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Thickness and marking quality of different occlusal contact registration strips

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www.scielo.br/jaos http://dx.doi.org/10.1590/1678-775720140117 Thickness and m arking quality of different occlusal cont act regist rat ion st rips Maria Fernanda de Souza Mauá Serapião TOLEDO1, Renata Pilli JÓIAS2, Yves Santini MARQUES-IASI1, Ana Christina Claro NEVES3, Sigmar de Mello RODE4 1- Private Practice, São Paulo, SP, Brazil. 2- Department of Bioscience and Oral Diagnosis, Oral Biopathology Graduate Program, School of Dentistry, Science and Technology Institute, Univ. Estadual Paulista (UNESP), São José dos Campos, SP, Brazil. 3- Department of Dentistry, University of Taubaté (UNITAU), Taubaté, SP, Brazil. 4- Department of Dental Materials and Prosthodontics, School of Dentistry, Science and Technology Institute, Univ. Estadual Paulista (UNESP), São José dos Campos, SP, Brazil. Corresponding address: Sigmar de Mello Rode - Departmento de Materiais Odontológicos e Prótese - Instituto de Ciência e Tecnologia - Univ. Estadual Paulista (UNESP) - R. Eng Francisco José Longo, 777 - Jardim São Dimas - São José dos Campos - SP - Brazil - 12245-000 - e-mail: sigmarrode@uol.com.br 6XEPLWWHG0DUFK0RGL¿FDWLRQ-XQH$FFHSWHG6HSWHPEHU ABSTRACT O bj ect ives: Evaluat e t he t hickness and t he m arking qualit y of different occlusal cont act regist rat ion st rips ( OCRS) and a possible correlat ion bet ween t hem . Mat erial and 0HWKRGV7KHIROORZLQJ2&56ZHUHVHOHFWHG$FFX¿OP,,%.%.%.%.%. and BK31. The t hickness was m easured in t hree point s of t he OCRS wit h an elect ronic m easuring device ( TESA) , and t he m ean was calculat ed. To produce t he m arks on t he st rips, com posit e resin specim ens were adapt ed t o a universal t est ing m achine ( Versat 2000) wit h 40 kgf load cell at a speed of 1.0 m m / m in. The m ark im ages were phot ographed wit h a st ereoscopic m icroscope ( St em i SV11) and processed and analyzed by t he 550- Leica Qwin ® DQDO\]HU 5HVXOWV 9DOXHV NjP  IRXQG LQ WKH st and 2 nd t hickness m easurem ent s were: $FFX¿OP,,DQG%.DQG%.DQG%.DQG 8.7; BK23 - 9.8 and 7.9; BK28 - 12.8 and 10.0; and BK31 - 8.4 and 8.0, respect ively. The m ean ( m m 2 YDOXHVIRXQGLQWKHPDUNDUHDVZHUH$FFX¿OP,,%.%. - 0.045; BK22 - 0.012; BK23 - 0.022; BK28 - 0.024; and BK31 - 0.024. The result s were subm it t ed t o t he Kruskal-Wallis ( p< 0.05) and Pearson’s correlat ion t est s. Conclusions: Only in t he 2 nd m easurem ent , t he OCRS t hickness observed was sim ilar t o t he value indicat ed E\WKHPDQXIDFWXUHUVWKH$FFX¿OP,,DQGWKH%.VWULSVVKRZHGWKHEHWWHUPDUNVDQG no correlat ion was found bet ween t he t hickness and t he m arking area. Keyw ords: Dent al occlusion. Occlusal adj ust m ent . Prosthodontics. Dentistry. Oral diagnosis. I N TROD UCTI ON posit ion are required 8 . I nappropriat e occlusal cont act s can be t riggered by occlusal int er fer ences, ser ious discr epancies bet ween cent ric relat ion ( CR) and cent ric occlusion ( CO) , loss of t eet h, absence of t ight cont act s, m alocclusion, bruxism , loss of vert ical dim ension, and increased t oot h m obilit y, am ong ot her fact ors. I n t h e p r esen ce of occl u sal d i sh ar m on y associat ed w it h t em porom andibular j oint s ( TMJ) p h y siolog ical an d f u n ct ion al im b alan ce, som e st ress m ay t rigger various delet erious effect s, such as t oot h m obilit y and/ or sensit iv it y, per iodont al problem s, incoordinat ion or hyperfunct ion of t he m ast icat or y m uscles, and lat eral occlusal loads, am ong ot hers3,5,7 . Wit h a v iew t o t h e p r eser v at ion of d en t al, periodont al, art icular, and m uscular healt h, it is im port ant t o observe adequat e st at ic and funct ional o ccl u si o n . I n st at i c o ccl u si o n , si m u l t an eo u s, bilat eral, and hom ogeneous cont act s should be observed bet ween t he m axillary and m andibular t eet h; absence of r ot at ion and cr ow ding, t ight cont act s, pr oper m esio- dist al cr ow n angulat ion and labio- lingual crown inclinat ion, and m oderat e over j et and over bit e 1 . Wit h r egar d t o funct ional m ovem ent s of t he j aw, canine guidance wit hout int erference or prem at ure cont act s on bot h sides and m ut ually prot ect ed occlusion in t he prot rusive J Appl Oral Sci. 516 2014;22(6):516-21 Thickness and marking quality of different occlusal contact registration strips and t he t ype of ink on it s surface are relat ed t o t he m arks it produces on t he t oot h surface. However, som e unfavorable aspect s have been perceived, such as m oist ur e failing ( saliva) , low elast icit y, WKLQ EDVH DQG UHODWLYHO\ LQÀH[LEOH EDVH PDWHULDO All t hese fact ors m ay result in large num bers of pseudocont act s9 . Silk st r ip s h av e b een con sid er ed t h e b est m at erial for t his purpose due t o t heir soft t ext ure, which does not produce pseudocont act s2 , alt hough t hey could be dam aged by saliva. I t is advisable t o st ore silk st rips in a cool and closed place9 . Plast ic st rips are t hinner m at erials t hat provide m ore accurat e occlusal cont act regist rat ions, which are m ore easily visualized. However, as t hey are not capable of regist ering low- pressure sit uat ions ( st raight and shiny surfaces) , t hey need t o be used under st rong pressure. Based on t he lack of st udies evaluat ing t he accu r acy o f m at er i al s u sed t o m ar k o ccl u sal cont act s, t he obj ect ives of t his st udy w ere: 1 evaluat e t he t hickness of different OCRS; 2 - assess t he qualit y of t he m arking each OCRS produces; and 3 – correlat e t he OCRS t hickness and t he qualit y of m arks. Having est ablished t he im port ance of balanced an d h ar m on iou s occlu sion , it is im p or t an t f or den t ist s t o per f or m r ou t in e an aly sis of den t al occlusion and check cont act bet ween t he m axillary and m andibular arches. For t his purpose, occlusal contact registration strips ( OCRS) are recom m ended f or r ecor d in g occlu sal con t act s, as an aid t o diagnosis, enabling proper t reat m ent planning and m inim izing or solving occlusal disorders. There is a large num ber of m at erials available IRUFKHFNLQJDQGGH¿QLQJRFFOXVDOFRQWDFWVVXFKDV waxes, carbon paper, m et al sheet s, plast ic sheet s, silk st rips, as well as different m et hods t o m easure t hese cont act s. Am ong t hese m et hods, t here are bot h qualit at ive and quant it at ive t ypes; t hus, t he form er t ype is used t o locat e t he cont act point s, and t he lat t er, t o set t heir sequence and densit y. The qualit at ive m et hod requires t he sam e m at erials as WKRVHLQGLFDWHGIRUWKHYHUL¿FDWLRQDQGLGHQWL¿FDWLRQ of cont act s, whereas in t he quant it at ive m et hod, t he “ T- scan” and “ phot o- occlusion” are used 9 . Som e fact ors need t o be considered in choosing t he m ost appropriat e OCRS for checking occlusal co n t a ct s, su ch a s t h i ck n e ss, st r e n g t h , a n d elast icit y 6 . I f t he OCRS is ext rem ely t hick, it m ay int er fer e w it h t he pat ient ’s occlusal per cept ion, ZKLFK UDQJHV IURP  WR  NjP 5HVLVWDQFH refers t o t he OCRS ability t o wit hst and t he m oist oral HQYLURQPHQWZLWKRXWLPSDLULQJLWVDELOLW\WRGH¿QH t he occlusal cont act s. The elast icit y is t he OCRS abilit y t o st ret ch wit hout t earing when it is pulled out of t he oral cavit y aft er checking t he cont act 4 . I t is recom m ended t hat t he OCRS should be used only once t o m ark t he cont act s, because, ot herwise, t he num ber of cont act s m ay decrease due t o t he m at erial det eriorat ion 9 . Carbon paper is a widely used m at erial because of it s low cost and ease of use of it s widt h, t hickness M ATERI AL AN D M ETH OD S Sa m ple Sam ples of seven t ypes of OCRS ( n= 10) were select ed ( Figure 1) and st andardized t o a lengt h of 5.5 cm and widt h of 3.0 cm . M e a su r in g t h e OCRS t h ick n e ss The t hickness evaluat ion was perform ed t hree t im es at t hree point s, one cent ral and t he ot hers at each ext rem it y, using t he elect ronic m easuring dev ice TESA ( TESA, Micr ont esa, Kem pt on Par k , Kind Brand Thickness Color $FFX¿OP,, Parkell, Farmingdale, N.Y., USA ȝP Black/red BK 20- Folha de Articulação $UWLIROXOWUD¿QD Bausch, Nashua, NH, USA ȝP Black (single-side) BK 21- Folha de Articulação $UWLIROXOWUD¿QD Bausch, Nashua, NH, USA ȝP Red (single-side) BK 22- Folha de Articulação $UWLIROXOWUD¿QD Bausch, Nashua, NH, USA ȝP Green (single-side) BK 23- Folha de Articulação $UWLIROXOWUD¿QD Bausch, Nashua, NH, USA ȝP Blue (single-side) BK 28- Folha de Articulação $UWLIROXOWUD¿QD Bausch, Nashua, NH, USA ȝP Black/red BK 31- Arti-fol Metallic Folha de Shimstock Bausch, Nashua, NH, USA ȝP Red (single-side) Figure 1- Occlusal contact registration strips (OCRS) used to record occlusal contacts J Appl Oral Sci. 517 2014;22(6):516-21 TOLEDO MFSMS, JÓIAS RP, MARQUES-IASI YS, NEVES ACC, RODE SM Spe cim e n Gaut eng, Sout h Africa) wit h a clock m icrom et er UHDGLQJVFDOHVNjPNjPNjPXP  NjP  DQG D IHHOHU LQ RUGHU WR REWDLQ WKH PHDQ t hickness values. For t he t hickness evaluat ion, each OCRS was SODFHG EHWZHHQ WKH IHHOHU DQG D ÀDW WDEOH DQG t he reading scale was set t o zero, so t hat all t he m easurem ent s were perform ed at t he sam e point , using t he sam e references for st andardizat ion. A u n iv er sal t est in g m ach in e ( Ver sat 2 0 0 0 , Panam bra Zwick/ Roell, São Bernardo do Cam po, SP, Brazil) wit h a 40 kgf load cell was used t o m ake t he OCRS m arks on t he specim ens. Aft er t his, t he second t hickness m easurem ent was evaluat ed at WKHVDPHSRLQWWKDWKDGUHFHLYHGWKH¿VWSXQFWXUH load , f ollow in g ex act ly t h e sam e p r ot ocol as GHVFULEHGLQWKH¿UVWPHDVXUHPHQW Th e sp ecim en s con sist ed of n y lon d ev ices m easur ing 3 cm in height x 5 cm in diam et er, w it h a cen t r al ch an n el of 3 cm d iam et er x 5 m m in widt h x 2 m m in dept h. To obt ain t he t en VSHFLPHQVWKHF\OLQGHUZDV¿OOHGZLWKFRPSRVLWH resin Filt ek Z350 ( 3M, ESPE, Sum aré, SP, Brasil) using t he incr em ent al t echnique and 30- second poly m er izat ion ( Ult ralux , Dabi At lant e, Ribeir ão Pret o, SP, Brazil) . Decreasing granulat ions of wat er abrasive papers were used t o polish t he specim ens. M a r k in g t h e con t a ct s The com pression t est was also perform ed wit h t he 2000 Versat m achine, using a 500 kg load cell and 40 kgf at a speed of 1 m m / m in. A st ainless st eel ball 2.5 m m in diam et er was coupled t o t he t op of t he m achine, t o m ake t he OCRS punct ure t oward t he specim en. I n t he specim ens, t here w ere predet erm ined areas where t he cont act s would be dist ribut ed on t he surface of t he com posit e resin t o enable t he area WREHLGHQWL¿HGDQGPHDVXUHG7KHUHZDVDQLQWHUYDO wit h a m inim um dist ance of 6.5 m m bet ween t he m arks ( Figure 2) . The bilat eral surface OCRS were t est ed on t he VDPH VLGH LQ WKH ¿UVW ¿YH VSHFLPHQV DQG RQ WKH RSSRVLWHVLGHLQWKHQH[W¿YHVSHFLPHQV Sca n n in g a n d im a ge a n a lysis The m arks were photographed with a stereoscopic PLFURVFRSH DW [ PDJQL¿FDWLRQ 6WHPL 69 Zeiss, Oberkochen, Baden-Württem berg, Germ any) , and t he im ages were processed wit h t he Qwin ® Leica 550 ( Leica Microsyst em s I m age Solut ions SA, Wet zlar, Hessen, Germ any) , in which t he higher int ensit y pix els w er e r ecognized by a soft w ar e and highlight ed in red, and t he area around t hem was also grouped. Finally, for each specim en, t he m arking area was calculat ed in m m 2 ( Figures 3a, b) . Figure 2- Contact marks Figure 3-$FFX¿OP,,PDUNEHIRUHDQGDIWHUWUHDWPHQWRIWKHDUHDE\/HLFDDQDO\]HU J Appl Oral Sci. 518 2014;22(6):516-21 0.1584 RESULTS 7KH WKLFNQHVV YDOXHV REWDLQHG LQ WKH ¿UVW DQG second m easurem ent s ( Table 1) were st at ist ically different . ANOVA showed t hat t he “ p” values were lower t han 0.05 in all groups ( Table 2) , m eaning t hat t here ZHUHVLJQL¿FDQWFKDQJHVLQDOOWKH2&56DIWHUWKH m arks were perform ed. 7KH 3HDUVRQ FRUUHODWLRQ FRHI¿FLHQW U 3HDUVRQ 0.3495) showed no correlat ion bet ween t he OCRS t hickness and t he area of t he m ark. I n t he diagram ( Figure 4) , hom ogeneous dispersion of t he point s in t he t wo OCRS t hickness t racks can be observed. Thus, t his dat a, com bined wit h t he low “ r ” value, indicat es t hat t here is no correlat ion. 0.6660 0.9877 0.2910 0.2771 Dat a an aly sis w as car r ied ou t by m ean s of t he D’Agost ino nor m alit y t est , and because t he dat a did n ot adh er e t o t h e n or m al cu r v e, t h e nonparam et ric Kruskal-Wallis t est was applied at a OHYHORIVLJQL¿FDQFHRI$129$IRUWZRSDLUHG sam ples was used t o visualize t he difference t hat RFFXUUHGEHWZHHQWKH¿UVWDQGWKHVHFRQGWKLFNQHVV m easu r em en t ( sig n if ican ce lev el of 5 % ) . Th e Pearson correlat ion t est was applied in order t o ¿QGRXWZKHWKHUWKHUHZDVDQ\SRVVLEOHFRUUHODWLRQ bet ween t he OCRS t hickness and t he area of t he m ark. 0.8714 0.0001* 0.0009* 0.0000* 0.0001* 0.0000* 0.0055 0.0000* 0.0000* 0.1452 10 D I SCUSSI ON The int er est in inv est igat ing t he OCRS w as aroused due t o t he lack of research and art icles relat ed t o t his t opic. There have been few st udies t hat have t est ed t he accuracy and r eliabilit y of t hese m at er ials and concer ning t he t echniques used for m arking occlusal cont act s, considering t hat t he occlusal analysis m ade wit h t hese OCRS is of param ount im port ance in m any dent al procedures. I t is well known t hat t he effect of t he pressure DUHDRQWKH2&56LVLQÀXHQFHGE\WKHUHVLOLHQFH and viscosit y of t he periodont al ligam ent , but t his in v it r o st udy pr ov ides an init ial under st anding regarding t he OCRS qualit y of m arking since t he load applied is pat t erned. Sam ples of t he OCRS select ed for t he survey pr esent ed t w o t ypes of base m at er ial ( silk and m et al) , four pigm ent colors ( blue, black, green, and red) and bilat eral st aining ( black/ red) when FRQVLGHULQJ$FFX¿OP,,DQG%. Depending on t he OCRS phy sical pr oper t ies ( t hickness, plast ic defor m at ion and r esist ance) , there m ay be interference in the m arking processes; t hus, a product m ay m ark a real cont act area; produce a false m ark, or m ark an area t hat has no occlusal cont act 10 . St udies have shown t hat t he *statistically different values 0.0763 0.0878 St a t ist ica l a n a lysis 0.8472 0.0015* 0.1081 - - - - 0.0719 - 0.0763 0.2007 0.7229 0.8593 0.7229 0.4409 9 0.0191 0.0357* 0.2414 0.0022* 0.0018* 0.0843 0.0110* 0.0005* 0.0004* 0.0000* 0.0001* 0.0030* 0.0000* 0.0000* 0.9631 8 0.3277 0.1484 0.0059* 0.1047 0.0334* 0.6029 0.3550 0.0038* 0.0259 0.0001* 0.0042* 0.0000* 0.0907 0.0000* 0.0003* 0.6886 7 0.5324 0.2704 - - - 0.8593 0.0004* 0.0009* 0.0000* 0.0001* 0.0000* 0.0052* 0.0000* 0.0000* 0.3053 6 - - - - - 0.0125* 0.0016* 0.0005* 0.0002* 0.0001* 0.0089* 0.0000* 0.0000* - 5 - - - - - - - - - - - - - - - - 0.0512 0.3353 0.2414 0.4363 0.1967 0.7432 0.5922 - 0.1837 0.0218* 0.1573 4 0.3164 2 3 0.5249 - - - - - 4 3 2 1 p - - - - - 6 5 - - 7 - 9 8 - 2nd 1st 2nd Measurement 1st 2nd Measurement 1st 2nd Measurement 1st 2nd Measurement 1st 2nd Measurement 1st 2nd Measurement 1st 2nd Measurement 1st 2nd Measurement Table 1-2FFOXVDOFRQWDFWUHJLVWUDWLRQVWULSV 2&56 WKLFNQHVV³S´YDOXHV VLJQL¿FDQFHOHYHO ±¿UVWDQGVHFRQGPHDVXUHPHQWV 1st Measurement Thickness and marking quality of different occlusal contact registration strips J Appl Oral Sci. 519 2014;22(6):516-21 TOLEDO MFSMS, JÓIAS RP, MARQUES-IASI YS, NEVES ACC, RODE SM Table 2-2FFOXVDOFRQWDFWUHJLVWUDWLRQVWULSV 2&56 WKLFNQHVV³S´YDOXHV VLJQL¿FDQFHOHYHO PDUNLQJDUHD p 1 2 3 4 5 6 7 8 9 2 0.1607 - - - - - - - - 3 0.3987 0.5763 - - - - - - - 4 0.7933 0.254 0.5606 - - - - - - 5 0.005* 0.1607 0.0498* 0.011* - - - - - 6 0.0744 0.7028 0.3471 0.128 0.3071 - - - - 7 0* 0.0008* 0.0001* 0* 0.0516 0.003* - - - 8 0.0003* 0.0262* 0.0054* 0.0008* 0.4117 0.0655 0.2605 - - 9 0.3108 0.6971 0.8654 0.4524 0.0731 0.4409 0.0002* 0.009* - 10 0.0004* 0.035* 0.0077* 0.0012* 0.4807 0.0843 0.2146 0.908 0.0125* *statistically different values t he m arker subst ance affect ed t he size of t he m ark. The r ed OCRS r egist er ed com parat iv ely lar ger m arks t han t he ot her st rips of sim ilar t hickness, w hile t hinner poly est er plast ic st r ips pr oduced sm aller m arks t han t he paper or silk t ypes9 . The correlat ion bet ween t he OCRS t hickness and t he area of t he m ark, by t he Pearson’s correlat ion t est , show ed t hat bot h t he t hicker and t hinner OCRS pr odu ced lar ger an d sm aller m ar k s, so t here was no correlat ion bet ween t he t hickness m easurem ent and t he m arking area. Thus, based RQWKHUHVXOWVLWLVQRWSRVVLEOHWRDI¿UPWKDWWKH t hicker t he st rip t he bigger t he m ark produced, in disagreem ent wit h ot her aut hors2,9 . Alt hough t he m et hodology of t his research is very sim ilar t o t hat described in t he lit erat ure 9 , t he result s were not coincident . The t wo m et hods are sim ilar, but wit h som e changes t hat could have caused t his difference. A possible hypot hesis m ight be: t he sur face m at er ial m ar k ing, t he m ar k ing analysis, t he num ber of m arkings of each OCRS on t he specim ens, and t he t ypes of OCRS st udied. Figure 4- Pearson’s correlation test dispersion diagram thickness x marking area equilibrium value of MeCpG steps (,+14 deg.) [31,44]. In comparison, methylation has a significantly lower stability cost when happening at major groove positions, such as 211 and 21 base pair from dyad (mutations 9 and 12), where the roll of the nucleosome bound conformation (+10 deg.) is more compatible with the equilibrium geometry of MeCpG steps. The nucleosome destabilizing effect of cytosine methylation increases with the number of methylated cytosines, following the same position dependence as the single methylations. The multiple-methylation case reveals that each major groove meth- PLOS Computational Biology | www.ploscompbiol.org 3 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning ylation destabilizes the nucleosome by around 1 kJ/mol (close to the average estimate of 2 kJ/mol obtained for from individual methylation studies), while each minor groove methylation destabilizes it by up to 5 kJ/mol (average free energy as single mutation is around 6 kJ/mol). This energetic position-dependence is the reverse of what was observed in a recent FRET/SAXS study [30]. The differences can be attributed to the use of different ionic conditions and different sequences: a modified Widom-601 sequence of 157 bp, which already contains multiple CpG steps in mixed orientations, and which could assume different positioning due to the introduction of new CpG steps and by effect of the methylation. The analysis of our trajectories reveals a larger root mean square deviation (RMSD) and fluctuation (RMSF; see Figures S2– S3 in Text S1) for the methylated nucleosomes, but failed to detect any systematic change in DNA geometry or in intermolecular DNA-histone energy related to methylation (Fig. S1B, S1C, S4–S6 in Text S1). The hydrophobic effect should favor orientation of the methyl group out from the solvent but this effect alone is not likely to justify the positional dependent stability changes in Figure 2, as the differential solvation of the methyl groups in the bound and unbound states is only in the order of a fraction of a water molecule (Figure S5 in Text S1). We find however, a reasonable correlation between methylation-induced changes in hydrogen bond and stacking interactions of the bases and the change in nucleosome stability (see Figure S6 in Text S1). This finding suggests that methylation-induced nucleosome destabilization is related to the poorer ability of methylated DNA to fit into the required conformation for DNA in a nucleosome. Changes in the elastic deformation energy between methylated and un-methylated DNA correlate with nucleosomal differential binding free energies To further analyze the idea that methylation-induced nucleosome destabilization is connected to a worse fit of methylated DNA into the required nucleosome-bound conformation, we computed the elastic energy of the nucleosomal DNA using a harmonic deformation method [36,37,44]. This method provides a rough estimate of the energy required to deform a DNA fiber to adopt the super helical conformation in the nucleosome (full details in Suppl. Information Text S1). As shown in Figure 2, there is an evident correlation between the increase that methylation produces in the elastic deformation energy (DDE def.) and the free energy variation (DDG bind.) computed from MD/TI calculations. Clearly, methylation increases the stiffness of the CpG step [31], raising the energy cost required to wrap DNA around the histone octamers. This extra energy cost will be smaller in regions of high positive roll (naked DNA MeCpG steps have a higher roll than CpG steps [31]) than in regions of high negative roll. Thus, simple elastic considerations explain why methylation is better tolerated when the DNA faces the histones through the major groove (where positive roll is required) that when it faces histones through the minor groove (where negative roll is required). Nucleosome methylation can give rise to nucleosome repositioning We have established that methylation affects the wrapping of DNA in nucleosomes, but how does this translate into chromatin structure? As noted above, accumulation of minor groove methylations strongly destabilizes the nucleosome, and could trigger nucleosome unfolding, or notable changes in positioning or phasing of DNA around the histone core. While accumulation of methylations might be well tolerated if placed in favorable positions, accumulation in unfavorable positions would destabilize the nucleosome, which might trigger changes in chromatin structure. Chromatin could in fact react in two different ways in response to significant levels of methylation in unfavorable positions: i) the DNA could either detach from the histone core, leading to nucleosome eviction or nucleosome repositioning, or ii) the DNA could rotate around the histone core, changing its phase to place MeCpG steps in favorable positions. Both effects are anticipated to alter DNA accessibility and impact gene expression regulation. The sub-microsecond time scale of our MD trajectories of methylated DNAs bound to nucleosomes is not large enough to capture these effects, but clear trends are visible in cases of multiple mutations occurring in unfavorable positions, where unmethylated and methylated DNA sequences are out of phase by around 28 degrees (Figure S7 in Text S1). Due to this repositioning, large or small, DNA could move and the nucleosome structure could assume a more compact and distorted conformation, as detected by Lee and Lee [29], or a slightly open conformation as found in Jimenez-Useche et al. [30]. Using the harmonic deformation method, we additionally predicted the change in stability induced by cytosine methylation for millions of different nucleosomal DNA sequences. Consistently with our calculations, we used two extreme scenarios to prepare our DNA sequences (see Fig. 3): i) all positions where the minor grooves contact the histone core are occupied by CpG steps, and ii) all positions where the major grooves contact the histone core are occupied by CpG steps. We then computed the elastic energy required to wrap the DNA around the histone proteins in unmethylated and methylated states, and, as expected, observed that methylation disfavors DNA wrapping (Figure 3A). We have rescaled the elastic energy differences with a factor of 0.23 to match the DDG prediction in figure 2B. In agreement with the rest of our results, our analysis confirms that the effect of methylation is position-dependent. In fact, the overall difference between the two extreme methylation scenarios (all-in-minor vs all-in-major) is larger than 60 kJ/mol, the average difference being around 15 kJ/ mol. We have also computed the elastic energy differences for a million sequences with CpG/MeCpG steps positioned at all possible intermediate locations with respect to the position (figure 3B). The large differences between the extreme cases can induce rotations of DNA around the histone core, shifting its phase to allow the placement of the methylated CpG steps facing the histones through the major groove. It is illustrative to compare the magnitude of CpG methylation penalty with sequence dependent differences. Since there are roughly 1.5e88 possible 147 base pairs long sequence combinations (i.e., (4n+4(n/2))/2, n = 147), it is unfeasible to calculate all the possible sequence effects. However, using our elastic model we can provide a range of values based on a reasonably large number of samples. If we consider all possible nucleosomal sequences in the yeast genome (around 12 Mbp), the energy difference between the best and the worst sequence that could form a nucleosome is 0.7 kj/mol per base (a minimum of 1 kJ/mol and maximum of around 1.7 kJ/mol per base, the first best and the last worst sequences are displayed in Table S3 in Text S1). We repeated the same calculation for one million random sequences and we obtained equivalent results. Placing one CpG step every helical turn gives an average energetic difference between minor groove and major groove methylation of 15 kJ/ mol, which translates into ,0.5 kJ/mol per methyl group, 2 kJ/ mol per base for the largest effects. Considering that not all nucleosome base pair steps are likely to be CpG steps, we can conclude that the balance between the destabilization due to CpG methylation and sequence repositioning will depend on the PLOS Computational Biology | www.ploscompbiol.org 4 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning Figure 3. Methylated and non-methylated DNA elastic deformation energies. (A) Distribution of deformation energies for 147 bplong random DNA sequences with CpG steps positioned every 10 base steps (one helical turn) in minor (red and dark red) and major (light and dark blue) grooves respectively. The energy values were rescaled by the slope of a best-fit straight line of figure 2, which is 0.23, to source of circulating FGF-21. The lack of association between circulating and muscle-expressed FGF-21 also suggests that muscle FGF-21 primarily works in a local manner regulating glucose metabolism in the muscle and/or signals to the adipose tissue in close contact to the muscle. Our study has some limitations. The number of subjects is small and some correlations could have been significant with greater statistical power. Another aspect is that protein levels of FGF-21 were not determined in the muscles extracts, consequently we cannot be sure the increase in FGF-21 mRNA is followed by increased protein expression. In conclusion, we show that FGF-21 mRNA is increased in skeletal muscle in HIV patients and that FGF-21 mRNA in muscle correlates to whole-body (primarily reflecting muscle) insulin resistance. These findings add to the evidence that FGF-21 is a myokine and that muscle FGF-21 might primarily work in an autocrine manner. Acknowledgments We thank the subjects for their participation in this study. Ruth Rousing, Hanne Willumsen, Carsten Nielsen and Flemming Jessen are thanked for excellent technical help. The Danish HIV-Cohort is thanked for providing us HIV-related data. PLOS ONE | www.plosone.org 6 March 2013 | Volume 8 | Issue 3 | e55632 Muscle FGF-21,Insulin Resistance and Lipodystrophy Author Contributions Conceived and designed the experiments: BL BKP JG. Performed the experiments: BL TH TG CF PH. Analyzed the data: BL CF PH. Contributed reagents/materials/analysis tools: BL. Wrote the paper: BL. References 1. Kharitonenkov A, Shiyanova TL, Koester A, Ford AM, Micanovic R, et al. (2005) FGF-21 as a novel metabolic regulator. J Clin Invest 115: 1627–1635. 2. Coskun T, Bina HA, Schneider MA, Dunbar JD, Hu CC, et al. (2008) Fibroblast growth factor 21 corrects obesity in mice. Endocrinology 149: 6018– 6027. 3. Xu J, Lloyd DJ, Hale C, Stanislaus S, Chen M, et al. 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Vienberg SG, Brons C, Nilsson E, Astrup A, Vaag A, et al. (2012) Impact of short-term high-fat feeding and insulin-stimulated FGF21 levels in subjects with low birth weight and controls. Eur J Endocrinol 167: 49–57. 13. Carr A, Samaras K, Burton S, Law M, Freund J, et al. (1998) A syndrome of peripheral lipodystrophy, hyperlipidaemia and insulin resistance in patients receiving HIV protease inhibitors. AIDS 12: F51–F58. 14. Haugaard SB, Andersen O, Dela F, Holst JJ, Storgaard H et al. (2005) Defective glucose and lipid metabolism in human immunodeficiency virus-infected patients with lipodystrophy involve liver, muscle tissue and pancreatic betacells. Eur J Endocrinol 152: 103–112. 15. Reeds DN, Yarasheski KE, Fontana L, Cade WT, Laciny E, et al. (2006) Alterations in liver, muscle, and adipose tissue insulin sensitivity in men with HIV infection and dyslipidemia. Am J Physiol Endocrinol Metab 290: E47–E53. 16. Meininger G, Hadigan C, Laposata M, Brown J, Rabe J, et al. 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(2007) Inhibition of lipolysis stimulates peripheral glucose uptake but has no effect on endogenous glucose production in HIV lipodystrophy. Diabetes 56: 2070–2077. 21. DeFronzo RA, Tobin JD, Andres R (1979) Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol 237: E214–E223. 22. Plomgaard P, Bouzakri K, Krogh-Madsen R, Mittendorfer B, Zierath JR, et al. (2005) Tumor necrosis factor-alpha induces skeletal muscle insulin resistance in healthy human subjects via inhibition of Akt substrate 160 phosphorylation. Diabetes 54: 2939–2945. 23. Thomas JA, Schlender KK, Larner J (1968) A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose. Anal Biochem 25: 486–499. 24. Haugaard SB, Andersen O, Madsbad S, Frosig C, Iversen J, et al. (2005) Skeletal Muscle Insulin Signaling Defects Downstream of Phosphatidylinositol 3-Kinase at the Level of Akt Are Associated With Impaired Nonoxidative Glucose Disposal in HIV Lipodystrophy. Diabetes 54: 3474–3483. 25. Boden G, Jadali F, White J, Liang Y, Mozzoli M, et al. (1991) Effects of fat on insulin-stimulated carbohydrate metabolism in normal men. J Clin Invest 88: 960–966. 26. Mashili FL, Austin RL, Deshmukh AS, Fritz T, Caidahl K, et al. (2011) Direct effects of FGF21 on glucose uptake in human skeletal muscle: implications for type 2 diabetes and obesity. Diabetes Metab Res Rev 27: 286–297. 27. Torriani M, Thomas BJ, Barlow RB, Librizzi J, Dolan S, et al. (2006) Increased intramyocellular lipid accumulation in HIV-infected women with fat redistribution. J Appl Physiol 100: 609–614. 28. Lee MS, Choi SE, Ha ES, An SY, Kim TH, et al. (2012) Fibroblast growth factor-21 protects human skeletal muscle myotubes from palmitate-induced insulin resistance by inhibiting stress kinase and NF-kappaB. Metabolism . 29. Tyynismaa H, Carroll CJ, Raimundo N, Ahola-Erkkila S, Wenz T, et al. (2010) Mitochondrial myopathy induces a starvation-like response. Hum Mol Genet 19: 3948–3958. 30. Maagaard A, Holberg-Petersen M, Kollberg G, Oldfors A, Sandvik L, et al. (2006) Mitochondrial (mt)DNA changes in tissue may not be reflected by depletion of mtDNA in peripheral blood mononuclear cells in HIV-infected patients. Antivir Ther 11: 601–608. 31. Payne BA, Wilson IJ, Hateley CA, Horvath R, Santibanez-Koref M, et al. (2011) Mitochondrial aging is accelerated by anti-retroviral therapy through the clonal expansion of mtDNA mutations. Nat Genet 43: 806–810. 32. Kliewer SA, Mangelsdorf DJ (2010) Fibroblast growth factor 21: from pharmacology to physiology. Am J Clin Nutr 91: 254S–257S. 33. Gallego-Escuredo JM, Domingo P, Gutierrez MD, Mateo MG, Cabeza MC, et al. (2012) Reduced Levels of Serum FGF19 and Impaired Expression of Receptors for Endocrine FGFs in Adipose Tissue From HIV-Infected Patients. J Acquir Immune Defic Syndr 61: 527–534. 34. Domingo P, Gallego-Escuredo JM, Domingo JC, Gutierrez MM, Mateo MG, et al. (2010) Serum FGF21 levels are elevated in association with lipodystrophy, insulin resistance and biomarkers of liver injury in HIV-1-infected patients. AIDS 24: 2629–2637. PLOS ONE | www.plosone.org 7 March 2013 | Volume 8 | Issue 3 | e55632
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