypes and haplotypes (p = 0.039 and 0.039, respectively). The lowest 25(OH)D concentrations were found in diplotype variants GC1S/2, GC1S/F, and GC2/2 (p = 0.033), but free and bioavailable 25(OH)D concentrations were highest in the GC2/2 variant after corrected with a genotype-specific binding coefficient (p < 0.001 in both groups). Surprisingly, one of the lowest PTH concentrations was also present in variant GC2/2 in diplotypes (p = 0.040 of the overall ANCOVA analysis of PTH). Among SNP rs705124, there was a difference only in PTH concentrations (p = 0.013). Conclusions Our findings indicate that genetic variation of the DBP coding gene, and free concentrations of 25(OH)D may be relevant when vitamin D status, metabolism, and action are investigated.