A population-based nested case control study on recurrent pneumonias in children with severe generalized cerebral palsy: ethical considerations of the design and representativeness of the study sample

BACKGROUND: In children with severe generalized cerebral palsy, pneumonias are a major health issue. Malnutrition, dysphagia, gastro-oesophageal reflux, impaired respiratory function and constipation are hypothesized risk factors. Still, no data are available on the relative contribution of these possible risk factors in the described population. This paper describes the initiation of a study in 194 children with severe generalized cerebral palsy, on the prevalence and on the impact of these hypothesized risk factors of recurrent pneumonias. METHODS/DESIGN: A nested case-control design with 18 months follow-up was chosen. Dysphagia, respiratory function and constipation will be assessed at baseline, malnutrition and gastro-oesophageal reflux at the end of the follow-up. The study population consists of a representative population sample of children with severe generalized cerebral palsy. Inclusion was done through care-centres in a predefined geographical area and not through hospitals. All measurements will be done on-site which sets high demands on all measurements. If these demands were not met in "gold standard" methods, other methods were chosen. Although the inclusion period was prolonged, the desired sample size of 300 children was not met. With a consent rate of 33%, nearly 10% of all eligible children in The Netherlands are included (n = 194). The study population is subtly different from the non-participants with regard to severity of dysphagia and prevalence rates of pneumonias and gastro-oesophageal reflux. DISCUSSION: Ethical issues complicated the study design. Assessment of malnutrition and gastro-oesophageal reflux at baseline was considered unethical, since these conditions can be easily treated. Therefore, we postponed these diagnostics until the end of the follow-up. In order to include a representative sample, all eligible children in a predefined geographical area had to be contacted. To increase the consent rate, on-site measurements are of first choice, but timely inclusion is jeopardized. The initiation of this first study among children with severe neurological impairment led to specific, unexpected problems. Despite small differences between participants and non-participating children, our sample is as representative as can be expected from any population-based study and will provide important, new information to bring us further towards effective interventions to prevent pneumonias in this population

A study of some fundamental physicochemical variables on the morphology of mesoporous silica nanoparticles MCM-41 type

[EN] All variables affecting the morphology of mesoporous silica nanoparticles (MSN) should be carefully analyzed in order to truly tailored design their mesoporous structure according to their final use. Although complete control on MCM-41 synthesis has been already claimed, reproducibility and repeatability of results remain a big issue due to the lack of information reported in literature. Stirring rate, reaction volume, and system configuration (i.e., opened or closed reactor) are three variables that are usually omitted, making the comparison of product characteristics difficult. Specifically, the rate of solvent evaporation is seldom disclosed, and its influence has not been previously analyzed. These variables were systematically studied in this work, and they were proven to have a fundamental impact on final particle morphology. Hence, a high degree of circularity (C = 0.97) and monodispersed particle size distributions were only achieved when a stirring speed of 500 rpm and a reaction scale of 500 mL were used in a partially opened system, for a 2 h reaction at 80 degrees C. Well-shaped spherical mesoporous silica nanoparticles with a diameter of 95 nm, a pore size of 2.8 nm, and a total surface area of 954 m(2) g(-1) were obtained. Final characteristics made this product suitable to be used in biomedicine and nanopharmaceutics, especially for the design of drug delivery systems.This study was funded partially by Departamento Administrativo de Ciencia Tecnología e Innovación–COLCIENCIAS (recipient, Angela A. Beltrán-Osuna); Ministerio de Economía y Competitividad, MINECO, research number MAT2016-76039-C4-1-R (Recipient, José L. Gómez-Ribelles); and Universidad Nacional de Colombia, grant number DIB201010021438 (Recipient, Jairo E. Perilla).Beltrán-Osuna, A.; Gómez Ribelles, JL.; Perilla-Perilla, JE. (2017). A study of some fundamental physicochemical variables on the morphology of mesoporous silica nanoparticles MCM-41 type. Journal of Nanoparticle Research. 19(12):1-14. https://doi.org/10.1007/s11051-017-4077-2S1141912Barrabino A (2011) Synthesis of mesoporous silica particles with control of both pore diameter and particle size. Master Thesis, Chalmers University of Technology, SwedenBastos FS, Lima OA, Filho CR, Fernandes LD (2011) Mesoporous molecular sieve MCM-41 synthesis from fluoride media. Brazilian. J Chem Eng 28:649–658Beck JS, Vartuli JC, Roth WJ, Leonowicz ME, Kresge CT, Schmitt KD, Chu CTW, Olson DH, Sheppard EW, McCullen SB, Higgins JB, Schlenker JL (1992) A new family of mesoporous molecular sieves prepared with liquid crystal templates. J Am Chem Soc 114(27):10834–10843. https://doi.org/10.1021/ja00053a020Beltrán-Osuna AA, Perilla JE (2016) Colloidal and spherical mesoporous silica particles: synthesis and new technologies for delivery applications. J Sol-Gel Sci Technol 77(2):480–496. https://doi.org/10.1007/s10971-015-3874-2Bernardos A, Mondragón L, Aznar E et al (2010) Enzyme-responsive intracellular controlled release using nanometric silica mesoporous supports capped with “saccharides”. ACS Nano 4(11):6353–6368. https://doi.org/10.1021/nn101499dBharti C, Nagaich U, Pal AK, Gulati N (2015) Mesoporous silica nanoparticles in target drug delivery system: a review. Int J Pharm Investig 5(3):124–133. https://doi.org/10.4103/2230-973X.160844Brevet D, Hocine O, Delalande A, Raehm L, Charnay C, Midoux P, Durand JO, Pichon C (2014) Improved gene transfer with histidine-functionalized mesoporous silica nanoparticles. Int J Pharm 471(1-2):197–205. https://doi.org/10.1016/j.ijpharm.2014.05.020Cai Q, Luo Z, Pang W et al (2001) Dilute solution routes to various controllable morphologies of MCM-41 silica with a basic medium. Chem Mater 13(2):258–263. https://doi.org/10.1021/cm990661zChakraborty I, Mascharak PK (2016) Mesoporous silica materials and nanoparticles as carriers for controlled and site-specific delivery of gaseous signaling molecules. Microporous Mesoporous Mater 234:409–419. https://doi.org/10.1016/j.micromeso.2016.07.028Chen L, Zhang Z, Yao X, Chen X, Chen X (2015a) Intracellular pH-operated mechanized mesoporous silica nanoparticles as potential drug carries. Microporous Mesoporous Mater 201:169–175. https://doi.org/10.1016/j.micromeso.2014.09.023Chen X, Yao X, Wang C, Chen L, Chen X (2015b) Mesoporous silica nanoparticles capped with fluorescence-conjugated cyclodextrin for pH-activated controlled drug delivery and imaging. Microporous Mesoporous Mater 217:46–53. https://doi.org/10.1016/j.micromeso.2015.06.012Chen Y, Chen H, Shi J (2013) In vivo bio-safety evaluations and diagnostic / therapeutic applications of chemically designed mesoporous silica nanoparticles. Adv Mater 25(23):3144–3176. https://doi.org/10.1002/adma.201205292Chen Y, Shi X, Han B, Qin H, Li Z, Lu Y, Wang J, Kong Y (2012) The complete control for the nanosize of spherical MCM-41. J Nanosci Nanotechnol 12(9):7239–7249. https://doi.org/10.1166/jnn.2012.6459Cheng Y-J, Zeng X, Cheng D-B, Xu XD, Zhang XZ, Zhuo RX, He F (2016) Functional mesoporous silica nanoparticles (MSNs) for highly controllable drug release and synergistic therapy. Colloids Surfaces B Biointerfaces 145:217–225. https://doi.org/10.1016/j.colsurfb.2016.04.051Crommelin DJA, Florence AT (2013) Towards more effective advanced drug delivery systems. Int J Pharm 454(1):496–511. https://doi.org/10.1016/j.ijpharm.2013.02.020Edler KJ (1997) Synthesis and characterisation of the mesoporous molecular sieve, MCM-41. Doctoral dissertation, The Australian National University, AustraliaGuo Z, Liu X-M, Ma L, Li J, Zhang H, Gao YP, Yuan Y (2013) Effects of particle morphology, pore size and surface coating of mesoporous silica on naproxen dissolution rate enhancement. Colloids Surf B Biointerfaces 101:228–235. https://doi.org/10.1016/j.colsurfb.2012.06.026Han N, Wang Y, Bai J, Liu J, Wang Y, Gao Y, Jiang T, Kang W, Wang S (2016) Facile synthesis of the lipid bilayer coated mesoporous silica nanocomposites and their application in drug delivery. Microporous Mesoporous Mater 219:209–218. https://doi.org/10.1016/j.micromeso.2015.08.006Hu X, Wang Y, Peng B (2014) Chitosan-capped mesoporous silica nanoparticles as pH-responsive nanocarriers for controlled drug release. Chem - An Asian J 9(1):319–327. https://doi.org/10.1002/asia.201301105Huh S, Wiench JW, Yoo J et al (2003) Organic functionalization and morphology control of mesoporous silicas via a co-condensation synthesis method. Chem Mater 15(22):4247–4256. https://doi.org/10.1021/cm0210041Ikari K, Suzuki K, Imai H (2006) Structural control of mesoporous silica nanoparticles in a binary surfactant system. Langmuir 22(2):802–806. https://doi.org/10.1021/la0525527Iliade P, Miletto I, Coluccia S, Berlier G (2012) Functionalization of mesoporous MCM-41 with aminopropyl groups by co-condensation and grafting: a physico-chemical characterization. Res Chem Intermed 38(3-5):785–794. https://doi.org/10.1007/s11164-011-0417-5IUPAC (1985) Reporting physisorption data for gas/solid systems. Pure Appl Chem 57:603–619IUPAC (2014) Compendium of chemical terminology-gold book, 2.3.3. International Union of Pure and Applied ChemistryKhezri K, Roghani-Mamaqani H, Sarsabili M, Sobani M, Mirshafiei-Langari SA (2014) Spherical mesoporous silica nanoparticles/tailor-made polystyrene nanocomposites by in situ reverse atom transfer radical polymerization. Polym Sci Ser B 56(6):909–918. https://doi.org/10.1134/S1560090414660026Kresge CT, Leonowicz ME, Roth WJ, Vartuli JC, Beck JS (1992) Ordered mesoporous molecular sieves synthesized by a liquid-crystal template mechanism. Nature 359(6397):710–712. https://doi.org/10.1038/359710a0Lelong G, Bhattacharyya S, Kline S, Cacciaguerra T, Gonzalez MA, Saboungi ML (2008) Effect of surfactant concentration on the morphology and texture of MCM-41 materials. J Phys Chem C 112(29):10674–10680. https://doi.org/10.1021/jp800898nLv X, Zhang L, Xing F, Lin H (2016) Controlled synthesis of monodispersed mesoporous silica nanoparticles: particle size tuning and formation mechanism investigation. Microporous Mesoporous Mater 225:238–244. https://doi.org/10.1016/j.micromeso.2015.12.024Mamaeva V, Sahlgren C, Lindén M (2013) Mesoporous silica nanoparticles in medicine: recent advances. Adv Drug Deliv Rev 65(5):689–702. https://doi.org/10.1016/j.addr.2012.07.018Manzano M, Aina V, Areán CO, Balas F, Cauda V, Colilla M, Delgado MR, Vallet-Regí M (2008) Studies on MCM-41 mesoporous silica for drug delivery: effect of particle morphology and amine functionalization. Chem Eng J 137(1):30–37. https://doi.org/10.1016/j.cej.2007.07.078Merkus HG (2009) Particle size measurements: fundamentals, practice, quality. Springer Science +Businees Media B.V, The NetherlandsMorishige K, Fujii H, Uga M, Kinukawa D (1997) Capillary critical point of argon, nitrogen, oxygen, ethylene, and carbon dioxide in MCM-41. Langmuir 13(13):3494–3498. https://doi.org/10.1021/la970079ude Padua Oliveira DC, de Barros ALB, Belardi RM et al (2016) Mesoporous silica nanoparticles as a potential vaccine adjuvant against Schistosoma mansoni. J Drug Deliv Sci Technol 35:234–240. https://doi.org/10.1016/j.jddst.2016.07.002Phillips E, Penate-Medina O, Zanzonico PB, Carvajal RD, Mohan P, Ye Y, Humm J, Gonen M, Kalaigian H, Schoder H, Strauss HW, Larson SM, Wiesner U, Bradbury MS (2014) Clinical translation of an ultrasmall inorganic optical-PET imaging nanoparticle probe. Sci Transl Med 6(260):260ra149. https://doi.org/10.1126/scitranslmed.3009524Qu F, Zhu G, Lin H, Zhang W, Sun J, Li S, Qiu S (2006) A controlled release of ibuprofen by systematically tailoring the morphology of mesoporous silica materials. J Solid State Chem 179(7):2027–2035. https://doi.org/10.1016/j.jssc.2006.04.002Rafi AA, Mahkam M, Davaran S, Hamishehkar H (2016) A smart pH-responsive nano-carrier as a drug delivery system: a hybrid system comprised of mesoporous nanosilica MCM-41 (as a nano-container) & a pH-sensitive polymer (as smart reversible gatekeepers): preparation, characterization and in vitro releas. Eur J Pharm Sci 93:64–73. https://doi.org/10.1016/j.ejps.2016.08.005Rouquerol J, Rouquerol F, Llewellyn P, et al (2014) Adsorption by powders and porous solids: principles, methodology and applications. Elsevier Ltd.Selvam P, Bhatia SK, Sonwane CG (2001) Recent advances in processing and characterization of periodic mesoporous MCM-41 silicate molecular sieves. Ind Eng Chem Res 40(15):3237–3261. https://doi.org/10.1021/ie0010666Shi YT, Cheng HY, Geng Y, Nan HM, Chen W, Cai Q, Chen BH, Sun XD, Yao YW, Li HD (2010) The size-controllable synthesis of nanometer-sized mesoporous silica in extremely dilute surfactant solution. Mater Chem Phys 120(1):193–198. https://doi.org/10.1016/j.matchemphys.2009.10.045Shibata H, Chiba Y, Kineri T, Matsumoto M, Nishio K (2010) The effect of heat treatment on the interplanar spacing of the mesostructure during the synthesis of mesoporous MCM-41 silica. Colloids Surfaces A Physicochem Eng Asp 358(1-3):1–5. https://doi.org/10.1016/j.colsurfa.2009.12.020Slowing II, Vivero-Escoto JL, Wu C-W, Lin VSY (2008) Mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers. Adv Drug Deliv Rev 60(11):1278–1288. https://doi.org/10.1016/j.addr.2008.03.012Sun R, Wang W, Wen Y, Zhang X (2015) Recent advance on mesoporous silica nanoparticles-based controlled release system: intelligent switches open up. Nano 5(4):2019–2053. https://doi.org/10.3390/nano5042019U.S. Department of Health & Human Services (2015) Cancer Nanotechnology PlanUkmar T, Maver U, Planinšek O, Kaučič V, Gaberšček M, Godec A (2011) Understanding controlled drug release from mesoporous silicates: theory and experiment. J Control Release 155(3):409–417. https://doi.org/10.1016/j.jconrel.2011.06.038Vallet-Regi M, Arcos Navarrete D (2016) Nanoceramics in clinical use, 1st edn. The Royal Society of Chemistry, CambridgeVallet-Regi M, Rámila A, Del Real RP, Pérez-Pariente J (2001) A new property of MCM-41: drug delivery system. Chem Mater 13(2):308–311. https://doi.org/10.1021/cm0011559Varga N, Benko M, Sebok D et al (2015) Mesoporous silica core-shell composite functionalized with polyelectrolytes for drug delivery. Microporous Mesoporous Mater 213:134–141. https://doi.org/10.1016/j.micromeso.2015.02.008Wang Y, Zhao Q, Han N, Bai L, Li J, Liu J, Che E, Hu L, Zhang Q, Jiang T, Wang S (2015) Mesoporous silica nanoparticles in drug delivery and biomedical applications. Nanomed Nanotechnol Biol Med 11(2):313–327. https://doi.org/10.1016/j.nano.2014.09.014Wanyika H, Gatebe E, Kioni P et al (2011) Synthesis and characterization of ordered mesoporous silica nanoparticles with tunable physical properties by varying molar composition of reagents. African J Pharm Pharmacol 5(21):2402–2410. https://doi.org/10.5897/AJPP11.592Wu SH, Mou CY, Lin HP (2013) Synthesis of mesoporous silica nanoparticles. Chem Soc Rev 42(9):3862–3875. https://doi.org/10.1039/c3cs35405aXu X, Lü S, Gao C, Wang X, Bai X, Gao N, Liu M (2015a) Facile preparation of pH-sensitive and self-fluorescent mesoporous silica nanoparticles modified with PAMAM dendrimers for label-free imaging and drug delivery. Chem Eng J 266:171–178. https://doi.org/10.1016/j.cej.2014.12.075Xu X, Lü S, Gao C, Wang X, Bai X, Duan H, Gao N, Feng C, Liu M (2015b) Polymeric micelle-coated mesop orous silica nanoparticle for enhanced fluorescent imaging and pH-responsive drug delivery. Chem Eng J 279:851–860. https://doi.org/10.1016/j.cej.2015.05.085Xu X, Lü S, Gao C, Feng C, Wu C, Bai X, Gao N, Wang Z, Liu M (2016) Self-fluorescent and stimuli-responsive mesoporous silica nanoparticles using a double-role curcumin gatekeeper for drug delivery. Chem Eng J 300:185–192. https://doi.org/10.1016/j.cej.2016.04.087Yang Y, Yu C (2015) Advances in silica based nanoparticles for targeted cancer therapy. Nanomedicine nanotechnology. Biol Med 12(2):317–332. https://doi.org/10.1016/j.nano.2015.10.018Zhang H, Tong C, Sha J, Liu B, Lü C (2015) Fluorescent mesoporous silica nanoparticles functionalized graphene oxide: a facile FRET-based ratiometric probe for Hg2+. Sensors Actuators B Chem 206:181–189. https://doi.org/10.1016/j.snb.2014.09.051Zhou C, Yan C, Zhao J, Wang H, Zhou Q, Luo W (2016) Rapid synthesis of morphology-controlled mesoporous silica nanoparticles from silica fume. J Taiwan Inst Chem Eng 62:307–312. https://doi.org/10.1016/j.jtice.2016.01.03

EGF functionalized polymer-coated gold nanoparticles promote EGF photostability and EGFR internalization for photothermal therapy

The application of functionalized nanocarriers on photothermal therapy for cancer ablation has wide interest. The success of this application depends on the therapeutic efficiency and biocompatibility of the system, but also on the stability and biorecognition of the conjugated protein. This study aims at investigating the hypothesis that EGF functionalized polymer -coated gold nanoparticles promote EGF photostability and EGFR internalization, making these conjugated particles suitable for photothermal therapy. The conjugated gold nanoparticles (100-200 nm) showed a plasmon absorption band located within the near infrared range (650-900 nm), optimal for photothermal therapy applications. The effects of temperature, of polymer-coated gold nanoparticles and of UVB light (295nm) on the fluorescence properties of EGF have been investigated with steady-state and time-resolved fluorescence spectroscopy. The fluorescence properties of EGF, including the formation of Trp and Tyr photoproducts, is modulated by temperature and by the intensity of the excitation light. The presence of polymeric-coated gold nanoparticles reduced or even avoided the formation of Trp and Tyr photoproducts when EGF is exposed to UVB light, protecting this way the structure and function of EGF. Cytotoxicity studies of conjugated nanoparticles carried out in normal-like human keratinocytes showed small, concentration dependent decreases in cell viability (0-25%). Moreover, conjugated nanoparticles could activate and induce the internalization of overexpressed Epidermal Growth Factor Receptor in human lung carcinoma cells. In conclusion, the gold nanoparticles conjugated with Epidermal Growth Factor and coated with biopolymers developed in this work, show a potential application for near infrared photothermal therapy, which may efficiently destroy solid tumours, reducing the damage of the healthy tissue.Support was provided by: Fundacao para a Ciencia e Tecnologia (FCT) for the financial support under the project reference PTDC/BBB-BMC/0611/2012 [https://www.fct.pt/apoios/projectos)]. The work at CBMA was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI) [https://www.fct.pt/apoios/projectos]; European Commission through the project H2020-644242-SAPHELY (https://saphely.eu/project.php) and the project H2020-634013-2-PHOCNOSIS [http://cordis.europa.eu/project/rcn/193268_en.html].The authors would like to thank Fundacao para a Ciencia e Tecnologia (FCT) for the financial support under the project reference PTDC/BBB-BMC/0611/2012. The work at CBMA was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI). The authors acknowledge the funding from the European Commission through the project H2020-644242-SAPHELY and the project H2020-634013-2-PHOCNOSIS. Finally, the authors would also like to thank the master student Joao Lopes from Universidade Lusofona (Portugal) for the help with in vitro cytotoxic assays. Isabel Correia acknowledges FCT for Investigator FCT contract.info:eu-repo/semantics/publishedVersio

A new class of glycomimetic drugs to prevent free fatty acid-induced endothelial dysfunction

Background: Carbohydrates play a major role in cell signaling in many biological processes. We have developed a set of glycomimetic drugs that mimic the structure of carbohydrates and represent a novel source of therapeutics for endothelial dysfunction, a key initiating factor in cardiovascular complications. Purpose: Our objective was to determine the protective effects of small molecule glycomimetics against free fatty acid­induced endothelial dysfunction, focusing on nitric oxide (NO) and oxidative stress pathways. Methods: Four glycomimetics were synthesized by the stepwise transformation of 2,5­dihydroxybenzoic acid to a range of 2,5­substituted benzoic acid derivatives, incorporating the key sulfate groups to mimic the interactions of heparan sulfate. Endothelial function was assessed using acetylcholine­induced, endotheliumdependent relaxation in mouse thoracic aortic rings using wire myography. Human umbilical vein endothelial cell (HUVEC) behavior was evaluated in the presence or absence of the free fatty acid, palmitate, with or without glycomimetics (1µM). DAF­2 and H2DCF­DA assays were used to determine nitric oxide (NO) and reactive oxygen species (ROS) production, respectively. Lipid peroxidation colorimetric and antioxidant enzyme activity assays were also carried out. RT­PCR and western blotting were utilized to measure Akt, eNOS, Nrf­2, NQO­1 and HO­1 expression. Results: Ex vivo endothelium­dependent relaxation was significantly improved by the glycomimetics under palmitate­induced oxidative stress. In vitro studies showed that the glycomimetics protected HUVECs against the palmitate­induced oxidative stress and enhanced NO production. We demonstrate that the protective effects of pre­incubation with glycomimetics occurred via upregulation of Akt/eNOS signaling, activation of the Nrf2/ARE pathway, and suppression of ROS­induced lipid peroxidation. Conclusion: We have developed a novel set of small molecule glycomimetics that protect against free fatty acidinduced endothelial dysfunction and thus, represent a new category of therapeutic drugs to target endothelial damage, the first line of defense against cardiovascular disease

Reference values of Forced Expiratory Volumes and pulmonary flows in 3–6 year children: a cross-sectional study

<p>Abstract</p> <p>Background</p> <p>The aims of this study were to verify the feasibility of respiratory function tests and to assess their validity in the diagnosis of respiratory disorders in young children.</p> <p>Methods</p> <p>We performed spirometry and collected information on health and parents' lifestyle on a sample of 960 children aged 3–6.</p> <p>Results</p> <p>The cooperation rate was 95.3%. Among the valid tests, 3 or more acceptable curves were present in 93% of cases. The variability was 5% within subjects in 90.8% of cases in all the parameters. We propose regression equations for FVC (Forced Vital Capacity), FEV1, FEV0.5, FEV0.75 (Forced Expiratory Volume in one second, in half a second and in 3/4 of a second), and for Maximum Expiratory Flows at different lung volume levels (MEF75, 50, 25). All parameters are consistent with the main reference values reported in literature. The discriminating ability of respiratory parameters versus symptoms always shows a high specificity (>95%) and a low sensitivity (<20%) with the highest OR (10.55; IC95% 4.42–25.19) for MEF75. The ability of FEV0.75 to predict FEV1 was higher than that of FEV0.50: FEV0.75 predicts FEV1 with a determination coefficient of 0.95.</p> <p>Conclusion</p> <p>Our study confirms the feasibility of spirometry in young children; however some of the current standards are not well suited to this age group. Moreover, in this restricted age group the various reference values have similar behaviour.</p